RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Reyanning (RYN) mixture is a traditional Chinese medicine composed of Taraxacum, Polygonum cuspidatum, Scutellariae Barbatae and Patrinia villosa and is used for the treatment of acute respiratory system diseases with significant clinical efficacy. AIM OF THE STUDY: Acute lung injury (ALI) is a common clinical disease characterized by acute respiratory failure. This study was conducted to evaluate the therapeutic effects of RYN on ALI and to explore its mechanism of action. MATERIALS AND METHODS: Ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the chemical components of RYN. 7.5 mg/kg LPS was administered to induce ALI in rats. RYN was administered by gavage at doses of 2 ml/kg, 4 ml/kg or 8 ml/kg every 8 h for a total of 6 doses. Observations included lung histomorphology, lung wet/dry (W/D) weight ratio, lung permeability index (LPI), HE staining, Wright-Giemsa staining. ELISA was performed to detect the levels of TNF-α, IL-6, IL-10, Arg-1,UDPG. Immunohistochemical staining detected IL-6, F4/80 expression. ROS, MDA, SOD, GSH/GSSG were detected in liver tissues. Multiple omics techniques were used to predict the potential mechanism of action of RYN, which was verified by in vivo closure experiments. Immunofluorescence staining detected the co-expression of CD86 and CD206, CD86 and P2Y14, CD86 and UGP2 in liver tissues. qRT-PCR detected the mRNA levels of UGP2, P2Y14 and STAT1, and immunoblotting detected the protein expression of UGP2, P2Y14, STAT1, p-STAT1. RESULTS: RYN was detected to contain 1366 metabolites, some of the metabolites with high levels have anti-inflammatory, antibacterial, antiviral and antioxidant properties. RYN (2, 4, and 8 ml/kg) exerted dose-dependent therapeutic effects on the ALI rats, by reducing inflammatory cell infiltration and oxidative stress damage, inhibiting CD86 expression, decreasing TNF-α and IL-6 levels, and increasing IL-10 and Arg-1 levels. Transcriptomics and proteomics showed that glucose metabolism provided the pathway for the anti-ALI properties of RYN and that RYN inhibited lung glycogen production and distribution. Immunofluorescence co-staining showed that RYN inhibited CD86 and UGP2 expressions. In vivo blocking experiments revealed that blocking glycogen synthesis reduced UDPG content, inhibited P2Y14 and CD86 expressions, decreased P2Y14 and STAT1 mRNA and protein expressions, reduced STAT1 protein phosphorylation expression, and had the same therapeutic effect as RYN. CONCLUSION: RYN inhibits M1 macrophage polarization to alleviate ALI. Blocking glycogen synthesis and inhibiting the UDPG/P2Y14/STAT1 signaling pathway may be its molecular mechanism.
Asunto(s)
Lesión Pulmonar Aguda , Lipopolisacáridos , Ratas , Animales , Lipopolisacáridos/toxicidad , Lipopolisacáridos/metabolismo , Interleucina-10/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Cromatografía Liquida , Interleucina-6/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Glucosa/farmacología , Uridina Difosfato Glucosa/uso terapéutico , Espectrometría de Masas en Tándem , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Pulmón , Macrófagos/metabolismo , ARN Mensajero/metabolismoRESUMEN
The pathological mechanism of cholestatic hepatic injury is associated with oxidative stress, hepatocyte inflammation, and dysregulation of hepatocyte transporters. Paeonia lactiflora Pall. and its compound can improve hepatic microcirculation, dilate bile duct, and promote bile flow, which is advantageous to ameliorate liver damage. Paeoniflorin (PEA), as the main efficacy component of Paeonia lactiflora Pall., has multiple pharmacological effects. PEA improves liver injury, but it remains obscure whether the protective action on [Formula: see text]-naphthalene isothiocyanate (ANIT)-induced cholestatic liver injury is dependent on the NF-E2 p45-related Factor 2 (Nrf2) signaling pathway. In this study, C57BL/6 mice were administrated with 80 mgâ kg[Formula: see text]â d[Formula: see text] ANIT followed by PEA (75, 150, and 300 mgâ kg[Formula: see text]â d[Formula: see text]) orally for 10 days, respectively. Tissue histology and liver function were detected, including serum enzymes, gallbladder (GB) weight, phenobarbital-induced sleeping time (PEN-induced ST), hepatic uridine di-phosphoglucuronosyltransferase (UDPG-T), malondialdehyde (MDA), and glutathione (GSH). The expressions of protein Nrf2, sodium taurocholate cotransporting polypeptide (Ntcp), and NADPH oxidase 4 (Nox4) were evaluated. Nrf2 plasmid or siRNA-Nrf2 transfection on LO2 cells and Nrf2-/- mice were used to explore the liver protective mechanism of PEA. Compared to ANIT-treated mice, PEA decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA), and phenobarbital-induced sleeping time. The bile secretion, hepatic UDPG-T, MDA, GSH, and liver histology were improved. The expressions of protein Nrf2 and Ntcp in liver tissues increased, but Nox4 decreased. After Nrf2 plasmid or small interfering RNA (siRNA)-Nrf2 transfection, the protective effects of PEA on LO2 cells were, respectively, strengthened or weakened. Moreover, PEA had no significant effects on ANIT-treated Nrf2-/- mice. Our results suggest that Nrf2 is essential for PEA protective effects on ANIT-induced liver injury.
Asunto(s)
Colestasis , Paeonia , 1-Naftilisotiocianato/toxicidad , Animales , Bilirrubina/metabolismo , Colestasis/metabolismo , Glucósidos , Glutatión/metabolismo , Isotiocianatos/farmacología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Monoterpenos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Fenobarbital/efectos adversos , ARN Interferente Pequeño/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Glucosa/farmacología , Uridina Difosfato Glucosa/uso terapéuticoRESUMEN
Nucleotide sugars are required for the synthesis of glycoproteins and glycolipids, which play crucial roles in many cellular functions such as cell communication and immune responses. Uridine diphosphate-glucose (UDP-Glc) was previously believed to be the only nucleotide sugar detectable in brain by 31 P-MRS. Using spectra of high SNR and high resolution acquired at 7 T, we showed that multiple nucleotide sugars are coexistent in brain and can be measured simultaneously. In addition to UDP-Glc, these also include UDP-galactose (UDP-Gal), -N-acetyl-glucosamine (UDP-GlcNAc) and -N-acetyl-galactosamine (UDP-GalNAc), collectively denoted as UDP(G). Coexistence of these UDP(G) species is evident from a quartet-like multiplet at -9.8 ppm (M-9.8 ), which is a common feature seen across a wide age range (24-64 years). Lineshape fitting of M-9.8 allows an evaluation of all four UDP(G) components, which further aids in analysis of a mixed signal at -8.2 ppm (M-8.2 ) for deconvolution of NAD+ and NADH. For a group of seven young healthy volunteers, the concentrations of UDP(G) species were 0.04 ± 0.01 mM for UDP-Gal, 0.07 ± 0.03 mM for UDP-Glc, 0.06 ± 0.02 mM for UDP-GalNAc and 0.08 ± 0.03 mM for UDP-GlcNA, in reference to ATP (2.8 mM). The combined concentration of all UDP(G) species (average 0.26 ± 0.06 mM) was similar to the pooled concentration of NAD+ and NADH (average 0.27 ± 0.06 mM, with a NAD+ /NADH ratio of 6.7 ± 2.1), but slightly lower than previously found in an older cohort (0.31 mM). The in vivo NMR analysis of UDP-sugar composition is consistent with those from tissue extracts by other modalities in the literature. Given that glycosylation is dependent on the availability of nucleotide sugars, assaying multiple nucleotide sugars may provide valuable insights into potential aberrant glycosylation, which has been implicated in certain diseases such as cancer and Alzheimer's disease.
Asunto(s)
Encéfalo/diagnóstico por imagen , Hexosas/metabolismo , Espectroscopía de Resonancia Magnética , Uridina Difosfato Glucosa/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Femenino , Humanos , Masculino , NAD/metabolismo , Fósforo , Procesamiento de Señales Asistido por Computador , Uridina Difosfato Glucosa/síntesis química , Uridina Difosfato Glucosa/química , Adulto JovenRESUMEN
Steviol glycosides from Stevia rebaudiana leaves are used in stevia-based sweeteners for their intense sweetness and low calories. Rebaudioside D is present in leaves in minute quantities (â¼0.4-0.5% w/w total dry weight), but it is â¼350 times sweeter than sucrose, and sweeter than the more abundant rebaudioside A and stevioside. In the present study, pathways for rebaudioside D synthesis and UDP-glucose recycling were developed by coupling recombinant UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Reaction parameters, including substrate ratio, sucrose concentration, temperature, crude extract concentration, and reaction time, were evaluated, and 17.4â¯g/l of rebaudioside D (yieldâ¯=â¯74.6%) was obtained from 20â¯g/l of rebaudioside A after 20â¯h, using UDP or UDP-glucose in recombinant cell crude extracts. Extending the reaction time generated rebaudioside M2 from further glycosylation of rebaudioside D. Km values for UGTSL2 indicated a higher affinity for rebaudioside D than for rebaudioside A.
Asunto(s)
Diterpenos de Tipo Kaurano/biosíntesis , Glicósidos/biosíntesis , Glicosiltransferasas/metabolismo , Uridina Difosfato Glucosa/metabolismo , Cromatografía Líquida de Alta Presión , Diterpenos de Tipo Kaurano/análisis , Diterpenos de Tipo Kaurano/química , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glicósidos/análisis , Glicósidos/química , Glicosiltransferasas/genética , Hojas de la Planta/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Solanum/enzimología , Espectrometría de Masa por Ionización de Electrospray , Stevia/metabolismo , TemperaturaRESUMEN
Lesion mimic mutants are valuable to unravel the mechanisms governing the programmed cell death (PCD) process. Uridine 5'-diphosphoglucose-glucose (UDPG) functions as a signaling molecule activating multiple pathways in animals, but little is known about its function in plants. Two novel allelic mutants of spl29 with typical PCD characters and reduced pollen viability were obtained by ethane methyl sulfonate mutagenesis in rice cv Kitaake. The enzymatic analyses showed that UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) irreversibly catalyzed the decomposition of UDPG. Its activity was severely destroyed and caused excessive UDPG accumulation, with the lesion occurrence associated with the enhanced caspase-like activities in spl29-2. At the transcriptional level, several key genes involved in endoplasmic reticulum stress and the unfolded protein response were abnormally expressed. Moreover, exogenous UDPG could aggravate lesion initiation and development in spl29-2. Importantly, exogenous UDPG and its derivative UDP-N-acetylglucosamine could induce reactive oxygen species (ROS) accumulation and lesion mimics in Kitaake seedlings. These results suggest that the excessive accumulation of UDPG, caused by the mutation of UAP1, was a key biochemical event resulting in the lesion mimics in spl29-2. Thus, our findings revealed that UDPG might be an important component involved in ROS accumulation, PCD execution and lesion mimicking in rice, which also provided new clues for investigating the connection between sugar metabolism and PCD process.
Asunto(s)
Apoptosis , Nucleotidiltransferasas/metabolismo , Oryza/fisiología , Especies Reactivas de Oxígeno/metabolismo , Uridina Difosfato Glucosa/metabolismo , Caspasas/metabolismo , Estrés del Retículo Endoplásmico , Mutación , Nucleotidiltransferasas/genética , Oryza/enzimología , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/enzimología , Polen/genética , Polen/fisiologíaRESUMEN
Nitric oxide (NO) is extensively involved in various growth processes and stress responses in plants; however, the regulatory mechanism of NO-modulated cellular sugar metabolism is still largely unknown. Here, we report that NO significantly inhibited monosaccharide catabolism by modulating sugar metabolic enzymes through S-nitrosylation (mainly by oxidizing dihydrolipoamide, a cofactor of pyruvate dehydrogenase). These S-nitrosylation modifications led to a decrease in cellular glycolysis enzymes and ATP synthase activities as well as declines in the content of acetyl coenzyme A, ATP, ADP-glucose and UDP-glucose, which eventually caused polysaccharide-biosynthesis inhibition and monosaccharide accumulation. Plant developmental defects that were caused by high levels of NO included delayed flowering time, retarded root growth and reduced starch granule formation. These phenotypic defects could be mediated by sucrose supplementation, suggesting an essential role of NO-sugar cross-talks in plant growth and development. Our findings suggest that molecular manipulations could be used to improve fruit and vegetable sweetness.
Asunto(s)
Arabidopsis/metabolismo , Monosacáridos/metabolismo , Óxido Nítrico/farmacología , Complejos de ATP Sintetasa/metabolismo , Adenosina Difosfato Glucosa/metabolismo , Adenosina Trifosfato/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Glucólisis/efectos de los fármacos , Mutación/genética , Nitrosación , Oxidación-Reducción , Fenotipo , Desarrollo de la Planta/efectos de los fármacos , Raíces de Plantas/anatomía & histología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Solubilidad , Almidón/metabolismo , Sacarosa/farmacología , Ácido Tióctico/análogos & derivados , Ácido Tióctico/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
Anionic lipids, sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), are major classes of the thylakoid membrane lipids in cyanobacteria and plant chloroplasts. PG is essential for growth and photosynthesis of cyanobacteria, algae and plants, but the requirement for SQDG differs even among cyanobacterial species. Although SQDG and PG can compensate each other in part presumably to maintain proper balance of anionic charge in lipid bilayers, the functional relationship of these lipids is largely unknown. In this study, we inactivated the sqdB gene, encoding a UDP-sulfoquinovose synthase and involved in SQDG biosynthesis, in Thermosynechococcus elongatus BP-1. In wild-type cells, PG accounted for only approximately 3.5 mol% of total membrane lipids, but its content was substantially increased along with complete loss of SQDG in the sqdB mutant. Under phosphate (Pi)-sufficient conditions, the growth rate and PSII activity were slightly lower in sqdB than in wild-type cells. In addition, the formation of PSI trimers and PSII dimers and energy transfer in phycobilisomes were perturbed in the mutant. Under Pi-deficient conditions, the growth of sqdB cells was severely impaired, with a decrease in PSII activity. PG supplementation could partially rescue the defective growth and PSII activity of Pi-deficient sqdB cells but fully recovered the impaired growth of the pgsA mutant of T. elongatus, which is deficient in PG biosynthesis. These data suggest that SQDG has a specific role in the growth and photosynthesis of T. elongatus, which cannot be complemented by PG, particularly under Pi-deficient conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cianobacterias/fisiología , Diglicéridos/metabolismo , Fosfatidilgliceroles/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Uridina Difosfato Glucosa/análogos & derivados , Proteínas Bacterianas/genética , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Mutación , Fosfatos/deficiencia , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Uridina Difosfato Glucosa/metabolismoRESUMEN
Nitrogen fixing legumes rely on phosphorus for nodule formation, nodule function and the energy costs of fixation. Phosphorus is however very limited in soils, especially in ancient sandstone-derived soils such as those in the Cape Floristic Region of South Africa. Plants growing in such areas have evolved the ability to tolerate phosphorus stress by eliciting an array of physiological and biochemical responses. In this study we investigated the effects of phosphorus limitation on N2 fixation and phosphorus recycling in the nodules of Virgilia divaricata (Adamson), a legume native to the Cape Floristic Region. In particular, we focused on nutrient acquisition efficiencies, phosphorus fractions and the exudation and accumulation of phosphatases. Our finding indicate that during low phosphorus supply, V. divaricata internally recycles phosphorus and has a lower uptake rate of phosphorus, as well as lower levels adenylates but greater levels of phosphohydrolase exudation suggesting it engages in recycling internal nodule phosphorus pools and making use of alternate bypass routes in order to conserve phosphorus.
Asunto(s)
Fabaceae/metabolismo , Fósforo/metabolismo , Nódulos de las Raíces de las Plantas/metabolismo , Suelo/química , Fosfatasa Ácida/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Biomasa , Espectroscopía de Resonancia Magnética , Metaboloma , Minerales/metabolismo , Fijación del Nitrógeno , Monoéster Fosfórico Hidrolasas/metabolismo , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
Genistin and daidzein exhibit a protective effect on DNA damage and inhibit cell proliferation. Glycosylation and malonylation of the compounds increase water solubility and stability. Constructed pET15b-GmIF7GT and pET28a-GmIF7MAT were used for the transformation of Escherichia coli and bioconversion of genistein and daidzein. To increase the availability of malonyl-CoA, a critical precursor of GmIF7MAT, genes for the acyl-CoA carboxylase α and ß subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinia were also introduced. Thus, the isoflavonoids were glycosylated at position 7 by 7-O-glycosyltranferase and were further malonylated at position 6(â³) of glucose by malonyl-CoA: isoflavone 7-O-glucoside-6(â³)-O-malonyltransferase both from Glycine max. Engineered E. coli produced 175.7 µM (75.90 mg/L) of genistin and 14.2 µM (7.37 mg/L) genistin 6''-O-malonate. Similar conditions produced 162.2 µM (67.65 mg/L) daidzin and 12.4 µM (6.23 mg/L) daidzin 6''-O-malonate when 200 µM of each substrate was supplemented in the culture. Based on our findings, we speculate that isoflavonoids and their glycosides may prove useful as anticancer drugs with added advantage of increased solubility, stability and bioavailability.
Asunto(s)
Escherichia coli/metabolismo , Isoflavonas/biosíntesis , Malonatos/metabolismo , Inhibidores de la Angiogénesis/biosíntesis , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Escherichia coli/genética , Ingeniería Genética , Glucósidos/biosíntesis , Glicosilación , Isoflavonas/química , Isoflavonas/farmacología , Malonil Coenzima A/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP-l-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP-l-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP-l-Rha/UDP-d-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP-l-Rha and UDP-d-Gal for matrix polysaccharide biosynthesis.
Asunto(s)
Arabidopsis/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Familia de Multigenes , Ramnosa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Arabidopsis/enzimología , Transporte Biológico , Cinética , Datos de Secuencia Molecular , Pectinas/metabolismo , Filogenia , Proteolípidos/metabolismo , Fracciones Subcelulares/metabolismo , Factores de TiempoRESUMEN
Crocin is an apocarotenoid glycosyl ester accumulating in fruits of Gardenia jasminoides and used as a food coloring and nutraceutical. For the first time, the two glucosyltransferases UGT75L6 and UGT94E5 that sequentially mediate the final glucosylation steps in crocin biosynthesis in G. jasminoides have been identified and functionally characterized. UGT75L6 preferentially glucosylates the carboxyl group of crocetin yielding crocetin glucosyl esters, while UGT94E5 glucosylates the 6' hydroxyl group of the glucose moiety of crocetin glucosyl esters. The expression pattern of neither UGT75L6 nor UGT94E5 correlated with the pattern of crocin accumulation in G. jasminoides.
Asunto(s)
Carotenoides/metabolismo , Colorantes de Alimentos/metabolismo , Gardenia/enzimología , Glucosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Uridina Difosfato Glucosa/metabolismo , Alquilación , Células Cultivadas , Suplementos Dietéticos , Frutas/enzimología , Gardenia/citología , Gardenia/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucósidos/metabolismo , Glucosiltransferasas/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Filogenia , Proteínas de Plantas/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Plantones/citología , Plantones/enzimología , Plantones/metabolismo , Especificidad por Sustrato , Vitamina A/análogos & derivadosRESUMEN
Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. [(2009) Proc Natl Acad Sci USA 106:13124-13129], who showed that (i) SUS activity in wild type (WT) leaves is too low to account for normal rate of starch accumulation in Arabidopsis, and (ii) different organs of the sus1/sus2/sus3/sus4 SUS mutant impaired in SUS activity accumulate WT levels of ADP-glucose, UDP-glucose, cellulose and starch. However, these authors assayed SUS activity under unfavorable pH conditions for the reaction. By using favorable pH conditions for assaying SUS activity, in this work we show that SUS activity in the cleavage direction is sufficient to support normal rate of starch accumulation in WT leaves. We also demonstrate that sus1/sus2/sus3/sus4 leaves display WT SUS5 and SUS6 expression levels, whereas leaves of the sus5/sus6 mutant display WT SUS1-4 expression levels. Furthermore, we show that SUS activity in leaves and stems of the sus1/sus2/sus3/sus4 and sus5/sus6 plants is â¼85% of that of WT leaves, which can support normal cellulose and starch biosynthesis. The overall data disprove Barratt et al. (2009) claims, and are consistent with the possible involvement of SUS in cellulose and starch biosynthesis in Arabidopsis.
Asunto(s)
Arabidopsis/enzimología , Celulosa/biosíntesis , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Mutación/genética , Almidón/biosíntesis , Adenosina Difosfato Glucosa/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Concentración de Iones de Hidrógeno/efectos de los fármacos , Cinética , Luz , Cloruro de Magnesio/farmacología , Extractos Vegetales/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
During the fermentation of Streptomyces hygroscopicus TL01 to produce validamycin A (18 g/L), a considerable amount of an intermediate validoxylamine A (4.0 g/L) is accumulated. Chemical or enzymatic hydrolysis of validamycin A was not observed during the fermentation process. Over-expression of glucosyltransferase ValG in TL01 did not increase the efficiency of glycosylation. However, increased validamycin A and decreased validoxylamine A production were observed in both the cell-free extract and fermentation broth of TL01 supplemented with a high concentration of UDP-glucose. The enzymatic activity of UDP-glucose pyrophosphorylase (Ugp) in TL01, which catalyzes UDP-glucose formation, was found to be much lower than the activities of other enzymes involved in the biosynthesis of UDP-glucose and the glucosyltransferase ValG. An ugp gene was cloned from S. hygroscopicus 5008 and verified to code for Ugp. In TL01 with an extra copy of ugp, the transcription of ugp was increased for 1.5 times, and Ugp activity was increased by 100%. Moreover, 22 g/L validamycin A and 2.5 g/L validoxylamine A were produced, and the validamycin A/validoxylamine A ratio was increased from 3.15 in TL01 to 5.75. These data prove that validamycin A biosynthesis is limited by the supply of UDP-glucose, which can be relieved by Ugp over-expression.
Asunto(s)
Inositol/análogos & derivados , Streptomyces/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/biosíntesis , Regulación hacia Arriba , Fermentación , Glucosiltransferasas/metabolismo , Glicosilación , Inositol/biosíntesis , Transcripción Genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , Uridina Difosfato Glucosa/metabolismoRESUMEN
Cellulose synthesis in plants is believed to be carried out by the plasma membrane-associated rosette structure which can be observed by electron microscopy. Despite decade-long speculation, it had not been demonstrated whether the rosette is the site of catalytic activity of cellulose synthesis. To determine the relationship between this structure and cellulose synthesis, we successfully isolated detergent-insoluble rosettes from the plasma membrane of bean epicotyls. However, the purified rosettes did not possess cellulose synthesis activity in vitro. Conversely, detergent-soluble granular particles of approximately 9.5-10 nm diameter were also isolated and exhibited UDP-glucose binding activity and possessed beta-1,4-glucan (cellulose) synthesis activity in vitro. The particle, referred to as the catalytic unit of cellulose synthesis, was enriched with a 78 kDa polypeptide which was verified as sucrose synthase like by mass spectrometry and immunoblotting. The catalytic units were able to bind to the rosettes and retained the cellulose synthesis activity in the presence of UDP-glucose or sucrose plus UDP when supplemented with magnesium. The incorporation of the catalytic unit into the rosette structure was confirmed by immunogold labeling with anti-sucrose synthase antibodies under an electron microscope. Our results suggest that the plasma membrane-associated rosette anchors the catalytic unit of cellulose synthesis to form the functional cellulose synthesis machinery.
Asunto(s)
Celulosa/biosíntesis , Fabaceae/enzimología , Glucosiltransferasas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Glucanos/biosíntesis , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sacarosa/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
(+)-Sesaminol 2-O-triglucoside is the most abundant water-soluble furofuran lignan in sesame seeds (Sesamum indicum) and is considered to be a beneficial compound for human health. The biosyntheses and physiological roles of lignan glycosides, however, remain elusive. Here we report the molecular identification and biochemical characterization of two Sesamum uridine diphosphate (UDP) glucose:lignan glucosyltransferases. Sesamum indicum UGT71A9 preferentially glucosylated at the 2-hydroxyl group of (+)-sesaminol, resulting in (+)-sesaminol 2-O-glucoside. Similarly, two UGT71A9 homologs from Sesamum radiatum (UGT71A10) and Sesamum alatum (UGT71A8) also showed (+)-sesaminol glucosylating activity, evidencing the functional conservation of (+)-sesaminol 2-O-glucosyltransferases in the Sesamum genus. In addition, S. indicum UGT94D1 specifically glucosylated at the 6'-hydroxyl group of the sugar moiety of (+)-sesaminol 2-O-glucoside but not at that of flavonoid glucosides. The gene expression patterns of UGT71A9 and UGT94D1 during seed development were correlated with the glucosylating activities toward (+)-sesaminol in planta, suggesting that the two lignan UDP-glycosyltransferases participate in the sequential glucosylation steps in the biosynthesis of (+)-sesaminol 2-O-triglucoside.
Asunto(s)
Dioxoles/metabolismo , Furanos/metabolismo , Glucosiltransferasas/metabolismo , Lignanos/metabolismo , Sesamum/enzimología , Cromatografía Líquida de Alta Presión , Dioxoles/química , Furanos/química , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucósidos/química , Glucósidos/metabolismo , Glucosiltransferasas/genética , Lignanos/química , Modelos Biológicos , Estructura Molecular , Filogenia , Extractos Vegetales/química , Extractos Vegetales/metabolismo , Sesamum/genética , Sesamum/metabolismo , Uridina Difosfato Glucosa/química , Uridina Difosfato Glucosa/metabolismoRESUMEN
The aim of this work was to study the effect of addition of different amino acids and sugar nucleotides as metabolic precursors on the production of scleroglucan. A maximum yield of 20.00 g/l and 22.32 g/l was obtained with optimized media supplemented with L-lysine (1.1 mM) and uridine mono-phosphate (UMP), respectively as compared to 16.52 g/l scleroglucan achieved with the control in the absence of metabolic precursors.
Asunto(s)
Hongos/metabolismo , Glucanos/biosíntesis , Aminoácidos/metabolismo , Factores de Tiempo , Uridina Difosfato Glucosa/metabolismo , Uridina Monofosfato/metabolismoRESUMEN
UDP-L-rhamnose is required for the biosynthesis of cell wall rhamnogalacturonan-I, rhamnogalacturonan-II, and natural compounds in plants. It has been suggested that the RHM2/MUM4 gene is involved in conversion of UDP-D-glucose to UDP-L-rhamnose on the basis of its effect on rhamnogalacturonan-I-directed development in Arabidopsis thaliana. RHM2/MUM4-related genes, RHM1 and RHM3, can be found in the A. thaliana genome. Here we present direct evidence that all three RHM proteins have UDP-D-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and UDP-4-keto-L-rhamnose 4-keto-reductase activities in the cytoplasm when expressed in the yeast Saccharomyces cerevisiae. Functional domain analysis revealed that the N-terminal region of RHM2 (RHM2-N; amino acids 1-370) has the first activity and the C-terminal region of RHM2 (RHM2-C; amino acids 371-667) has the two following activities. This suggests that RHM2 converts UDP-d-glucose to UDP-L-rhamnose via an UDP-4-keto-6-deoxy-D-glucose intermediate. Site-directed mutagenesis of RHM2 revealed that mucilage defects in MUM4-1 and MUM4-2 mutant seeds of A. thaliana are caused by abolishment of RHM2 enzymatic activity in the mutant strains and furthermore, that the GXXGXX(G/A) and YXXXK motifs are important for enzymatic activity. Moreover, a kinetic analysis of purified His(6)-tagged RHM2-N protein revealed 5.9-fold higher affinity of RHM2 for UDP-D-glucose than for dTDP-D-glucose, the preferred substrate for dTDP-D-glucose 4,6-dehydratase from bacteria. RHM2-N activity is strongly inhibited by UDP-L-rhamnose, UDP-D-xylose, and UDP but not by other sugar nucleotides, suggesting that RHM2 maintains cytoplasmic levels of UDP-D-glucose and UDP-L-rhamnose via feedback inhibition by UDP-L-rhamnose and UDP-D-xylose.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Pared Celular/metabolismo , Genoma de Planta/fisiología , Complejos Multienzimáticos/metabolismo , Ramnosa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Secuencias de Aminoácidos/genética , Arabidopsis/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , Pectinas/biosíntesis , Pectinas/química , Pectinas/genética , Ramnosa/química , Ramnosa/genética , Saccharomyces cerevisiae/genética , Semillas/química , Semillas/enzimología , Semillas/genética , Uridina Difosfato Glucosa/química , Uridina Difosfato Glucosa/genética , Uridina Difosfato Xilosa/química , Uridina Difosfato Xilosa/genética , Uridina Difosfato Xilosa/metabolismoRESUMEN
Whether the hexosamine biosynthesis pathway acts as a nutrient-sensing pathway is still unclear. Glucose is directed into this pathway by GFAT. Because the activity of GFAT is tightly regulated, we examined whether UDP-hexosamine levels can increase significantly and dose-dependently in response to elevated glucose concentrations. In glucosamine-treated 3T3-L1 adipocytes, inhibition of insulin-stimulated glucose uptake was highly correlated with UDP-hexosamine levels (r = -0.992; p < 0.0001 for UDP-GlcNAc and r = -0.996; p < 0.0001 for UDP-GalNAc). Incubation of 3T3-L1 adipocytes with 0.1 microM insulin for 24 h in medium containing 1 and 5 mM glucose increased the rate of glucose uptake by 365% and 175% compared to untreated cells, respectively. This increase was not observed when the cells were incubated for 24 h with insulin in medium containing 10 or 25 mM glucose. However, treatment of cells with insulin and 1, 5, 10, or 25 mM glucose resulted in similar increases in levels of UDP-GlcNAc and UDP-GalNAc that always amounted to approx 30-40% above baseline values. This led us to conclude that despite exposure of adipocytes to conditions of extreme and prolonged glucose disposal, the increases in cellular UDP-hexosamines were minimal and not dependent on the extracellular glucose concentration. Taken together, our results are in line with the hypothesis that in glucosamine-treated adipocytes UDP-hexosamines influence insulin-stimulated glucose uptake. However, our observations in glucose-treated adipocytes argue against the possibility that UDP-hexosamines function as a nutrient-sensor, and question the role of the hexosamine biosynthesis pathway in the pathogenesis of insulin resistance.
Asunto(s)
Adipocitos/metabolismo , Hexosaminas/metabolismo , Células 3T3-L1 , Animales , Desoxiglucosa/metabolismo , Glucosamina/farmacología , Glucosa/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Ratones , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Corynebacterium glutamicum CCTCC M201005 produces a novel polygalacturonic acid bioflocculant, REA-11, consisting of galacturonic acid as the main structural unit. A biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 was proposed. Evidence for the biosynthetic pathway was provided by: (1) analyzing the response upon addition of UDP-glucose to the culture medium; (2) detecting the presence of several key intermediates in the pathway; and (3) correlating the activities of several key enzymes involved in the pathway with the yields of polygalacturonic acid. The production of polygalacturonic acid was improved by 24%, while the activities of UDP-galactose epimerase and UDP-galactose dehydrogenase were improved by 200% and 50%, respectively, upon addition of 100 microM UDP-glucose. In addition, the key intermediates in the proposed biosynthetic pathway, such as UDP-glucose, UDP-galactose, and UDP-glucuronic acid, were detected in cell-free extracts. Furthermore, the activities of UDP-glucose pyrophosphorylase (R2=0.97), UDP-galactose epimerase (R2=0.75) and UDP-galactose dehydrogenase (R2=0.89) were well correlated with the yields of polygalacturonic acid when different sugars were used as sole carbon sources. Therefore, the biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 starts from phosphate-1-glucose, which was then converted to UDP-glucose by UDP-pyrophosphorylase. Predominantly, the UDP-glucose was converted to UDP-galactose by UDP-galactose epimerase; the latter was further converted to UDP-galacturonic acid by UDP-galactose dehydrogenase, which was presumably polymerized to polygalacturonic acid bioflocculant REA-11 by an unknown glucosyltransferase and a polymerase.
Asunto(s)
Corynebacterium/metabolismo , Corynebacterium/fisiología , Pectinas/metabolismo , Corynebacterium/enzimología , Corynebacterium/crecimiento & desarrollo , Medios de Cultivo , Floculación , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Azúcares de Uridina Difosfato/metabolismoRESUMEN
To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.