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1.
J Agric Food Chem ; 67(42): 11665-11674, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31588753

RESUMO

A long-term high-fat diet (HFD) can cause a range of health problems. Gut microbiota plays a decisive role in the development of HFD-associated inflammation, involved in function of T cells. This study was designed to probe the regulative effects of dietary stachyose, a functional oligosaccharide, on HFD-induced weight gain, inflammation, gut microbiota dysbiosis, and T cell abnormality in C57Bl/6 mice. Mice were divided into three groups which received normal chow, HFD and HFD plus stachyose (400 mg/kg), respectively. Results showed that administration of stachyose diminished the HFD-induced upregulation of serum TNF-α level and elevation of peripheral blood leukocyte populations to alleviate the HFD-caused colonic and hepatic inflammation in mice. Analysis of gut microbiota revealed that stachyose improved the intestinal homeostasis of HFD-fed mice by improving the bacterial diversity with the increases in the relative abundances of the Prevotellaceae_NK3B31_group, Parasutterella, Christensenellaceae_R-7_group, and Anaerovorax, as well as the fecal level of butanoic acid, while decreasing the ratio of Firmicutes-to-Bacteroidetes and the abundances of the Lachnospiraceae_NK4A136_group, Desulfovibrio, Anaerotruncus, Mucispirillum, Roseburia, and Odoribacter. Flow cytometric analysis showed that stachyose antagonized the HFD-induced decrease of peripheral CD4+ T cell population in mice. Conclusively, these findings suggest that long-term consumption of stachyose can ameliorate the HFD-associated colonic and hepatic inflammation and its complications by modulating gut microbiota.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Colo/imunologia , Disbiose/dietoterapia , Microbioma Gastrointestinal , Fígado/imunologia , Oligossacarídeos/metabolismo , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Colo/microbiologia , Dieta Hiperlipídica/efeitos adversos , Disbiose/imunologia , Disbiose/microbiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
2.
J Agric Food Chem ; 67(40): 11158-11166, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31537069

RESUMO

There are ongoing interests in improving the galactooligosaccharide (GOS) synthesis efficiency of ß-galactosidase by protein engineering. In this study, an intelligent double-hydrophobic amino acid scanning strategy was proposed and employed to target nine residues forming the glycon-binding site (-1 subsite) of ß-galactosidase Bgal1-3. Two mutants C510V and H512I with significantly improved GOS synthesis efficiency were obtained. When 40% (w/v) lactose was used as a substrate, Bgal1-3 reached a maximum GOS yield of 45.3% at 16 h, while the mutants reached higher yields in a much shorter time (59.1% at 10 h for C510V, 51.5% at 2 h for H512I). When skim milk was treated with these enzymes, more GOS was produced (19.9 g/L for C510V, 12.7 g/L for H512I) than that for Bgal1-3 (10.3 g/L) at a lactose conversion of 90%. These results validated hydrophobicity scanning as an efficient method to engineer ß-galactosidases into promising catalysts for the preparation of GOS and GOS-enriched milk.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Galactose/química , Oligossacarídeos/química , beta-Galactosidase/química , Sequência de Aminoácidos , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Galactose/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lactose/química , Oligossacarídeos/metabolismo , Engenharia de Proteínas , Alinhamento de Sequência , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
3.
Microb Cell Fact ; 18(1): 159, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542050

RESUMO

BACKGROUND: Xylanases randomly cleave the internal ß-1,4-glycosidic bonds in the xylan backbone and are grouped into different families in the carbohydrate-active enzyme (CAZy) database. Although multiple xylanases are detected in single strains of many filamentous fungi, no study has been reported on the composition, synergistic effect, and mode of action in a complete set of xylanases secreted by the same microorganism. RESULTS: All three xylanases secreted by Penicillium chrysogenum P33 were expressed and characterized. The enzymes Xyl1 and Xyl3 belong to the GH10 family and Xyl3 contains a CBM1 domain at its C-terminal, whereas Xyl2 belongs to the GH11 family. The optimal temperature/pH values were 35 °C/6.0, 50 °C/5.0 and 55 °C/6.0 for Xyl1, Xyl2, and Xyl3, respectively. The three xylanases exhibited synergistic effects, with the maximum synergy observed between Xyl3 and Xyl2, which are from different families. The synergy between xylanases could also improve the hydrolysis of cellulase (C), with the maximum amount of reducing sugars (5.68 mg/mL) observed using the combination of C + Xyl2 + Xyl3. Although the enzymatic activity of Xyl1 toward xylan was low, it was shown to be capable of hydrolyzing xylooligosaccharides into xylose. Xyl2 was shown to hydrolyze xylan to long-chain xylooligosaccharides, whereas Xyl3 hydrolyzed xylan to xylooligosaccharides with a lower degree of polymerization. CONCLUSIONS: Synergistic effect exists among different xylanases, and it was higher between xylanases from different families. The cooperation of hydrolysis modes comprised the primary mechanism for the observed synergy between different xylanases. This study demonstrated, for the first time, that the hydrolysates of GH11 xylanases can be further hydrolyzed by GH10 xylanases, but not vice versa.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/enzimologia , Polissacarídeos/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glucuronatos/metabolismo , Temperatura Alta , Hidrólise , Família Multigênica , Oligossacarídeos/metabolismo , Penicillium chrysogenum/química , Penicillium chrysogenum/genética , Domínios Proteicos , Xilanos/metabolismo
4.
J Agric Food Chem ; 67(38): 10702-10712, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31490688

RESUMO

Human milk oligosaccharides are complex carbohydrates with multibiofunctional health benefits to newborns. Human milk free oligosaccharides (HMOs) are well characterized. However, changes in the N/O-glycome during lactation are poorly reported. Herein, we qualitatively and quantitatively investigated N/O-glycome profiles and their alteration in human milk at different lactation stages. N-Glycans were mainly fucosylated and nonsialylated, nonfucosylated throughout lactation. O-Glycans mainly consisted of sialylated and nonsialylated, nonfucosylated in colostrum and transitional milk, and fucosylated and nonfucosylated, nonsialylated in mature milk. Fucosylated and sialylated N-glycans gradually decreased and increased, respectively, as lactation progressed; O-glycans showed the reverse. Interestingly, changes in HMO abundance decreased during lactation, complementing HMG N/O-glycome changes. In conclusion, temporal HMG glycosylation changes provide the groundwork for developing infant formula that is closer to breast milk at different lactation stages.


Assuntos
Glicoproteínas/química , Lactação , Leite Humano/química , Adulto , Colostro/química , Feminino , Glicoproteínas/metabolismo , Glicosilação , Humanos , Espectrometria de Massas , Leite Humano/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo
5.
J Agric Food Chem ; 67(38): 10744-10755, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31525900

RESUMO

We previously reported that ß-glucosidase BGL1 at low concentration (15 µg mL-1) from Coprinopsis cinerea exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 µg mL-1) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 µg mL-1) showed transglycosylation but BGL1 at high concentration (200 µg mL-1) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a ß(1 → 3) or ß(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-ß-d-gentiobiosyl-d-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.


Assuntos
Agaricales/enzimologia , Dissacarídeos/metabolismo , Proteínas Fúngicas/química , Oligossacarídeos/metabolismo , beta-Glucosidase/química , Agaricales/química , Agaricales/genética , Sequência de Aminoácidos , Dissacarídeos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Hidrólise , Cinética , Estrutura Molecular , Oligossacarídeos/química , Especificidade por Substrato , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
6.
J Agric Food Chem ; 67(37): 10373-10379, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453692

RESUMO

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase, which is an important enzyme for efficient saccharification of agarose or preparation of bioactive oligosaccharides from agarose. Although many ß-agarases have been reported and characterized, there are only a few studies on α-agarases. Here, we cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately 200 kDa belonging to glycoside hydrolase family 96 from Catenovulum agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37 °C and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as agarobiose (A2), agarotetraose (A4), and agarohexaose (A6), in which A4 is the dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose (A3) and A6 into A2 and A4 but cannot act on A2, A3, or A4. This is the first report to characterize the α-agarase action on AOSs in detail. Therefore, CaLJ96 has potential for the manufacture of bioactive AOSs.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Alteromonadaceae/química , Alteromonadaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sefarose/química , Sefarose/metabolismo , Especificidade por Substrato
7.
Food Chem ; 298: 124999, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31261010

RESUMO

Glycoside hydrolase family 8 (GH8) includes endoglucanases, lichenases, chitosanases and xylanases, which are essential for polysaccharides breakdown. In this work, we studied a thermally stable GH8 from the cellulose synthase complex of Enterobacter sp. R1, for deconstruction of ß-glucans. The biochemical characterization of the recombinant GH8ErCel showed high specificity towards barley ß-glucan and lichenan and lower activity on carboxymethylcellulose and swollen cellulose, yielding different length oligosaccharides. By molecular modeling, six conserved subsites for glucose binding and some possible determinants for its lack of xylanase and chitosanase activity were identified. GH8ErCel was active at a broad range of pH and temperature and presented remarkable stability at 60 °C. Additionally, it hydrolyzed ß-glucan from oat and wheat brans mainly to tri- and tetraoligosaccharides. Therefore, GH8ErCel may be a good candidate for enzymatic deconstruction of ß-glucans at high temperature in food and feed industries, including the production of prebiotics and functional foods.


Assuntos
Celulase/química , Celulase/metabolismo , Celulose/metabolismo , Enterobacter/enzimologia , beta-Glucanas/metabolismo , Argentina , Carboximetilcelulose Sódica/metabolismo , Celulase/genética , Enterobacter/genética , Enterobacter/isolamento & purificação , Estabilidade Enzimática , Glucanos/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Microbiologia do Solo , Especificidade por Substrato , Temperatura Ambiente , beta-Glucanas/química
8.
Bioresour Technol ; 289: 121755, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31301946

RESUMO

A novel approach was proposed for the production of xylooligosaccharides by direct pre-hydrolysis using gluconic acid as catalyst. Maximum xylooligosaccharides (degree of polymerization 2-6) yield of 53.2% could be obtained in 60 min through 5% gluconic acid hydrolysis of sugarcane bagasse at 150 °C. Furthermore, the yield of glucose from solids following gluconic acid hydrolysis treatment was 86.2% after fed-batch enzymatic hydrolysis with 10% solids loading. Results indicated that gluconic acid pretreatment combined with enzymatic hydrolysis could be successfully applied to sugarcane bagasse substrate. Subsequently, glucose could be efficiently bio-oxidized to gluconic acid by Gluconobacter oxydans ATCC 621H with 93.1% yield, and sugarcane bagasse derived gluconic acid has been proved to be an effective catalyst for xylooligosaccharides production. In this study, xylooligosaccharides production from sugarcane bagasse by gluconic acid hydrolysis demonstrated a great potential with respect to the production of these probiotics around the world.


Assuntos
Celulose/química , Gluconatos/metabolismo , Glucuronatos/metabolismo , Oligossacarídeos/metabolismo , Saccharum/química , Biocatálise , Glucose/metabolismo , Hidrólise
9.
Int J Nanomedicine ; 14: 4517-4528, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354271

RESUMO

Purpose: We developed a contrast agent for targeting E-selectin expression. We detected the agent using magnetic resonance imaging (MRI) in vivo in nude mice that had undergone nasopharyngeal carcinoma (NPC) metastasis. Methods: Sialyl Lewis X (sLeX) was conjugated with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. Hydrodynamic size, polydispersity index, and ζ-potential of USPIO-polyethylene glycol (PEG) nanoparticles and USPIO-PEG-sLeX nanoparticles were measured. Component changes in nanoparticles of USPIO, USPIO-PEG, and USPIO-PEG-sLeX were analyzed by thermogravimetric analysis and Fourier-transform infrared spectroscopy. A model of NPC metastasis to inguinal lymph nodes in nude mice was used to investigate characteristics of the USPIO-PEG-sLeX nanoparticles in vivo. We investigated the ability of the T2* value, change in T2* value (ΔT2* value), and enhancement rate (ER) to assess accumulation of USPIO-PEG-sLeX nanoparticles quantitatively in mice of a metastasis group and control group. Four MRI scans were undertaken for each mouse. The first scan (t0) was done before administration of USPIO-PEG-sLeX nanoparticles (0.1 mL) via the tail vein. The other scans were carried out at 0 (t1), 1 (t2), and 2 hours (t3) postinjection. The mean optical density was used to reflect E-selectin expression. Results: sLeX was labeled onto USPIO successfully. In vivo, there were significant interactions between the groups and time for T2* values after administration of USPIO-PEG-sLeX nanoparticles. Six parameters (T2* at t2, ΔT2* at t1, ΔT2* at t2, ER at t1, ER at t2, and ER at t3) were correlated with the mean optical density. Conclusion: USPIO-PEG-sLeX nanoparticles can be used to assess E-selectin expression quantitatively. Use of such molecular probes could enable detection of early metastasis of NPC, more accurate staging, and treatment monitoring.


Assuntos
Dextranos/química , Selectina E/metabolismo , Nanopartículas de Magnetita/química , Animais , Linhagem Celular Tumoral , Dextranos/ultraestrutura , Difusão Dinâmica da Luz , Feminino , Metástase Linfática , Imagem por Ressonância Magnética , Nanopartículas de Magnetita/ultraestrutura , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas/patologia , Metástase Neoplásica , Oligossacarídeos/metabolismo , Polietilenoglicóis/química , Eletricidade Estática , Termodinâmica
10.
Food Chem ; 297: 124945, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253310

RESUMO

Almond shell, a by-product obtained from the nut industry, was valorised into low degree of polymerisation xylooligosaccharides using alkaline pretreatment and enzymatic hydrolysis. The effect of particle size on hemicellulose recovery upon pretreatment was studied using 1 and 2 M NaOH. It was observed that particle size significantly influences hemicellulose recovery, as particles below 120 µm resulted in near complete recovery at 2 M NaOH. Enzymatic hydrolysis of hemicellulose was optimised using response surface methodology, to obtain efficient xylooligosaccharides production at low enzyme dose and high substrate concentration. For higher XOS yield, an enzyme dose of 10 U and substrate concentration <2% was optimal. The in-vitro human faecal fermentation study revealed no significant difference in gas and short chain fatty acid level among substrates evaluated. It was observed that short chain oligosaccharides produce higher level of acetate than medium chain oligosaccharides.


Assuntos
Fezes/microbiologia , Pentoses/química , Polissacarídeos/metabolismo , Técnicas de Cultura Celular por Lotes , Biomassa , Cromatografia Líquida de Alta Pressão , Endo-1,4-beta-Xilanases/metabolismo , Ácidos Graxos Voláteis/análise , Ácidos Graxos Voláteis/metabolismo , Fezes/química , Gases/química , Glucuronatos/análise , Glucuronatos/metabolismo , Humanos , Hidrólise , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Tamanho da Partícula , Polissacarídeos/química , Hidróxido de Sódio/química
11.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 805-815, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31222999

RESUMO

Hyaluronic acid (HA) is widely used in many fields, such as medicine, cosmetics and food. The bioactivity of HA depends on its molecular weight (Mw). Owing to the important physiological activities and special physiological functions, HA oligosaccharides have important application prospects in medicine fields. Streptococcus zooepidemicus has wide applications in commercial production of HA, due to its short fermentation cycle and strong production intensity. In order to efficiently synthesize HA oligosaccharides and solve the dissolved oxygen in the fermentation process, in this study, we overexpressed HA synthase (HasA) and introduced and optimized the leech hyaluronidase LHAase in Streptococcus zooepidemicus WSH-24. As a result, HA oligosaccharides were efficiently produced with improved dissolved oxygen. After 24 h, HA oligosaccharides production intensity reached to 294.2 mg/(L·h), and the concentration accumulated to 0.97 g/L in flask cultures, which was 1.82 times of the wild strain. Impressively, HA oligosaccharides were increased to 7.06 g/L in 3 L fermentor. The constructed Streptococcus zooepidemicus strain for producing HA oligosaccharides would have broad application prospects.


Assuntos
Microbiologia Industrial , Oligossacarídeos , Streptococcus equi/genética , Reatores Biológicos , Fermentação , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/genética , Ácido Hialurônico/metabolismo , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Streptococcus equi/metabolismo
12.
Food Chem ; 295: 311-319, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174763

RESUMO

A novel gene aga3027 from the genome of Flammeovirga sp. OC4, isolated from the deep sea, was screened and expressed in Escherichia coli BL21. This gene encoded the genetic information of a potential agarase that consists of 851 amino acids and belongs to 16 ß-agarase family of glycoside hydrolase. Purified recombinant Aga3027 demonstrated the maximum activity of agarase at 40 °C and pH 9.0, displaying excellent thermostability and pH-stability. The agarase retained more than 80% of its maximum activity after incubation at 30-40 °C for 48 h, or after incubation at pH 6.0-9.0 for 60 min, which indicated that this agarase was suitable for industrial applications. Silica gel chromatography was used to purify the hydrolysates of agar treated by agarase from the recombinant Aga3027. The hydrolysates were identified as neoagarotetraose and neoagarohexaose by thin layer chromatography and further confirmed by ion chromatography.


Assuntos
Ágar/química , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Ágar/metabolismo , Bacteroidetes/enzimologia , Bacteroidetes/genética , Cromatografia em Camada Delgada , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosídeos/metabolismo , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Oligossacarídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
13.
Life Sci ; 231: 116589, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31226416

RESUMO

AIMS: Despite the protective effect of galacto-oligosaccharides (GOS) on human colon has been widely-reported, the mechanism of its beneficial effect is still unclear. This paper aims to reveal the internal mechanism underlined the anti-colitis effect of GOS by studying its regulatory effect on miRNAs. MAIN METHODS: An in vitro model of colitis was constructed by using human colon epithelial FHC cells and lipopolysaccharide (LPS). An in vivo colitis model was established as well, by injecting Rag2-/- Sprague-Dawley (SD) rats with helicobacter hepaticus. The effects of GOS pre-treatment on these two models were tested, and the miRNAs involved in these effects were studied. KEY FINDINGS: The expression of miR-19b, miR-590-5p and miR-495 was up-regulated, and the expression of miR-29a, miR-31 and miR-142-5p was down-regulated by GOS treatment in both normal and LPS-stimulated FHC cells. Among which, miR-19b was the most varied miRNA. GOS pre-treatment significantly attenuated LPS-induced cell injury, as evidenced by the increase of cell viability, the decrease of apoptosis, as well as the suppressed release of TNF-α, IFN-γ and IL-1ß. GOS pre-treatment could also prevent Rag2-/- rats against helicobacter hepaticus injection induced diarrhea and inflammation, as the body weight and colon organ weight were recovered, diarrhea score was declined, and the release of pro-inflammatory cytokines was inhibited. The in vitro and in vivo effects of GOS abovementioned were all impeded when miR-19b was silenced. SIGNIFICANCE: In vitro and in vivo experiments showed that GOS have certain anti-colitis effect, and this effect may be achieved by up-regulating miR-19b.


Assuntos
MicroRNAs/genética , Oligossacarídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colite/genética , Colite/fisiopatologia , Colo/efeitos dos fármacos , Colo/metabolismo , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , MicroRNAs/metabolismo , Oligossacarídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima
14.
Nat Commun ; 10(1): 2406, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160622

RESUMO

Organ-specific colonization suggests that specific cell-cell recognition is essential. Yet, very little is known about this particular interaction. Moreover, tumor cell lodgement requires binding under shear stress, but not static, conditions. Here, we successfully isolate the metastatic populations of cancer stem/tumor-initiating cells (M-CSCs). We show that the M-CSCs tether more and roll slower than the non-metastatic (NM)-CSCs, thus resulting in the preferential binding to the peritoneal mesothelium under ascitic fluid shear stress. Mechanistically, this interaction is mediated by P-selectin expressed by the peritoneal mesothelium. Insulin-like growth factor receptor-1 carrying an uncommon non-sulfated sialyl-Lewisx (sLex) epitope serves as a distinct P-selectin binding determinant. Several glycosyltransferases, particularly α1,3-fucosyltransferase with rate-limiting activity for sLex synthesis, are highly expressed in M-CSCs. Tumor xenografts and clinical samples corroborate the relevance of these findings. These data advance our understanding on the molecular regulation of peritoneal metastasis and support the therapeutic potential of targeting the sLex-P-selectin cascade.


Assuntos
Líquido Ascítico , Carcinoma/secundário , Adesão Celular , Hidrodinâmica , Células-Tronco Neoplásicas/metabolismo , Oligossacarídeos/metabolismo , Neoplasias Ovarianas/patologia , Selectina-P/metabolismo , Neoplasias Peritoneais/secundário , Animais , Carcinoma/metabolismo , Linhagem Celular Tumoral , Epitélio/metabolismo , Feminino , Fucosiltransferases/metabolismo , Células HEK293 , Humanos , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/metabolismo , Peritônio/metabolismo , Receptor IGF Tipo 1/metabolismo , Estresse Mecânico
15.
Fish Shellfish Immunol ; 91: 202-208, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31085328

RESUMO

This study investigated the effect of individual and combination of dietary pre- and probiotics (ß-glucan, 3 mg/g; mannan oligosaccharide (MOS), 4 mg/g; and Lactobacillus plantarum; 108 CFU/mg diet) on growth performance, blood immune parameters, expression of immune related genes, and intestinal microbial of Caspian trout (Salmo trutta caspius). On the basis of feeding with immunostimulant diets, the fish were assigned into eight groups denoted as: control (basal diet), bß (basal diet + ß-glucan), bM (basal diet + MOS), bLp (basal diet + L. plantarum), bßLp (basal diet + ß-glucan + L. plantarum), bMLp (basal diet + MOS + L. plantarum), bMß (basal diet + MOS + ß-glucan), and bMßLp (basal diet + MOS + ß-glucan + L. plantarum). All of the immunostimulant diets, in general, reduced feed intake (FI) and food conversion ratio (FCR) and increased WG, PER, and final weight. Condition factor (CF) demonstrated the lowest level in the experimental group received bMßLp. Total lipid increased in the fish received the additives, especially bM and bMß. Ash content demonstrated significant increase in the fish fed on bß and bMßLp, whereas moisture content was reduced in the group fed with L. plantarum-supplemented diet. All immunostimulant diets enhanced the activity and levels of lysozyme, Immunoglobulin M (IgM), and serum alternative complement activity (ACH50); the highest value for these indices was observed in the groups fed with bMß, bMßLp, and bßLp. bMß-treated fish group displayed the highest cortisol and glucose levels. bM diet induced the highest mRNA transcription of TNF-α1 in head kidney, whereas bLp, bMß, and bMßLp showed no effect. IL1ß exhibited the greatest up-regulation, about 8.75 fold change, in response to the diet supplemented only with ß-glucan. bßLp and bß significantly enhanced the relative IL-8 mRNA expression in the head kidney (about 2.75 and 1.9 folds, respectively), yet in response to bMßLp treatment it showed a decrease of about 5.7 times lower than the control group. In addition, intestinal population of L. plantarum showed the highest loads in the groups fed on the diets which were treated with the probiotic. Taken together, combinational use of these immunostimulants enhanced humoral innate immune system, whereas their individual and combinational application could increase and decrease the transcription of inflammation-related genes, respectively.


Assuntos
Microbioma Gastrointestinal , Lactobacillus plantarum/química , Mananas/metabolismo , Truta/genética , Truta/imunologia , beta-Glucanas/metabolismo , Ração Animal/análise , Animais , Dieta/veterinária , Intestinos/microbiologia , Mananas/administração & dosagem , Oligossacarídeos/administração & dosagem , Oligossacarídeos/metabolismo , Prebióticos/administração & dosagem , Probióticos/administração & dosagem , Probióticos/química , Probióticos/farmacologia , Distribuição Aleatória , Truta/crescimento & desenvolvimento , Truta/microbiologia , beta-Glucanas/administração & dosagem
16.
J Appl Microbiol ; 127(2): 520-532, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31099168

RESUMO

AIMS: A thermostable endo-mannanase from the fungus Talaromyces cellulolyticus was identified to facilitate manno-oligosaccharide preparation from Konjac (Amorphophallus konjac) flour. METHODS AND RESULTS: A putative endo-1,4-ß-mannanase from the T. cellulolyticus was obtained and efficiently expressed by improving its gene dosage in the genome of the host. After cultivation in a bench-top bioreactor for about 120 h, the protein content and enzyme activity of mannanase increased to 3·4 g l-1 and 17 500 U ml-1 respectively. Enzymatic characterization showed that this enzyme has an optimal temperature of 80°C, optimal pH of 5·0. Under the optimized hydrolysis conditions of pH 5·0, 70°C, and an enzyme concentration of 200 U l-1 solution, this enzyme could efficiently hydrolyse 0·5% konjac flour into manno-oligosaccharides (MOSs) with the degree of polymerization range from 3 to 7. The possible mechanism by which the enzyme produced MOSs was also discussed. CONCLUSION: Talaromyces cellulolyticus endo-mannanase is thermostable and has a broad pH range adaptability. Method of improving the dosage of mannanase gene in the genome could realized its high-level impression. This enzyme could efficiently hydrolyse konjac flour into manno-oligosaccharide products. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has enriched endo-mannanase resources, facilitated its bulk production and provided a strong reference for its application in manno-oligosaccharide preparation from the natural glucomannan of konjac flour.


Assuntos
Amorphophallus/química , Manosidases/metabolismo , Oligossacarídeos/metabolismo , Talaromyces/enzimologia , Estabilidade Enzimática , Farinha , Concentração de Íons de Hidrogênio , Hidrólise , Mananas/metabolismo , Manosidases/genética , Polimerização , Temperatura Ambiente
17.
Carbohydr Polym ; 217: 90-97, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31079689

RESUMO

Pectin oligosaccharides (POSs) have prebiotic and antiglycation activities in vitro, but the specific structure-activity relationship is unclear. In this study, POSs were obtained by enzymatic and ultrasound-assisted enzymatic degradation of pectin polysaccharide (PPS), respectively. Based on the chemical characterization, the antiglycation in vitro and prebiotic activities of POSs were compared and the structure-activity relationship was studied. The results showed that the antiglycation activity of POSs in vitro was proportional to the galacturonic acid content and GalA:Rha molar ratios except for the low molecular weight POSs (LM-POSs), and inversely proportional to its branching degree, such as Ara:Rha and Gal:Rha molar ratios. In addition, it was also found that the prebiotic activity of POSs was positively correlated with Ara:Rha and Gal:Rha molar ratios in molecule composition and the neutral sugar content, especially galactose and arabinose. The degree of esterification (DE) was less important for both antiglycation and prebiotic activity of POSs. These results provided an important theoretical basis for POSs application in food.


Assuntos
Actinidia/química , Frutas/química , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Oligossacarídeos/metabolismo , Pectinas/metabolismo , Prebióticos , Adulto , Fezes/microbiologia , Feminino , Fermentação , Microbioma Gastrointestinal/fisiologia , Humanos , Hidrólise , Masculino , Estrutura Molecular , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Pectinas/química , Pectinas/isolamento & purificação , Poligalacturonase/química , Relação Estrutura-Atividade
18.
Food Chem ; 294: 293-301, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126466

RESUMO

To construct a high-performance engineered endo-inulinase for fructo-oligosaccharides (FOS) production from inulin, an inulin binding module (IBM) was fused into either N- or C-terminal of an endo-inulinase. After heterologous expression, purification and characterization, the C-terminal fusion one (Eninu-IBM) with better activity, thermostability and inulin binding ability was employed for high-temperature in situ inulin hydrolysis in a 10-L fermentor. During this process, Eninu-IBM was first efficiently produced by the yeast cells at 28 °C for 96 h, and subsequently 1600 g unsterilized inulin per liter fermentation liquor was directly supplemented into the bioreactor for FOS production at 60 °C for 2 h. Finally, high purity of FOS (91.4%) were obtained with FOS titer, yield and productivity of 717.3 g/L, 0.912 gFOS/gInulin and 358.6 g/L/h, respectively. The in vitro prebiotic assay indicated that the final FOS products with main polymerization degrees of 3-5 were preferably fermented by beneficial bifidobacteria and lactobacilli.


Assuntos
Glicosídeo Hidrolases/metabolismo , Inulina/metabolismo , Oligossacarídeos/metabolismo , Reatores Biológicos , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Prebióticos , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Temperatura Ambiente
19.
Prep Biochem Biotechnol ; 49(8): 744-758, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050587

RESUMO

Lignocellulosic biomass (LB) is the renewable feedstock for the production of fuel/energy, feed/food, chemicals, and materials. LB could also be the versatile source of the functional oligosaccharides, which are non-digestible food ingredients having numerous applications in food, cosmetics, pharmaceutical industries, and others. The burgeoning functional food demand is expected to be more than US$440 billion in 2022. Because of higher stability at low pH and high temperature, oligosaccharides stimulate the growth of prebiotic bifidobacteria and lactic acid bacteria. Xylooligosaccharides (XOS) are major constituents of oligosaccharides consisting of 2-7 xylose monomeric units linked via ß-(1,4)-linkages. XOS can be obtained from various agro-residues by thermochemical pretreatment, enzymatic or chemoenzymatic methods. While thermochemical methods are fast, reproducible, enzymatic methods are substrate specific, costly, and produce minimum side products. Enzymatic methods are preferred for the production of food grade and pharmaceutically important oligosaccharides. XOS are potent prebiotics having antioxidant properties and enhance the bio-adsorption of calcium and improving bowel functions, etc. LB can cater to the increasing demand of oligosaccharides because of their foreseeable amount and the advancements in technology to recover oligosaccharides. This paper summarizes the methods for oligosaccharides production from LB, classification, and benefits of oligosaccharides on human health.


Assuntos
Biotecnologia/métodos , Glucuronatos/metabolismo , Lignina/metabolismo , Oligossacarídeos/metabolismo , Biomassa , Fibras na Dieta/análise , Humanos , Plantas/enzimologia , Plantas/metabolismo , Xilosidases/metabolismo
20.
Eur J Med Chem ; 177: 212-220, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31146126

RESUMO

Pathogenic micro-organisms utilize protein receptors (lectins) in adhesion to host tissues, a process that in some cases relies on the interaction between lectins and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analysed by X-ray crystallography the structures of the LecB lectin from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 4% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we propose a general strategy which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.


Assuntos
Lectinas/metabolismo , Oligossacarídeos/metabolismo , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Cristalografia por Raios X , Glucosamina/química , Glucosamina/metabolismo , Lectinas/química , Lectinas/genética , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Oligossacarídeos/química , Ligação Proteica , Pseudomonas aeruginosa/química
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