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1.
Nat Commun ; 10(1): 4065, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492838

RESUMO

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per-O-acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N-azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O-propionylated GalNAz (1,3-Pr2GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG.


Assuntos
Acetilglucosamina/metabolismo , Monossacarídeos/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Receptores Estrogênicos/metabolismo , Animais , Azidas/química , Azidas/metabolismo , Linhagem Celular Tumoral , Autorrenovação Celular , Células Cultivadas , Glicosilação , Células HeLa , Hexosaminas/metabolismo , Humanos , Células MCF-7 , Camundongos , Monossacarídeos/química , Células-Tronco Embrionárias Murinas/citologia , Células NIH 3T3 , Células-Tronco Pluripotentes/citologia , Processamento de Proteína Pós-Traducional
2.
Biotechnol Lett ; 41(10): 1187-1200, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418101

RESUMO

OBJECTIVES: Bifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate. RESULTS: A new BP-2 strain that produces alginate lyase was screened and identified from rotted Sargassum. A new alginate lyase, Alg17B, belonging to the polysaccharide lyase family 17, was isolated and purified from BP-2 fermentation broth by freeze-drying, dialysis, and ion exchange chromatography. The enzymatic properties of the purified lyase were investigated. The molecular weight of Alg17B was approximately 77 kDa, its optimum reaction temperature was 40-45 °C, and its optimum reaction pH was 7.5-8.0. The enzyme was relatively stable at pH 7.0-8.0, with a temperature range of 25-35 °C, and the specific activity of the purified enzyme reached 4036 U/mg. A low Na+ concentration stimulated Alg17B enzyme activity, but Ca2+, Zn2+, and other metal ions inhibited it. Substrate specificity analysis, thin-layer chromatography, and mass spectrometry showed that Alg17B is an alginate lyase that catalyses the hydrolysis of sodium alginate, polymannuronic acid (polyM) and polyguluronic acid to produce monosaccharides and low molecular weight oligosaccharides. Alg17B is also bifunctional, exhibiting both endolytic and exolytic activities toward alginate, and has a wide substrate utilization range with a preference for polyM. CONCLUSIONS: Alg17B can be used to saccharify the main carbohydrate, alginate, in the ethanolic production of brown algae fuel as well as in preparing and researching oligosaccharides.


Assuntos
Organismos Aquáticos/enzimologia , Gammaproteobacteria/enzimologia , Polissacarídeo-Liase/isolamento & purificação , Polissacarídeo-Liase/metabolismo , Sargassum/microbiologia , Alginatos/metabolismo , Ácido Algínico/metabolismo , Organismos Aquáticos/classificação , Organismos Aquáticos/genética , Organismos Aquáticos/isolamento & purificação , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Monossacarídeos/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeos Bacterianos/metabolismo , Especificidade por Substrato , Temperatura Ambiente
3.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426432

RESUMO

Sugar transporters of the SWEET family mediate cross membrane movement of mono- and disaccharides and play vital roles in diverse physiological and pathophysiological processes, including sink-source relationship, pathogen responses, reproductive growth, and development. However, it remains to be determined how these transporters function in non-module plants of agricultural significance, given the evolutionarily diverse traits. In this study, we combined transcriptome analysis, rapid amplification of cDNA ends-cloning (RACE-cloning), expression profiling, and heterologous functional assay to identify SWEET genes that may have potential roles during flower opening and sexual reproduction in Jasminum sambac . During the anthesis, the floral organs of J. sambac express seven SWEET homologous genes from all four clades of the family. JsSWEET9 and 2 are significantly upregulated when flowers are fully opened, up to 6- and 3-fold compared to unopened buds, respectively. The other transporters, JsSWEET1, 5, 10, and 17 are also accumulated slightly at stage associated with fragrance release, whereas only the vacuole transporter JsSWEET16 showed small decrease in transcript level after anthesis. The JsSWEET5, a clade II member, is capable to complement yeast cell uptake on most tested sugar substrates with a preference for hexoses, while the clade I transporter JsSWEET1 mediates merely galactose import when expressed in yeast. Our results provide first evidence for further investigation on sugar transport and allocation during flowering and reproductive processes in J. sambac.


Assuntos
Flores/genética , Jasminum/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Plantas/genética , Clonagem Molecular , Dissacarídeos/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Jasminum/crescimento & desenvolvimento , Jasminum/metabolismo , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/análise , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Monossacarídeos/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo
4.
PLoS One ; 14(5): e0217435, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31120985

RESUMO

Sudangrass, Sorghum sudanense (Piper) Stapf, is a vigorous forage crop that has also been used for biogas, paper, and electricity production. Due to the large biomass yields achieved by sudangrass and the large area of potential growth in Argentina seven sudangrass accessions from a collection of S. sudanense were analyzed to evaluate their potential as feedstocks for lignocellulosic bioethanol production, and to assess whether there is an association between the response to biotic and abiotic stresses and the composition of the biomass. The biomass composition was analyzed for major cell wall polymers, monosaccharides, and elemental composition. On average, 68% of stem lignocellulosic biomass was comprised of matrix polysaccharides and crystalline cellulose, representing a potential source of sugars for bioethanol production. Xylose was the predominant matrix polysaccharide monosaccharide comprising, on average, 45% of the total sugars, followed by arabinose, glucose, galactose, galacturonic acid, mannose, glucuronic acid, and fucose. Rhamnose was not detected in any of the biomasses analyzed. Silica was the most abundant element in sudangrass stem, followed by chloride, calcium, phosphorus and sulfur. We performed saccharification analyses after pretreatments. Alkaline pretreatment was more effective than water pretreatment. Sodium hydroxide pretreatment exposed different levels of recalcitrance among sudangrass accessions, whereas the water pretreatment did not. Phenological traits were also evaluated, showing significant variability among accessions. The comparison of major cell wall polymers and monosaccharide composition between tolerant and susceptible accessions to abiotic and biotic stresses suggests an association between the composition of the biomass and the response to stress.


Assuntos
Fontes de Energia Bioelétrica , Biomassa , Etanol/metabolismo , Lignina/metabolismo , Sorghum/fisiologia , Argentina , Parede Celular/química , Parede Celular/metabolismo , Lignina/análise , Monossacarídeos/análise , Monossacarídeos/metabolismo , Polissacarídeos/análise , Polissacarídeos/metabolismo , Sorghum/química , Estresse Fisiológico
5.
Gastroenterol Nurs ; 42(2): 150-158, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30946302

RESUMO

Patients with irritable bowel syndrome (IBS) suffer from abdominal pain, bloating, and abnormal defecation. Reducing the dietary intake of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) has been shown to be beneficial in reducing IBS symptoms. However, diet modification plays an important role in the composition of colonic microbiota. Currently, the effects of a FODMAP diet on the composition of the gut microbiome are not known. We conducted a systematic review to determine (1) the effectiveness of low-FODMAPs diet to reduce symptoms of patients with IBS and (2) the association between a low-FOMAPs diet and the composition of gut microbiome. Four electronic databases were searched using key words "IBS" or "irritable bowel syndrome," and "FODMAP" or "FODMAPs" or "fermentable oligosaccharides, disaccharides, monosaccharides, and polyols," and "microbiome." Two reviewers (H.S. and Y.T.L.) selected and reviewed articles according to our inclusion criteria. A total of 87 articles were reviewed and 7 met inclusion criteria. Based on the systematic review, low FODMAPs appear to reduce gastrointestinal symptoms for a least a subset of patients with IBS. However, due to the heterogeneity of reviewed studies, the influence on patients' gut microbiome composition and/or microbiota metabolites requires additional studies.


Assuntos
Dieta com Restrição de Carboidratos/métodos , Microbioma Gastrointestinal , Síndrome do Intestino Irritável/dietoterapia , Dissacarídeos/metabolismo , Feminino , Fermentação/fisiologia , Humanos , Síndrome do Intestino Irritável/diagnóstico , Masculino , Monossacarídeos/metabolismo , Oligossacarídeos/metabolismo , Polímeros/metabolismo , Prognóstico , Ensaios Clínicos Controlados Aleatórios como Assunto , Índice de Gravidade de Doença , Resultado do Tratamento
6.
Bioresour Technol ; 281: 239-249, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30825827

RESUMO

Biomass grown in wastewater treatment photobioreactors is a cheap raw material with high contents of carbohydrates, proteins and lipids. This work studies the production of fermentable monosaccharides from three biomasses grown in piggery wastewater (P), domestic wastewater (W) and synthetic medium (S) by applying chemical pretreatment and enzymatic hydrolysis, using a Taguchi design. ANOVA identified temperature, chemical reagent type and chemical reagent concentration as significant operational parameters. However, the biomass concentration, pretreatment time, enzyme dosage and enzymatic hydrolysis time had no remarkable effect. The bacterial content of the biomass had no relevant impact on carbohydrate and protein solubilisation but had a remarkable effect on the degradation of the released carbohydrates (57, 60 and 37% for P, W and S), while also affecting lipid solubilisation. Pretreatment with HCl 2 M at 120 °C resulted the optimal conditions, achieving a monosaccharide recovery of 53, 59 and 80% for P, W and S biomasses, respectively.


Assuntos
Biomassa , Fermentação , Monossacarídeos/metabolismo , Fotobiorreatores , Águas Residuárias/química , Metabolismo dos Carboidratos , Carboidratos , Hidrólise , Temperatura Ambiente
7.
Methods Mol Biol ; 1954: 237-243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864136

RESUMO

The glycosyltransferases (GTs) are an important subclass of enzymes that catalyze the biosynthesis of glycosidic bonds in oligosaccharides, polysaccharides and glycoconjugates by transferring a sugar residue from a donor substrate to an acceptor substrate. The membrane-associated GTs play a vital role in the biosynthesis of bacterial cell-wall polysaccharides. Characterization and quantification of GT activities is important for studies of biosynthesis of polysaccharides, drug target development, and production of bacterial products. In this chapter, colorimetric assays for the measurement of GT activities will be presented. Assays for GTs acting on monosaccharide-derivatives are based on the cleavage of unreacted glycosyl-p-nitrophenol acceptors followed by detection of p-nitrophenolate. GT reactions coupled with phosphatases and detection of inorganic phosphate are suitable for most GTs. These assays permit convenient quantification of GT activities and kinetics without the use of radioactive sugars.


Assuntos
Proteínas de Bactérias/metabolismo , Colorimetria/métodos , Ensaios Enzimáticos/métodos , Glicosiltransferases/metabolismo , Neisseria meningitidis/enzimologia , Proteínas de Bactérias/análise , Glicosiltransferases/análise , Humanos , Cinética , Infecções Meningocócicas/microbiologia , Monossacarídeos/metabolismo , Neisseria meningitidis/química , Nitrofenóis/análise , Nitrofenóis/metabolismo , Especificidade por Substrato
8.
J Pregnancy ; 2019: 9514546, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30854239

RESUMO

Objective: To examine the expression of hypoxia-inducible factor-1α (HIF-1α), TfR1, and TfR1-attached terminal monosaccharides in placentas of women with IDAP and severe preeclampsia. Methods: TfR1 and HIF-1α were detected by western blot. Immunoadsorption of TfR1 was performed to characterize the terminal monosaccharides by specific lectin binding. Results: There was no difference in the expression of TfR1 and HIF-1α between groups. Lectin blot analysis pointed out an overexpression of galactose ß1-4 N-acetylglucosamine (Gal-GlcNAc) and mannose in severe preeclampsia. Conclusion: The increase in Gal-GlcNAc may be due to the increased presence of antennary structures and the mannose glycans of TfR1 may indicate the presence of misfolded or incomplete proteins. These findings may be associated with the low expression of placental TfR1 in women with preeclampsia.


Assuntos
Acetilglucosamina/genética , Acetilglucosamina/metabolismo , Anemia Ferropriva/genética , Anemia Ferropriva/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Complicações Hematológicas na Gravidez/genética , Complicações Hematológicas na Gravidez/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Adolescente , Adulto , Feminino , Expressão Gênica , Glicosilação , Humanos , Manose/genética , Manose/metabolismo , Monossacarídeos/genética , Monossacarídeos/metabolismo , Gravidez , Adulto Jovem
9.
Appl Biochem Biotechnol ; 188(4): 977-990, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30761446

RESUMO

In this study, bioethanol was produced from the seaweed Gelidium amansii as biomass through separate hydrolysis and fermentation (SHF) processes. The SHF processes examined in this study include thermal acid hydrolysis pretreatment, enzymatic saccharification, detoxification, and fermentation. Thermal acid hydrolysis pretreatment was conducted using H2SO4, with a slurry content of 8-16% and treatment time of 15-75 min. The optimal conditions for thermal acid hydrolysis pretreatment were 12% (w/v) seaweed slurry content and 180 mM H2SO4 at 121 °C for 45 min, at which 26.1 g/L galactose and 6.8 g/L glucose were produced. A monosaccharide (mainly glucose) was also obtained from the enzymatic saccharification of thermal acid hydrolysate using 16 U/mL Celluclast 1.5 L enzyme at 45 °C for 36 h. Detoxification was performed using the adsorption method with activated carbon, the overliming method with Ca (OH)2, and the ion exchange method with polyethyleneimine. Among those detoxification methods, activated carbon showed the best performance for hydroxymethylfurfural removal. Ethanol fermentation was performed using 12% (w/v) seaweed hydrolysate with Saccharomyces cerevisiae adapted to galactose as well as various detoxification treatments.


Assuntos
Alga Marinha/metabolismo , Adsorção , Biomassa , Fermentação/fisiologia , Furaldeído/análogos & derivados , Furaldeído/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Hidrólise , Monossacarídeos/metabolismo
10.
Food Chem ; 285: 204-212, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30797336

RESUMO

Prebiotic fructooligosaccharides (FOS) are currently obtained by enzymatic reaction with fructosyltransferases (FTFs) using sucrose as both donor and acceptor. In these reactions glucose results as the most abundant by-product, arising from each fructosyl transfer event and, together with fructose, because of the inherent hydrolytic activity of the FTFs. As FOS are mainly used as prebiotic in nutraceutical foods, the reduction or total elimination of monosaccharides is required. In this work the selective elimination of monosaccharides from a synthetic FOS mixture was achieved through the selective complexation of glucose and fructose with phenyl boronic acid (PBAc) followed by ethyl-acetate extraction. The process was applied to a complex mixture of FOS obtained in an enzymatic synthesis reaction containing 40% glucose, 15.8% fructose and 35% of FOS, elimination of the sugars was achieved through 3:1 molar reactions, resulting in a levan-type FOS product with 97% purity.


Assuntos
Ácidos Borônicos/metabolismo , Monossacarídeos/metabolismo , Oligossacarídeos/isolamento & purificação , Acetatos/química , Ácidos Borônicos/química , Cromatografia em Camada Delgada , Escherichia coli/metabolismo , Frutose/química , Glucose/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Extração Líquido-Líquido , Monossacarídeos/química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Prebióticos/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
11.
Nat Commun ; 10(1): 407, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679446

RESUMO

Plants are dependent on controlled sugar uptake for correct organ development and sugar storage, and apoplastic sugar depletion is a defense strategy against microbial infections like rust and mildew. Uptake of glucose and other monosaccharides is mediated by Sugar Transport Proteins, proton-coupled symporters from the Monosaccharide Transporter (MST) superfamily. We present the 2.4 Å structure of Arabidopsis thaliana high affinity sugar transport protein, STP10, with glucose bound. The structure explains high affinity sugar recognition and suggests a proton donor/acceptor pair that links sugar transport to proton translocation. It contains a Lid domain, conserved in all STPs, that locks the mobile transmembrane domains through a disulfide bridge, and creates a protected environment which allows efficient coupling of the proton gradient to drive sugar uptake. The STP10 structure illuminates fundamental principles of sugar transport in the MST superfamily with implications for both plant antimicrobial defense, organ development and sugar storage.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Monossacarídeos/metabolismo , Açúcares/metabolismo , Simportadores/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Transporte Biológico/fisiologia , Glucose/metabolismo , Transporte de Íons/fisiologia , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/genética , Conformação Proteica , Simportadores/genética , Xenopus
12.
Phytomedicine ; 55: 255-263, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668437

RESUMO

BACKGROUND: We previously showed that 3-O-ß-D-glucopyranosyl-(3R)-hydroxybutanolide (kinsenoside), a major compound of Anoectochilus formosanus, increased lipolysis through an AMP-activated protein kinase (AMPK)-dependent pathway. PURPOSE: To extend our previous finding, we investigated the in vivo and in vitro effects of kinsenoside on lipolysis and the involvement of cyclic AMP (cAMP)-dependent protein kinase A (PKA) and AMPK in kinsenoside-mediated lipolysis. STUDY DESIGN/METHODS: Mice were fed a high-fat diet for six weeks to induce lipid deposition and then treated with 50 and 100  mg/kg kinsenoside for two weeks. The coordination of PKA and AMPK activation in lipolysis in C3H10T1/2 adipocytes was evaluated in vitro by using PKA and AMPK's corresponding inhibitors, oil-red O staining, a glycerol production assay, and Western blot analysis. RESULTS: Kinsenoside reduced body weight, fat pad mass, and hepatic lipid accumulation in obese mice, and concurrently increased the induction and activation of hormone-sensitive lipase (HSL), perilipin, adipose triglyceride lipase (ATGL), and carnitine palmitoyltransferase I (CPT1). Kinsenoside concentration-dependently increased PKA activation by increasing the phosphorylation of Ser/Thr-PKA substrates in vitro. These increases were accompanied by a reduction in fat accumulation. Using H89 and Rp-8-Br-cAMPs to inhibit PKA reduced the release of glycerol but did not alter the activation of peroxisome proliferator-activated receptor alpha or the expression of CPT1 or ATGL. By contrast, compound C, an AMPK inhibitor, inhibited CPT1 and ATGL expression in kinsenoside-treated C3H10T1/2 adipocytes. In addition, H89 caused the reactivation of AMPK downstream targets by increasing the levels of the active form of pAMPK-Thr172, suggesting that PKA negatively modulates AMPK activity. CONCLUSION: Kinsenoside increased HSL activation through PKA-mediated phosphorylation at Ser660/563 and concomitantly increased perilipin activation in lipolysis. These lipolytic effects of kinsenoside were validated using 6-Bnz-cAMPs, a PKA agonist. In this study, we demonstrated that in addition to AMPK, PKA also plays a crucial role in kinsenoside-mediated lipolysis.


Assuntos
4-Butirolactona/análogos & derivados , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Lipólise/efeitos dos fármacos , Monossacarídeos/metabolismo , Extratos Vegetais/metabolismo , Esterol Esterase/metabolismo , 4-Butirolactona/metabolismo , Animais , Masculino , Camundongos , Orchidaceae/química , Extratos Vegetais/química
13.
J Microbiol Biotechnol ; 29(3): 410-418, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30518022

RESUMO

To investigate a novel ß-glucosidase from Bifidobacterium breve ATCC 15700 (BbBgl) to produce compound K (CK) via ginsenoside F2 by highly selective and efficient hydrolysis of the C-3 glycoside from ginsenoside Rd, the BbBgl gene was cloned and expressed in E. coli BL21. The recombinant BbBgl was purified by Ni-NTA magnetic beads to obtain an enzyme with specific activity of 37 U/mg protein using pNP-Glc as substrate. The enzyme activity was optimized at pH 5.0, 35°C, 2 or 6 U/ml, and its activity was enhanced by Mn2+ significantly. Under the optimal conditions, the half-life of the BbBgl is 180 h, much longer than the characterized ß-glycosidases, and the Km and Vmax values are 2.7 mM and 39.8 µmol/mg/min for ginsenoside Rd. Moreover, the enzyme exhibits strong tolerance against high substrate concentration (up to 40 g/l ginsenoside Rd) with a molar biotransformation rate of 96% within 12 h. The good enzymatic properties and gram-scale conversion capacity of BbBgl provide an attractive method for large-scale production of rare ginsenoside CK using a single enzyme or a combination of enzymes.


Assuntos
Bifidobacterium breve/metabolismo , Ginsenosídeos/metabolismo , Glucose/metabolismo , Monossacarídeos/metabolismo , beta-Glucosidase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bifidobacterium breve/genética , Biotransformação , Clonagem Molecular , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Ginsenosídeos/biossíntese , Ginsenosídeos/química , Glicosídeos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificação
14.
Int J Biol Macromol ; 125: 27-34, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30521907

RESUMO

BSH-1 is an O­acetyl-arabinoxylan obtained from bamboo shavings. This study investigated its fermentation behavior by human colonic microbiota in vitro. Results showed that BSH-1 remarkably modulated the composition of human colonic microbiota, mainly by increasing the growth of potential beneficial genera (i.e. Bifidobacterium, Lactobacillus, Bacteroides, Prevotella_7, Parabacteroides) and by decreasing the growth of potential harmful genera (i.e. Fusobacterium, Lachnospiraceae_UCG-008, Bilophila and Desulfovibrio). BSH-1 significantly promoted the production of short-chain fatty acids, especially acetic, propionic and n-butyric acids. After 48 h fermentation, the concentration of n-butyric acid in BSH-1 fermentation culture was increased by 2.41 times compared to the blank. During fermentation, the activity of acetyl xylan esterase, arabinofuranosidase, xylanase and xylosidase was enhanced. Moreover, free arabinose, xylose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose were detected. These results suggest that BSH-1 could potentially be a functional ingredient to improve gut health.


Assuntos
Fermentação , Microbioma Gastrointestinal , Sasa , Xilanos/metabolismo , Metabolismo dos Carboidratos , Carboidratos/química , Colo/microbiologia , Ácidos Graxos Voláteis/biossíntese , Fezes/microbiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Estrutura Molecular , Monossacarídeos/química , Monossacarídeos/metabolismo , Xilanos/química
15.
J Basic Microbiol ; 59(3): 256-266, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30589089

RESUMO

Arabitol has several applications in food and pharmaceutical industries as a natural sweetener, dental caries inhibitor, and texturing agent. Newly isolated yeast strains from seawater, sugarcane plantation soil samples, and Zygosaccharomyces rouxii 2635 from MTCC were tested for arabitol production. The yield of arabitol was found to be higher in seawater isolate (24.6 g L-1 ) compared to two soil isolates (22.5 g L-1 ) and Z. rouxii (19.4 g L-1 ). Based on ITS 26S rDNA sequence analysis, the seawater isolate was identified as Pichia manchurica. In the present study, the effect of different substrates, trace elements, nitrogen sources, pH, and temperature on arabitol production was examined. Three different carbon sources viz. glucose, arabinose, and galactose were studied. Glucose was determined to be the best substrate for arabitol production (27.6 g L-1 ) followed by arabinose (13.7 g L-1 ) and galactose (7.7 g L-1 ). Maximum production of arabitol was observed at pH 6.0 (34.7 g L-1 ). In addition, arabitol production was high (35.7 g L-1 ) at temperature of 30 °C. Among the different concentrations of ammonium sulfate tested (3, 4.5, 6, 7.5, and 9 g L-1 ) concentration of 6 g L-1 resulted in higher arabitol Individual metal ions had no effect on arabitol production by this strain as compared to control. Results obtained in this study identify ways for improved arabitol production with natural isolates using microbial processes.


Assuntos
Pichia/metabolismo , Álcoois Açúcares/metabolismo , Sulfato de Amônio/química , Concentração de Íons de Hidrogênio , Monossacarídeos/metabolismo , Filogenia , Pichia/classificação , Pichia/crescimento & desenvolvimento , Pichia/isolamento & purificação , RNA Ribossômico/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Especificidade por Substrato , Temperatura Ambiente
16.
Aliment Pharmacol Ther ; 49(2): 124-139, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30589971

RESUMO

BACKGROUND: Despite the efficacy of a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) for patients with irritable bowel syndrome, many questions remain unanswered with respect to its clinical implementation. AIM: To review literature to identify, synthesise, and provide direction for future research in the implementation and evaluation of the low FODMAP diet. METHODS: Bibliographical searches were performed in Ovid Medline, CINAHL, Scopus and PubMed from database commencement until September 2018, with search terms focused on the population (irritable bowel syndrome) and intervention of interest (FODMAP). RESULTS: Predictors of response to a low FODMAP diet remain under investigation, with preliminary data supporting faecal microbiota or faecal volatile organic compound profiling. Training of clinicians, and standards for the education of patients about the phases of a low FODMAP diet, as well as the role of Apps, require formal evaluation. There are limited data on the longer term efficacy and safety of the low FODMAP diet with respect to sustained symptom control, effect on quality of life and healthcare utilisation, nutritional adequacy, precipitation of disordered eating behaviours, effects on faecal microbiota and metabolomic markers, and subsequent translation to clinical effects. CONCLUSIONS: Many gaps in implementation of the low FODMAP diet in clinical practice, as well as long-term safety and efficacy, remain for further investigation.


Assuntos
Dieta com Restrição de Carboidratos/métodos , Dissacarídeos/deficiência , Síndrome do Intestino Irritável/dietoterapia , Monossacarídeos/deficiência , Oligossacarídeos/deficiência , Polímeros , Dissacarídeos/metabolismo , Fermentação/fisiologia , Microbioma Gastrointestinal/fisiologia , Humanos , Síndrome do Intestino Irritável/diagnóstico , Síndrome do Intestino Irritável/metabolismo , Monossacarídeos/metabolismo , Oligossacarídeos/metabolismo , Polímeros/metabolismo , Estudos Prospectivos , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos
17.
Sci Rep ; 8(1): 14297, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30250238

RESUMO

Non-alcoholic fatty liver disease (NAFLD) has emerged as a public health concern as reflected in its widespread distribution in the general population. Yet, treatment options are scarce which is at least in part due to lack of reliable human in vitro disease models. Here, we report a human hepatic 3D spheroid system cultured under defined chemical conditions that has the potential to mimic steatotic conditions in a reversible manner, useful for identification of novel drug treatment conditions. Primary human hepatocytes (PHH) from different donors were cultured as spheroid microtissues in physiological in vivo -like culture conditions. Hepatic steatosis was induced over the course of three weeks in culture by supplementing the culture medium with pathophysiological concentrations of free fatty acids, carbohydrates and insulin. Effects of steatosis in the 3D system were evaluated on transcriptional, metabolomic and lipidomic levels. Free fatty acids on one hand as well as a combination of insulin and monosaccharides, promoted lipid accumulation in hepatocytes and increased expression of lipogenic genes, such as fatty acid synthase. This milieu also promoted development of insulin resistance within 2 weeks as manifested by an increase in gluconeogenic and insulin resistance markers, which are observed in type 2 diabetes mellitus and metabolic syndrome. Induced steatosis was reversible after withdrawal of lipogenic substrates and a further reduction in cellular fat content was observed following treatment with different antisteatotic compounds, such as metformin, glucagon, olaparib and antioxidants. Taken together, these results demonstrate that the 3D hepatic spheroids can serve as a valuable, HTS compatible model for the study of liver steatosis and facilitate translational discovery of novel drug targets.


Assuntos
Fígado Gorduroso/patologia , Resistência à Insulina , Fígado/patologia , Modelos Biológicos , Esferoides Celulares/patologia , Adulto , Células Cultivadas , Ácidos Graxos/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/metabolismo , Insulina/farmacologia , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Fígado/metabolismo , Masculino , Metabolômica , Pessoa de Meia-Idade , Monossacarídeos/metabolismo , Esferoides Celulares/metabolismo , Xenobióticos/metabolismo , Adulto Jovem
18.
J Struct Biol ; 204(3): 498-506, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30244006

RESUMO

Substrate-binding proteins (SBPs) are periplasmic proteins consisting of two α/ß domains joined by a hinge region with specificity towards cognate ligands. Based on three-dimensional fold, sugar-specific SBPs have been classified into cluster B and cluster D-I. The analysis of sequences and structures of sugar-binding pocket of cluster D-I SBPs revealed the presence of extra residues on two loops (L1, L2) and a helix (H1) in few members of this family, that binds specifically to monosaccharides. Presence of conserved histidine in L2 and tryptophan in H1 can be considered as the identity marks for the cluster D-I monosaccharide-binding SBPs. A glucose binding protein (ppGBP) from Pseudomonas putida CSV86 was found to contain a structural fold similar to oligosaccharide-binding cluster D-I SBPs, but functionally binds to only glucose due to constriction of its binding pocket mainly by L2 (375-382). ppGBP with partial deletion of L2 (ppGBPΔL2) was created, crystallized and biochemical characterization was performed. Compared to wild type ppGBP, the ppGBPΔL2 structure showed widening of the glucose-binding pocket with ∼80% lower glucose binding. Our results show that the substrate specificity of SBPs can be altered by modulating the size of the binding pocket. Based on this, we propose a sub classification of cluster D-I SBPs into (i) cluster D-I(a)-monosaccharide-binding SBPs and (ii) cluster D-I(b)-oligosaccharide-binding SBPs. This study also provides the direct structural and functional correlation indicating that divergence of proteins may occur through insertions or deletions of sequences in the already existing SBPs leading to evolution at the functional level.


Assuntos
Proteínas de Bactérias/metabolismo , Glucose/metabolismo , Monossacarídeos/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Evolução Molecular , Glucose/química , Ligantes , Modelos Moleculares , Monossacarídeos/química , Mutação , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Filogenia , Conformação Proteica , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética
19.
Mol Cell Proteomics ; 17(11): 2107-2118, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30072579

RESUMO

Pseudomyxoma peritonei (PMP) is a subtype of mucinous adenocarcinoma that most often originates from the appendix, and grows in the peritoneal cavity filling it with mucinous ascites. KRAS and GNAS mutations are frequently found in PMP, but other common driver mutations are infrequent. As altered glycosylation can promote carcinogenesis, we compared N-linked glycan profiles of PMP tissues to those of normal appendix. Glycan profiles of eight normal appendix samples and eight low-grade and eight high-grade PMP specimens were analyzed by mass spectrometry. Our results show differences in glycan profiles between PMP and the controls, especially in those of neutral glycans, and the most prominent alteration was increased fucosylation. We further demonstrate up-regulated mRNA expression of four fucosylation-related enzymes, the core fucosylation performing fucosyltransferase 8 and three GDP-fucose biosynthetic enzymes in PMP tissues when compared with the controls. Up-regulated protein expression of the latter three enzymes was further observed in PMP cells by immunohistochemistry. We also demonstrate that restoration of fucosylation either by salvage pathway or by introduction of an expression of intact GDP-mannose 4,6-dehydratase enhance expression of MUC2, which is the predominant mucin molecule secreted by the PMP cells, in an intestinal-derived adenocarcinoma cell line with defective fucosylation because of deletion in the GDP-mannose 4,6-dehydratase gene. Thus, altered glycosylation especially in the form of fucosylation is linked to the characteristic mucin production of PMP. Glycomic data are available via ProteomeXchange with identifier PXD010086.


Assuntos
Fucose/metabolismo , Glicômica/métodos , Pseudomixoma Peritoneal/metabolismo , Apêndice/microbiologia , Apêndice/patologia , Linhagem Celular Tumoral , Glicosilação , Guanosina Difosfato/metabolismo , Humanos , Monossacarídeos/metabolismo , Mucina-2/metabolismo , Polissacarídeos/metabolismo , Análise de Componente Principal , Especificidade por Substrato
20.
Plant Sci ; 274: 121-128, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30080595

RESUMO

Durum wheat is naturally more susceptible to Fusarium graminerum infection in comparison to common wheat. The improvement of durum wheat resistance against F. graminearum is a challenge due to the lack of resistance sources in its gene pool. FHB-resistance factors were introduced in durum wheat by generating recombinant inbred lines (RILs), obtained by crossing the hexaploid resistant accession 02-5B-318 with the susceptible durum wheat cv. Saragolla. In this work we explored the possible contribution of cell wall (CW) in RILs with improved FHB resistance. We thoroughly studied CW components, mycotoxins content and the expression of related genes in different RILs selected for their extremely high and low resistance to FHB. Differences were found in resistant and susceptible lines in the degree of pectin methylesterification and in deoxynivalenol (DON) accumulation after fungal infection. Genes involved in biochemical modification of CW structure (WheatPme-1, Glu-1) and mycotoxins accumulation (ns-LTP-1) were analyzed as putative candidates for FHB resistance. Our results indicate that durum wheat plants with cell wall structure and gene response acquired from common wheat displayed an increased resistance to FHB.


Assuntos
Parede Celular/metabolismo , Resistência à Doença/fisiologia , Fusarium , Doenças das Plantas/microbiologia , Triticum/microbiologia , Parede Celular/fisiologia , Resistência à Doença/genética , Lignina/metabolismo , Monossacarídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Triticum/genética , Triticum/fisiologia
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