RESUMEN
In an experimental model of Alzheimer's disease in mice, oral administration of trehalose disaccharide reduces neuroinflammation assessed by the expression level of microglia activation marker Iba1 and affects the neutrophil degranulation activity. A potential anti-inflammatory effect of 4% trehalose solution associated with a decrease in the activity of leukocyte elastase in plasma was revealed.
Asunto(s)
Enfermedad de Alzheimer , Modelos Animales de Enfermedad , Microglía , Trehalosa , Animales , Trehalosa/farmacología , Trehalosa/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Microglía/efectos de los fármacos , Microglía/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Proteínas de Unión al Calcio/metabolismo , Elastasa de Leucocito/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Disacáridos/farmacología , Antiinflamatorios/farmacologíaRESUMEN
The aim of this study was to provide insight into high-energy phosphate compound concentration dynamics under realistic clinical cold-storage conditions using the Celsior solution in seven heart grafts discarded from transplantation. The hearts of seven local donors (three males, four females, age 37 ± 17 years, height 175 ± 5 cm, weight 75 ± 9 kg) initially considered for transplantation and eventually discarded were submitted to a Magnetic Resonance Spectroscopy observation in a clinical Magnetic Resonance Imaging scanner over at least 9 h. The grafts remained in their sterile container at 4°C during the entire examination. Hence, Phosphocreatine (PCr), adenosine triphosphate (ATP), inorganic phosphate (Pi) and intracellular pH were recorded non-destructively at a 30-minute interval. With the ischemic time Ti, the concentration ratios decreased at PCr/ATP = 1.68-0.0028·Tis, Pi/ATP = 1.38 + 0.0029·Tis, and intracellular pH at 7.43-0.0012·Tis. ATP concentration remained stable for at least 9 h and did not decrease as long as phosphocreatine was detectable. Acidosis remained moderate. In addition to the standard parameters assessed at the time of retrieval, Magnetic Resonance Spectroscopy can provide an assesment of the metabolic status of heart grafts before transplantation. These results show how HEPC metabolites deplete during cold storage. Although many parameters determine graft quality during cold storage, the dynamics of HEPC and intracellular pH may be helpful in the development of strategies aiming at extending the ischemic time.
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Adenosina Trifosfato , Disacáridos , Electrólitos , Glutamatos , Glutatión , Trasplante de Corazón , Histidina , Manitol , Soluciones Preservantes de Órganos , Preservación de Órganos , Fosfatos , Humanos , Femenino , Masculino , Adenosina Trifosfato/metabolismo , Adulto , Persona de Mediana Edad , Preservación de Órganos/métodos , Espectroscopía de Resonancia Magnética , Concentración de Iones de Hidrógeno , Fosfocreatina/metabolismo , Adulto Joven , Criopreservación , Imagen por Resonancia MagnéticaRESUMEN
Glycosyltransferases are nature's key biocatalysts for the formation of glycosidic bonds. Discovery and characterization of new synthetically useful glycosyltransferases are critical for the development of efficient enzymatic and chemoenzymatic strategies for producing complex carbohydrates and glycoconjugates. Herein we report the identification of Pasteurella multocida PmNatB as a bifunctional single-catalytic-domain glycosyltransferase with both ß1-3-galactosyltransferase and ß1-3-N-acetylgalactosaminyltransferase activities. It is a novel glycosyltransferase for constructing structurally diverse GalNAcß3Galα/ßOR and Galß3GalNAcα/ßOR disaccharides in one-pot multienzyme systems with in situ generation of UDP-sugars.
Asunto(s)
Disacáridos , N-Acetilgalactosaminiltransferasas , Pasteurella multocida , Pasteurella multocida/enzimología , Disacáridos/química , Disacáridos/síntesis química , Disacáridos/biosíntesis , N-Acetilgalactosaminiltransferasas/metabolismo , N-Acetilgalactosaminiltransferasas/química , Galactosiltransferasas/metabolismoRESUMEN
Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.
Asunto(s)
Proteínas Bacterianas , Paenibacillus , Saccharum , Xilanos , Xilosa , Xilosidasas , Xilanos/metabolismo , Paenibacillus/metabolismo , Paenibacillus/enzimología , Proteínas Bacterianas/metabolismo , Saccharum/metabolismo , Saccharum/química , Xilosidasas/metabolismo , Xilosa/metabolismo , Reactores Biológicos/microbiología , Fibras de la Dieta/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Disacáridos/metabolismo , Glicósido Hidrolasas/metabolismoRESUMEN
Tamarixetin, a natural dietary flavone, exhibits remarkable potential for the treatment of ischemic stroke. The present article aimed to explore the impact of tamarixetin on ischemic stroke and elucidate the underlying mechanisms. Effects of tamarixetin on ischemic stroke were evaluated in rats using the middle cerebral artery occlusion and reperfusion (MCAO/R) model, by assessing the neurological deficit scores, brain water content, brain infraction, and neuronal damage. The levels of proinflammatory cytokines, NLRP3 inflammasome activation, reactive oxygen species (ROS) production, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression were measured in MCAO/R rats and lipopolysaccharide-stimulated cells. Tamarixetin administration improved the neurological dysfunction and neuronal loss in MCAO/R rats. In addition, tamarixetin reduced microglial hyperactivation and proinflammatory cytokines expression in vivo and in vitro. Tamarixetin attenuated NF-κB p65 phosphorylation and promoter activity, reduced NLRP3 expression and caspase-1 cleavage, and downregulated IL-1ß and IL-18 secretions to suppress NLRP3 inflammasome activation. The levels of superoxide anion, hydrogen peroxide, and ROS were also suppressed by tamarixetin. The downregulation of NADP+ and NADPH levels, and gp91phox expression indicated the ameliorative effects of tamarixetin on NADPH oxidase activation. In the gp91phox knockdown cells treated with lipopolysaccharide, the effects of tamarixetin on NADPH oxidase activation, ROS generation, and NLRP3 inflammasome activation were diminished. Moreover, tamarixetin protects neurons against microglial hyperactivation in vitro. Our findings support the potential of tamarixetin as a therapeutic agent for ischemic stroke, and its mechanism of action involves the inhibition of NADPH oxidase-NLRP3 inflammasome signaling.
Asunto(s)
Disacáridos , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Daño por Reperfusión , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ratas , Daño por Reperfusión/tratamiento farmacológico , Masculino , Inflamasomas/metabolismo , Disacáridos/farmacología , Especies Reactivas de Oxígeno/metabolismo , NADPH Oxidasa 2/metabolismo , NADPH Oxidasas/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Modelos Animales de Enfermedad , Isquemia Encefálica/tratamiento farmacológico , Quercetina/análogos & derivadosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Liuwei dihuang pills is a famous Traditional Chinese Medicine with various anti-cancer properties. Over 50 pharmaceutical manufacturers produce Liuwei dihuang pills in China and an estimated millions of people around the world orally take it every day. D-glucaro-1,4-lactone (1,4-GL) was quantified to be about 12.0 mg/g in Liuwei dihuang pills and a primary bioactive component of it inhibiting the activity of ß-glucuronidase in vivo. 1,4-GL can prevent and effectively inhibit various types of cancer. However, its exact mechanism of action remains unknown. The study would justify the traditional usage of Liuwei dihuang pills against cancers. AIM OF THE STUDY: 1,4-GL, a bioactive ingredient derived from Liuwei dihuang pills, a famous Traditional Chinese Medicine, could delay the progression of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. The mechanism underpinning the effect, however, remains poorly understood. MATERIALS AND METHODS: Healthy and HCC rats were treated with or without 1,4-GL (40.0 mg/kg) and 1HNMR-based metabonomic analysis was employed. 10 metabolites in uric acid pathway were quantitatively determined by UPLC-MS/MS. The expression of xanthine dehydrogenase (XDH), SLC2A9 mRNA, and SLC2A9 protein was determined using RT-qPCR and Western Blot. The effect of 1,4-GL on HCC-LM3 cells was verified in vitro. The alterations of ROS activity, SLC2A9 and XDH gene levels were observed in NCTC-1469 cells induced by lipopolysaccharide (LPS) after 1,4-GL treatment. RESULTS: After the intervention of 1,4-GL, improved pathological morphology, liver lesions in HCC rats was observed with restored serum levels of AFP, AST, ALP, γ-GGT and Fisher's ratio. Hepatic metabonomics revealed that puring metabolism were significantly regulated by 1,4-GL in HCC rats. Uric acid, xanthine and hypoxanthine levels were quantified by UPLC-MS/MS and found to be nearly restored to control levels after 1,4-GL treatment in HCC rats. Changes in xanthine oxidase activity, XDH mRNA expression, and SLC2A9 mRNA and protein expression were also reversed. 1,4-GL treatment in LM3 HCC cells were consistent with the results in vivo. Furthermore, oxidative stress indicators such as T-SOD, GSH, CAT and MDA in serum and liver were improved after HCC rats treated with 1,4-GL. In vitro, 1,4-GL was observed to reduce lipopolysaccharide-induced ROS levels in NCTC-1469 cells with enhanced mRNA and protein expression of SLC2A9 and decreased mRNA level of XDH. CONCLUSION: The protective effects of 1,4-GL against DEN-induced HCC by reducing uric acid and ROS levels due to down-regulation of uric acid production and up-regulation of SLC2A9 expressions. 1,4-GL may represent a novel treatment that improves recovery from HCC by targeting uric acid-ROS pathway.
Asunto(s)
Carcinoma Hepatocelular , Dietilnitrosamina , Especies Reactivas de Oxígeno , Ácido Úrico , Animales , Dietilnitrosamina/toxicidad , Ácido Úrico/sangre , Masculino , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratas Sprague-Dawley , Lactonas/farmacología , Línea Celular Tumoral , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Transducción de Señal/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Disacáridos/farmacologíaRESUMEN
Carbohydrates have various biological functions that are based on their structures. However, the composition and the glycosidic-bond linkage and configuration of carbohydrates present challenges for their characterization. Furthermore, isomeric features contribute to the formation of intramolecular hydrogen bonds, which influence the flexibility and dynamics of carbohydrates. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) enables the analysis of protein dynamics by monitoring deuterium labeling after HDX for different lengths of time. In-electrospray ionization (in-ESI) HDX-MS has been used to rapidly label solvated carbohydrates with labeling occurring during desolvation of ESI droplets. Therefore, HDX-labeling times can be altered by changing the spray-solvent conductivity, which changes the initial size of ESI droplets and their resulting lifetimes. Here, we utilize in-ESI HDX-MS to characterize nine isomeric disaccharides with different monosaccharide compositions and glycosidic-bond linkages and configurations. We compared both the relative D-uptake of isomers at individual conductivities, or HDX-labeling times, and the trends associated with labeling at multiple conductivities. Interestingly, the relative D-uptake trends were correlated to isomeric features that affect disaccharide flexibility, including formation of intramolecular hydrogen bonds. Among the isomeric features studied, linkage was observed to have a significant influence on relative D-uptake with (1-3)-linked disaccharides having more change in relative D-uptake with changing conductivity compared to other linkages. Overall, this research illustrates how in-ESI HDX-MS can be applied to structurally characterize disaccharides with distinct isomeric features. Furthermore, this work shows that in-ESI HDX-MS can be used to monitor the dynamics of solvated molecules with rapidly exchanging functional groups.
Asunto(s)
Disacáridos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Ionización de Electrospray/métodos , Disacáridos/química , Solventes/química , Enlace de Hidrógeno , IsomerismoRESUMEN
BACKGROUND: A diet low in fermentable oligo-, di-, monosaccharides and polyols (LFD) improves symptoms in patients with irritable bowel syndrome (IBS). Previous studies have focused on patients with IBS and diarrhea (IBS-D). It is unclear whether LFD is effective for IBS with constipation (IBS-C) or IBS with mixed bowel habits (IBS-M). This open-label, real-world study evaluates the relative effectiveness of the LFD among IBS subtypes. METHODS: This study analyzes data from a service that provides low-FODMAP meals to individuals with IBS. Participants met with a registered dietitian and completed the IBS symptom severity survey (IBS-SSS) before and after undergoing a 2-4-week period of FODMAP restriction. The primary endpoint was the proportion of participants with ≥50-point decrease in IBS-SSS between the three IBS subtypes. KEY RESULTS: After FODMAP restriction, 90% of participants with IBS-D, 75% with IBS-C, and 84% with IBS-M met the primary endpoint (p = 0.045). Similar improvement was seen for a 100-point decrease, but the difference between IBS subtypes was not significant (p = 0.46). After FODMAP restriction, all groups had statistically significant improvement in total IBS-SSS as well as individual symptom categories. Improvement in IBS-SSS subcategories was similar among the groups except for the categories of bloating severity (IBS-M had greatest improvement) and bowel movement satisfaction (IBS-C had less improvement). CONCLUSION & INFERENCES: Though the proportion of responders was highest for IBS-D and lowest for IBS-C, the LFD led to robust improvement in overall symptoms in all IBS subtypes. Key individual symptoms also showed significant improvements in all IBS subtypes.
Asunto(s)
Fermentación , Síndrome del Colon Irritable , Monosacáridos , Polímeros , Humanos , Síndrome del Colon Irritable/dietoterapia , Femenino , Masculino , Persona de Mediana Edad , Adulto , Polímeros/uso terapéutico , Dieta Baja en Carbohidratos/métodos , Oligosacáridos , Disacáridos/uso terapéutico , Resultado del Tratamiento , Anciano , Estreñimiento/tratamiento farmacológico , Estreñimiento/dietoterapia , Diarrea/dietoterapia , Diarrea/tratamiento farmacológicoRESUMEN
Galactooligosaccharides (GOS) are important prebiotics with function closely related to their structure. However, a comprehensive overview of the structure-function relationship is still limited due to the challenge in characterizing multiple isomers in GOS. This study presents a strategy of combining both hydrophilic interaction liquid chromatography (HILIC) retention time and tandem mass spectrometry (MS/MS) fragmentation pattern to distinguish α/ß-linkages and linkage positions of disaccharide isomers in GOS through HILIC-MS/MS analysis. The results indicated that the ratio of m/z 203.0524 to m/z 365.1054 could distinguish α/ß-linkages, while the ratios of m/z 347.0947 to m/z 365.1054, m/z 245.0642 to m/z 365.1054 and HILIC retention time could distinguish (1 â 2), (1 â 3), (1 â 4) and (1 â 6) linkages. The above rules enabled effective characterization of disaccharides in GOS-containing food samples, including milk powder, rice flour, drink, yogurt. This method can be used in the quality control of GOS and future research on the structure-specific health effects of GOS.
Asunto(s)
Disacáridos , Oligosacáridos , Espectrometría de Masas en Tándem , Disacáridos/química , Oligosacáridos/química , Animales , Prebióticos/análisis , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Leche/química , Oryza/química , Yogur/análisisRESUMEN
Described is chitinase immobilization on magnetic nanoparticles (MNPs) as biocompatible support for enzymatic production of di-N-acetyl chitobiose from chitin waste. Chitinase immobilization was feasible with an immobilization yield of 88.9 ± 1.6 % with 97.8 ± 1.0 % retention of activity and compared to free enzyme work, immobilization conferred better thermal and storage stability. As practical benefit the attachment to magnetic nanocarriers enabled easy enzyme recovery after repeated application runs and thus sustainable reuse. In fixed state chitinase retained a remarkable 39.7 ± 2.6 % of the starting activity after 16 reaction cycles. Furthermore, immobilized chitinase showed higher catalytic activity than free chitinase in converting shrimp shells and squid-pens chitins into di-N-acetyl chitobiose in a single-step reaction. The final yield of purified compound was 37.0 ± 1.2 % from shrimp shells and 61.1 ± 0.5 % from squid-pens chitin. In conclusion, an efficient MNP-based chitinase immobilization system with the potential for large-scale production was developed.
Asunto(s)
Quitina , Quitinasas , Disacáridos , Enzimas Inmovilizadas , Reciclaje , Enzimas Inmovilizadas/metabolismo , Enzimas Inmovilizadas/química , Quitina/química , Quitinasas/metabolismo , Animales , Residuos , Biocatálisis , Decapodiformes , Temperatura , Estabilidad de Enzimas , Nanopartículas de Magnetita/química , Alimento Perdido y DesperdiciadoRESUMEN
Xyloglucan in plant cell walls has complex side-chain structures; Aspergillus oryzae produces various enzymes to degrade and assimilate xyloglucan. In this study, we identified and characterized α-1,2-l-fucosidase (AfcA) which is involved in xyloglucan degradation in A. oryzae. AfcA expression was induced in the presence of xyloglucan oligosaccharides. AfcA showed specific activity toward α-(1â2)-linked l-fucopyranosyl residues attached to the side chains of xyloglucan oligosaccharides and milk oligosaccharides, but not toward α-(1â3)-, α-(1â4)-, and α-(1â6)-linked l-fucopyranosyl residues. As fucopyranosyl residues in the side chains of xyloglucan oligosaccharides prevent the degradation of xyloglucan oligosaccharides by isoprimeverose-producing oligoxyloglucan hydrolase and ß-galactosidase, the cooperative action of AfcA, isoprimeverose-producing oligoxyloglucan hydrolase, and ß-galactosidase play a key role in degrading fucosylated xyloglucan in A. oryzae.
Asunto(s)
Aspergillus oryzae , Glucanos , Xilanos , alfa-L-Fucosidasa , Xilanos/metabolismo , Xilanos/química , Glucanos/metabolismo , Glucanos/química , Aspergillus oryzae/enzimología , Aspergillus oryzae/metabolismo , alfa-L-Fucosidasa/metabolismo , alfa-L-Fucosidasa/química , alfa-L-Fucosidasa/genética , Oligosacáridos/metabolismo , Oligosacáridos/química , beta-Galactosidasa/metabolismo , beta-Galactosidasa/química , Especificidad por Sustrato , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Pared Celular/metabolismo , DisacáridosRESUMEN
The disaccharide (ß-D-glucopyranosyluronic acid)-(1â4)-ß-D-glucopyranoside represents a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 3. A conjugate of the disaccharide with BSA (di-BSA conjugate) adjuvanted with aluminum hydroxide induced - in contrast to the non-adjuvanted conjugate - IgG1 antibody production and protected mice against S. pneumoniae serotype 3 infection after intraperitoneal prime-boost immunization. Adjuvanted and non-adjuvanted conjugates induced production of Th1 (IFNγ, TNFα); Th2 (IL-5, IL-13); Th17 (IL-17A), Th1/Th17 (IL-22), and Th2/Th17 cytokines (IL-21) after immunization. The concentration of cytokines in mice sera was higher in response to the adjuvanted conjugate, with the highest level of IL-17A production after the prime and boost immunizations. In contrast, the non-adjuvanted conjugate elicited only weak production of IL-17A, which gradually decreased after the second immunization. After boost immunization of mice with the adjuvanted di-BSA conjugate, there was a significant increase in the number of CD45+/CD19+ B cells, TCR+ γδ T cell, CD5+ Ð1 cells, and activated cells with MHC II+ expression in the spleens of the mice. IL-17A, TCR+ γδ T cells, and CD5+ Ð1 cells play a crucial role in preventing pneumococcal infection, but can also contribute to autoimmune diseases. Immunization with the adjuvanted and non-adjuvanted di-BSA conjugate did not elicit autoantibodies against double-stranded DNA targeting cell nuclei in mice. Thus, the molecular and cellular markers associated with antibody production and protective activity in response to immunization with the di-BSA conjugate adjuvanted with aluminum hydroxide are IL-17A, TCR+ γδ T cells, and CD5+ Ð1 cells against the background of increasing MHC II+ expression.
Asunto(s)
Interleucina-17 , Vacunas Neumococicas , Albúmina Sérica Bovina , Streptococcus pneumoniae , Animales , Interleucina-17/inmunología , Interleucina-17/metabolismo , Streptococcus pneumoniae/inmunología , Ratones , Albúmina Sérica Bovina/inmunología , Vacunas Neumococicas/inmunología , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/prevención & control , Disacáridos/inmunología , Cápsulas Bacterianas/inmunología , Polisacáridos Bacterianos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Femenino , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Linfocitos Intraepiteliales/inmunología , Serogrupo , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismoRESUMEN
The biological activities of heparan sulfate (HS) are intimately related to their molecular weights, degree and pattern of sulfation and homogeneity. The existing methods for synthesizing homogeneous sugar chains of low dispersity involve multiple steps and require stepwise isolation and purification processes. Here, we designed a mesoporous metal-organic capsule for the encapsulation of glycosyltransferase and obtained a microreactor capable of enzymatically catalyzing polymerization reactions to prepare homogeneous heparosan of low dispersity, the precursor of HS and heparin. Since the sugar chain extension occurs in the pores of the microreactor, low molecular weight heparosan can be synthesized through space-restricted catalysis. Moreover, the glycosylation co-product, uridine diphosphate (UDP), can be chelated with the exposed metal sites of the metal-organic capsule, which inhibits trans-cleavage to reduce the molecular weight dispersity. This microreactor offers the advantages of efficiency, reusability, and obviates the need for stepwise isolation and purification processes. Using the synthesized heparosan, we further successfully prepared homogeneous 6-O-sulfated HS of low dispersity with a molecular weight of approximately 6 kDa and a polydispersity index (PDI) of 1.032. Notably, the HS generated exhibited minimal anticoagulant activity, and its binding affinity to fibroblast growth factor 1 was comparable to that of low molecular weight heparins.
Asunto(s)
Heparitina Sulfato , Polimerizacion , Heparitina Sulfato/química , Anticoagulantes/química , Anticoagulantes/farmacología , Anticoagulantes/síntesis química , Peso Molecular , Porosidad , Humanos , Disacáridos/química , Glicosiltransferasas/metabolismo , Glicosiltransferasas/químicaRESUMEN
Arabinoxylan is a major hemicellulose in the sugarcane plant cell wall with arabinose decorations that impose steric restrictions on the activity of xylanases against this substrate. Enzymatic removal of the decorations by arabinofuranosidases can allow a more efficient arabinoxylan degradation by xylanases. Here we produced and characterized a recombinant Bifidobacterium longum arabinofuranosidase from glycoside hydrolase family 43 (BlAbf43) and applied it, together with GH10 and GH11 xylanases, to produce xylooligosaccharides (XOS) from wheat arabinoxylan and alkali pretreated sugarcane bagasse. The enzyme synergistically enhanced XOS production by GH10 and GH11 xylanases, being particularly efficient in combination with the latter family of enzymes, with a degree of synergism of 1.7. We also demonstrated that the enzyme is capable of not only removing arabinose decorations from the arabinoxylan and from the non-reducing end of the oligomeric substrates, but also hydrolyzing the xylan backbone yielding mostly xylobiose and xylose in particular cases. Structural studies of BlAbf43 shed light on the molecular basis of the substrate recognition and allowed hypothesizing on the structural reasons of its multifunctionality.
Asunto(s)
Bifidobacterium longum , Celulosa , Endo-1,4-beta Xilanasas , Glucuronatos , Glicósido Hidrolasas , Oligosacáridos , Saccharum , Xilanos , Oligosacáridos/química , Oligosacáridos/metabolismo , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/química , Glucuronatos/metabolismo , Glucuronatos/química , Endo-1,4-beta Xilanasas/metabolismo , Endo-1,4-beta Xilanasas/química , Xilanos/metabolismo , Xilanos/química , Saccharum/química , Saccharum/metabolismo , Celulosa/química , Celulosa/metabolismo , Bifidobacterium longum/enzimología , Bifidobacterium longum/metabolismo , Hidrólisis , Especificidad por Sustrato , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , DisacáridosRESUMEN
Xylanase is the most important hydrolase in the xylan hydrolase system, the main function of which is ß-1,4-endo-xylanase, which randomly cleaves xylans to xylo-oligosaccharides and xylose. Xylanase has wide ranging of applications, but there remains little research on the cold-adapted enzymes required in some low-temperature industries. Glycoside hydrolase family 8 (GH8) xylanases have been reported to have cold-adapted enzyme activity. In this study, the xylanase gene dgeoxyn was excavated from Deinococcus geothermalis through sequence alignment. The recombinant xylanase DgeoXyn encodes 403 amino acids with a theoretical molecular weight of 45.39 kDa. Structural analysis showed that DgeoXyn has a (α/α)6-barrel fold structure typical of GH8 xylanase. At the same time, it has strict substrate specificity, is only active against xylan, and its hydrolysis products include xylobiose, xylotrinose, xytetranose, xylenanose, and a small amount of xylose. DgeoXyn is most active at 70 â and pH 6.0. It is very stable at 10, 20, and 30 â, retaining more than 80% of its maximum enzyme activity. The enzyme activity of DgeoXyn increased by 10% after the addition of Mn2+ and decreased by 80% after the addition of Cu2+. The Km and Vmax of dgeox were 42 mg/ml and 20,000 U/mg, respectively, at a temperature of 70 â and pH of 6.0 using 10 mg/ml beechwood xylan as the substrate. This research on DgeoXyn will provide a theoretical basis for the development and application of low-temperature xylanase.
Asunto(s)
Deinococcus , Endo-1,4-beta Xilanasas , Estabilidad de Enzimas , Xilanos , Deinococcus/enzimología , Deinococcus/genética , Especificidad por Sustrato , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/metabolismo , Xilanos/metabolismo , Frío , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Concentración de Iones de Hidrógeno , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/química , Secuencia de Aminoácidos , Hidrólisis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Clonación Molecular , Cinética , Peso Molecular , DisacáridosRESUMEN
The Gal(α1-3)Gal is the terminal disaccharide unit of the α-Gal epitope [Gal(α1-3)Gal(ß1-4)GlcNAc], an exogenous antigenic determinant with several clinical implications, found in all non-primate mammals and in several dangerous pathogens, including certain protozoa and mycobacteria. Its absence in humans makes the α-Gal epitope an interesting target for several infectious diseases. Here we present the development of a macrocyclic tweezers-shaped receptor, resulting from the combination of the structural features of two predecessors belonging to the family of diaminocarbazole receptors, which exhibits binding properties in the low millimolar range toward the Gal(α1-3)Gal disaccharide of the α-Gal antigen.
Asunto(s)
Disacáridos , Disacáridos/química , Humanos , Epítopos/química , Compuestos Macrocíclicos/química , Biomimética , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismoRESUMEN
The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.
Asunto(s)
Endo-1,4-beta Xilanasas , Proteínas Recombinantes , Xilanos , Especificidad por Sustrato , Hidrólisis , Xilanos/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Clonación Molecular , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Glucuronatos/metabolismo , Estabilidad de Enzimas , Cinética , Peso Molecular , Oligosacáridos/metabolismo , DisacáridosRESUMEN
OBJECTIVES: Small fiber neuropathy presents a significant diagnostic and therapeutic challenge. To solve this challenge, efforts have been made to identify autoantibodies associated with this condition. Previous literature has often considered tri-sulfated heparin disaccharide (TS-HDS) and fibroblast growth factor receptor 3 (FGFR3) as a singular seropositive group and/or focused primarily on symptomatic associations. METHODS: One hundred seventy-two small fiber neuropathy patients with a Washington University Sensory Neuropathy panel were selected for TS-HDS seropositivity, FGFR-3 seropositivity, and seronegative controls. Data were collected to on the demographic, symptomatic, and laboratory profiles of each subgroup. RESULTS: Percent female (P = 0.0043), frequency of neuropathic pain symptoms (P = 0.0074), and erythrocyte sedimentation rate (P = 0.0293), vitamin D (P < 0.0001), and vitamin B12 (P = 0.0033) differed between the groups. Skin biopsy was more frequently normal within both the FGFR-3 and the TS-HDS cohort (P = 0.0253). CONCLUSIONS: TS-HDS and FGFR-3 display a distinct phenotype from both controls and one another. Immunoglobulin M (IgM) against FGFR-3 and IgM against TS-HDS may be individually valuable markers for the development of distinct clinical phenotypes.
Asunto(s)
Autoanticuerpos , Conducción Nerviosa , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Neuropatía de Fibras Pequeñas , Humanos , Femenino , Neuropatía de Fibras Pequeñas/diagnóstico , Persona de Mediana Edad , Masculino , Conducción Nerviosa/fisiología , Anciano , Adulto , Autoanticuerpos/sangre , Heparina/análogos & derivados , Inmunoglobulina M/sangre , Heparitina Sulfato/sangre , Estudios de Conducción Nerviosa , DisacáridosRESUMEN
OBJECTIVES: To demonstrate treatment efficacy on composite and non-length-dependent (NLD) punch biopsy specimens from intravenous immunoglobulin (IVIG) in pure small-fiber neuropathy (SFN) with trisulfated heparin disaccharide (TS-HDS), fibroblast growth factor-3 (FGFR-3), or Plexin D1 antibodies. SFN has an increasing prevalence, and over 30% of cases may be immune-mediated. TS-HDS, FGFR-3, and Plexin D1 autoantibodies have been shown to be present in 44%-55% of cryptogenic SFN cases, suggesting an immune mechanism. Reports have shown IVIG to be effective for this condition, but some controversy exists based on length-dependent (LD) post-IVIG treatment data in a recent trial. METHODS: In a retrospective review, all pure SFN cases tested for the 3 antibodies from January 2021 to May 2022 were tabulated, and patients who underwent IVIG treatment were separated and analyzed for changes in epidermal nerve fiber density (ENFD) on skin biopsy, as well as SFN-specific questionnaire and pain scores. RESULTS: Ninety-one patients with pure SFN had antibody testing. Sixty of these (66%) were seropositive, and 31 (34%) were seronegative. Seventeen seropositive patients (13 female patients, 4 male patients, 6 FGFR-3, 2 TS-HDS, 4 Plexin D1, 2 with all 3 antibodies, 1 with FGFR-3 and Plexin D1, 1 with FGFR-3 and TS-HDS, and 1 with TS-HDS and Plexin D1) underwent IVIG treatment. Of these, 2 patients stopped treatment due to side effects, and the remaining 15 completed at least 6 months of IVIG. Of these, 12 had a post-IVIG skin biopsy, and of these, 11 (92%) had a 55.1% improved mean composite ENFD (P = 0.01). NLD-ENFD specimens improved by 42.3% (P = 0.02), and LD-ENFD specimens improved by 99.7% (P = 0.01). Composite ENFD in Plexin D1-SFN patients improved by 139% (P = 0.04). In addition, 14 patients had questionnaires pre-IVIG/post-IVIG, and average pain decreased by 2.7 (P = 0.002). CONCLUSIONS: IVIG shows disease-modifying effect in immune SFN with novel antibodies, especially Plexin D1-SFN, as well as significantly improved pain. NLD-ENFD should be examined as well as LD-ENFD to see this effect. Further randomized controlled trials looking at NLD-ENFD as well as LD-ENFD improvement, along with pain and SFN-specific questionnaires, are needed to confirm these findings.
Asunto(s)
Autoanticuerpos , Inmunoglobulinas Intravenosas , Piel , Neuropatía de Fibras Pequeñas , Humanos , Neuropatía de Fibras Pequeñas/tratamiento farmacológico , Femenino , Masculino , Persona de Mediana Edad , Inmunoglobulinas Intravenosas/uso terapéutico , Estudios Retrospectivos , Autoanticuerpos/sangre , Adulto , Anciano , Biopsia , Piel/patología , Heparina/uso terapéutico , Heparina/análogos & derivados , Proteínas del Tejido Nervioso/inmunología , Resultado del Tratamiento , Receptores de Superficie Celular , Factores Inmunológicos/uso terapéutico , DisacáridosRESUMEN
This study aims to reveal the dynamics of the HPLC fingerprint, chromaticity values, and main chemical components of Mori Cortex during the stir-frying process. The fingerprints of raw and processed products of Mori Cortex were established. The content of mulberroside A, oxyresveratrol, kuwanon G, and kuwanon H in the samples and the chromaticity values of the samples were determined. Furthermore, the similarity evaluation of fingerprints and the correlation analysis between fingerprints and chromaticity values were carried out. The results showed that the fingerprints of raw and processed products of Mori Cortex had high similarity, and the overall changes in the content of the main chemical components in the stir-frying process were similar. According to the experience, when the stir-frying is moderate, the total chromaticity value difference |ΔE~*_(ab)| is above 1.5. With the extension of stir-frying time, the L~* and E~*_(ab) values keep decreasing, and the a~* value keeps increasing. The results of the correlation analysis between fingerprints and chromaticity values showed that peaks 1(5-hydroxy maltol), 2(mulberroside A), 3, 4, 6, 7, 11(oxyresveratrol), 14, 17(kuwanon G), and 18(kuwanon H) had significant correlations with the chromaticity values. Quantitative analysis of the four components with higher content showed that the content of the four components decreased to varying degrees when the stir-frying was excessive. In addition, 5-hydroxy maltol was produced after stir-frying of Mori Cortex, and the fingerprint and chromaticity values showed regular changes during the stir-frying process. The chromaticity can be included in the evaluation of the stir-frying process of Mori Cortex, which provides a reference for standardizing the quality of stir-fried Mori Cortex.