Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-κ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.     

An inhibitor of c-Jun NH2-terminal kinase, SP600125, protects mice from D-galactosamine/lipopolysaccharide-induced hepatic failure by modulating BH3-only proteins

Fulminant hepatic failure (FHF) is a dramatic clinical syndrome characterized by massive hepatocyte apoptosis and very high mortality. The c-Jun-N-terminal kinase (JNK) pathway is an important stress-responsive kinase activated by several forms of liver injury. The aim of this study is to assess the role of JNK during D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury, an experimental model of FHF, using SP600125, a small molecule JNK-specific inhibitor. Mice were given an intraperitoneal dose of GalN (800 microg/g body weight)/LPS (100 ng/g body weight) with and without subcutaneous SP600125 (50 mg/kg body weight) treatment (at 6 and 2 h before and 2 h after GalN/LPS administration). GalN/LPS treatment induced sustained JNK activation. Administration of SP600125 diminished JNK activity, suppressed lethality and the elevation of both serum alanine aminotransferase and aspartate aminotransferase, but had no effect on serum tumor necrosis factor-alpha, and reduced hepatocyte apoptosis after GalN/LPS administration. In support of the role of JNK in promoting the mitochondria-mediated apoptosis pathway, SP600125 prevented cytochrome c release, caspase-9 and caspase-3 activity. Moreover, SP600125 downregulated the mRNA and protein expression of Bad in the early periods following GalN/LPS injection and prevented Bid cleavage in the late periods. These results confirm the role of JNK as a critical apoptotic mediator in GalN/LPS-induced FHF. SP600125 has the potential to protect FHF by downregulating Bad and inhibiting Bid cleavage.     

Metabolic profiling reveals key metabolic features of renal cell carcinoma

Recent evidence suggests that metabolic changes play a pivotal role in the biology of cancer and in particular renal cell carcinoma (RCC). Here, a global metabolite profiling approach was applied to characterize the metabolite pool of RCC and normal renal tissue. Advanced decision tree models were applied to characterize the metabolic signature of RCC and to explore features of metastasized tumours. The findings were validated in a second independent dataset. Vitamin E derivates and metabolites of glucose, fatty acid, and inositol phosphate metabolism determined the metabolic profile of RCC. α‐tocopherol, hippuric acid, myoinositol, fructose‐1‐phosphate and glucose‐1‐phosphate contributed most to the tumour/normal discrimination and all showed pronounced concentration changes in RCC. The identified metabolic profile was characterized by a low recognition error of only 5% for tumour versus normal samples. Data on metastasized tumours suggested a key role for metabolic pathways involving arachidonic acid, free fatty acids, proline, uracil and the tricarboxylic acid cycle. These results illustrate the potential of mass spectroscopy based metabolomics in conjunction with sophisticated data analysis methods to uncover the metabolic phenotype of cancer. Differentially regulated metabolites, such as vitamin E compounds, hippuric acid and myoinositol, provide leads for the characterization of novel pathways in RCC.     

Comparative Proteomic Analyses of the Liver in D‐Galactosamine‐Sensitized Mice Treated with Different Toll‐Like Receptor Agonists

Acute liver failure (ALF) is a severe consequence of abrupt hepatocyte injury and has lethal outcomes. Three toll‐like receptor agonists, including polyinosinic‐polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS), and cytosine‐phosphate‐guanine (CpG) DNA, cause acute and severe hepatitis, respectively, in D‐galactosamine (D‐GalN)‐sensitized mice. However, the molecular differences among three ALF models (LPS/D‐GalN, poly(I:C)/D‐GalN, and CpG DNA/D‐GalN), are unclear. Here, tandem mass tag based quantitative proteomic analyses of three ALF mouse models are performed. 52 common differentially expressed proteins (DEPs) are identified, in three ALF groups, compared to the control. Gene ontology analyses show that among the common DEPs, ten proteins are involved in immune system process, and 39 proteins in metabolic process. Among 80,195, and 23 specifically‐expressed proteins in poly(I:C)/D‐GalN, LPS/D‐GalN, and CpG DNA/D‐GalN groups, LPS/D‐GalN‐specific proteins are mostly distributed in the endoplasmic reticulum and more enriched in metabolic pathways, whereas poly (I:C)/D‐GalN‐specific proteins are mainly in the membrane and CpG DNA/D‐GalN‐specific proteins are related to the ribosome structural composition. In conclusion, the common and specific DEPs in three ALF mouse models at molecular level are identified; and determined a close‐to‐complete reference map of mouse liver proteins which will be useful for clinical diagnosis and treatment of liver failure in humans.     

Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern

In this second of two companion articles, we compare the mass isotopomer distribution of metabolites of liver gluconeogenesis and citric acid cycle labeled from NaH(13)CO(3) or dimethyl [1,4-(13)C(2)]succinate. The mass isotopomer distribution of intermediates reveals the reversibility of the isocitrate dehydrogenase + aconitase reactions, even in the absence of a source of alpha-ketoglutarate. In addition, in many cases, a number of labeling incompatibilities were found as follows: (i) glucose versus triose phosphates and phosphoenolpyruvate; (ii) differences in the labeling ratios C-4/C-3 of glucose versus (glyceraldehyde 3-phosphate)/(dihydroxyacetone phosphate); and (iii) labeling of citric acid cycle intermediates in tissue versus effluent perfusate. Overall, our data show that gluconeogenic and citric acid cycle intermediates cannot be considered as sets of homogeneously labeled pools. This probably results from the zonation of hepatic metabolism and, in some cases, from differences in the labeling pattern of mitochondrial versus extramitochondrial metabolites. Our data have implications for the use of labeling patterns for the calculation of metabolic rates or fractional syntheses in liver, as well as for modeling liver intermediary metabolism.     

A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos

Developing oilseeds synthesize large quantities of triacylglycerol from sucrose and hexose. To understand the fluxes involved in this conversion, a quantitative metabolic flux model was developed and tested for the reaction network of glycolysis and the oxidative pentose phosphate pathway (OPPP). Developing Brassica napus embryos were cultured with [U-13C6]glucose, [1-13C]glucose, [6-13C]glucose, [U-13C12]sucrose, and/or [1,2-13C2]glucose and the labeling patterns in amino acids, lipids, sucrose, and starch were measured by gas chromatography/mass spectrometry and NMR. Data were used to verify a reaction network of central carbon metabolism distributed between the cytosol and plastid. Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.     

Chemical characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil-Felix test, were characterized by chemical analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To separate the O-polysaccharide, core-oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G-50 column. The core-oligosaccharides contained L-glycero-D-mannoheptose, D-glycero-D-mannoheptose, glucose (Glc), galactose, 3-deoxy-D-mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN-phosphate, and three fatty acids (myristic, plamitic, and beta-hydroxymyristic acids). However, the O-polysaccharides of OX2- and OXK-LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19-LPS seemed to lack O-polysaccharide.     

Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations

An experimental set-up for acquiring metabolite and transient (13)C-labeling data in mammalian cells is presented. An efficient sampling procedure was established for hepatic cells cultured in six-well plates as a monolayer attached to collagen, which allowed simultaneous quenching of metabolism and extraction of the intracellular intermediates of interest. Extracellular concentrations of glucose, amino acids, lactate, pyruvate, and urea were determined by GC-MS procedures and were used for estimation of metabolic uptake and excretion rates. Sensitive LC-MS and GC-MS methods were used to quantify the intracellular intermediates of tricarboxylic acid cycle, glycolysis, and pentose phosphate pathway and for the determination of isotopomer fractions of the respective metabolites. Mass isotopomer fractions were determined in a transient (13)C-labeling experiment using (13)C-labeled glucose as substrate. The absolute amounts of intracellular metabolites were obtained from a non-labeled experiment carried out in exactly the same way as the (13)C-labeling experiment, except that the media contained naturally labeled glucose only. Estimation of intracellular metabolic fluxes from the presented data is addressed in part II of this contribution.     

Evolution of intrahepatic carbon, nitrogen, and energy metabolism in a D-galactosamine-induced rat liver failure model

A clearer picture of the hepatic metabolic pathways affected by fulminant hepatic failure (FHF) would help develop nutritional support and nonsurgical therapies for FHF. We characterized the evolution of hepatic metabolism in a rat model of FHF using an isolated perfused liver system together with a mass-balance model of intermediary metabolism. Principal component analysis (PCA) was used to identify potential new sensitive markers for FHF. To induce FHF, rats were given two D-galactosamine injections under fasting conditions. Controls were fasted only. Livers were harvested 1, 4, 8, and 12 h later and perfused with Eagle minimal essential medium supplemented with amino acids and bovine serum albumin, and equilibrated with 95% O2/5% CO2. At the 1 h time point, lactate release increased concomitant with a decrease in gluconeogenesis, TCA cycle and mitochondrial electron transport fluxes. At 4 h, amino acid metabolism and urea cycle fluxes were significantly depressed. By 8 h, gluconeogenesis had switched to glycolysis. By 12 h, amino acid metabolism was broadly inhibited, and there was a net release of many amino acids. Mass-balance analysis shows that the main source of ATP production in the FHF liver gradually changed from mitochondrial oxidative phosphorylation to glycolysis. PCA suggests that a linear combination of glucose, lactate, and glutamine concentrations in arterial plasma is a sensitive marker for FHF. We conclude that D-galactosamine causes early mitochondrial dysfunction while glycolytic ATP synthesis remains functional. Markers that are indirectly linked to these pathways may be used to evaluate the progression of FHF.     

Metabolic analysis of guava (Psidium guajava L.) fruits at different ripening stages using different data-processing approaches

Gas chromatography coupled with time-of-flight mass spectrometry and principal component analysis were used to obtain the metabolite profiles of guava (Psidium guajava) fruits. Results with two types of data-processing software, ChromaTOF and AMDIS, were compared to explain the differences between the samples. There were some differences in score and loading plot patterns of PCA as well as in the composition of the metabolites. However, little difference was observed in the type of metabolites detected and identified using either type of software. Both the flesh and peel of premature and mature white guava fruits were compared for the analysis of the metabolite profiles. Malic acid, aspartic acid, and glucose were the major metabolites distinguishing the different parts of guava fruits in the PCA loading plot. In addition, the metabolic profiles of the fruits revealed significant changes in some metabolites during ripening. The major components contributing to the separation were serine, citric acid, fructose, sucrose, and some unknowns. In particular, sucrose, fructose, serine and citric acid were related to the ripening of guava fruits. Fructose and sucrose were increased whereas citric acid was decreased during guava fruit ripening.     

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.     

Identification of optimal classification functions for biological sample and state discrimination from metabolic profiling data

MOTIVATIONS: Classification of biological samples for diagnostic purposes is a difficult task because of the many decisions involved on the number, type and functional manipulations of the input variables. This study presents a generally applicable strategy for systematic formulation of optimal diagnostic indexes. To this end, we develop a novel set of computational tools by integrating regression optimization, stepwise variable selection and cross-validation algorithms. RESULTS: The proposed discrimination methodology was applied to plasma and tissue (liver) metabolic profiling data describing the time progression of liver dysfunction in a rat model of acute hepatic failure generated by d-galactosamine (GalN) injection. From the plasma data, our methodology identified seven (out of a total of 23) metabolites, and the corresponding transform functions, as the best inputs to the optimal diagnostic index. This index showed better time resolution and increased noise robustness compared with an existing metabolic index, Fischer's BCAA/AAA molar ratio, as well as indexes generated using other commonly used discriminant analysis tools. Comparison of plasma and liver indexes found two consensus metabolites, lactate and glucose, which implicate glycolysis and/or gluconeogenesis in mediating the metabolic effects of GalN.     

Dynamic Patterns of serum metabolites in fulminant hepatic failure pigs

Fulminant hepatic failure (FHF) is still an intractable disease associated with serious metabolic disorder. Investigating the dynamic changes of serum metabolites during the development of FHF would facilitate revealing the pathogenesis and also promote its treatment. Therefore, this study characterized the dynamic metabonome of serum from FHF Pigs using ultra performance liquid chromatography?Cmass spectrometry. Based on multiple statistical analysis of the resulting dataset, three types of up-regulated and one type of down-regulated patterns were delineated. Each pattern demonstrated distinct trends at different stages during the whole process of FHF, implying the differential clinical significance of them. Specifically, aromatic amino acids (Pattern 1) and lysophosphatidylcholines (LPCs) (Pattern 4) might be good markers for evaluating the severity of FHF, while some conjugated bile acids, long chain acylcarnitines (Pattern 2) and Glycocholic acid (Pattern 3) could indicate liver injury in the early stage. Inspired from the PCA plot that the pathogenetic condition of FHF aggravated with sampling time, a linear discriminant analysis (LDA) model based on phenylalanine and LPC 18:1 were further constructed for evaluating the severity of FHF. The leave-one-out cross-validation accuracy of 91.67% for the training set and the prediction accuracy of 92.31% for the external validation set confirmed its excellent performance. In conclusion, findings obtained from the present study, including four types of Dynamic Patterns of serum metabolites during FHF development and an LDA model for evaluating the severity of FHF, will be of great help to the research and management of FHF in the future.     

Metabolic signatures altered by in vitro temperature stress in <Emphasis Type="Italic">Ajuga bracteosa</Emphasis> Wall. ex. Benth.

To elucidate how biosynthesis of plant metabolites is affected by temperature, metabolite profiles from in vitro regenerated plants raised under different temperature regimes of 10, 15 °C, 20 °C, 25 °C and 30 °C were obtained using electrospray ionization mass spectrometry (ESI-MS), and principal component analysis (PCA) was carried out to identify key metabolites. Several bin masses were detected by PCA loading scatter plots which separated the samples. In-house bin program selectively manifested the putative known metabolites depending on % total ions count and intensity of selected bins in the plant samples. Total phenolic and flavonoid content were harvested to highest levels (12.9 mg GAE/g DW and 9.3 mg QE/g DW), respectively, at 15 °C. Besides, pinoresinol (lignan), some of the vital amino acids such as serine, methionine, histidine and glutamine were found to be at higher amount in plants raised at 15 °C. Significant phenylpropanoids like cinnamic acid, caffeic acid and quercitol were detected at a higher concentration in plants raised at 15 °C as compared to other treatments. However, phosphoenolpyruvate, and oxalosuccinate (intermediates of the pentose phosphate pathway) were accumulated the most in plants raised at 30 °C and they were detected with lowest values at 10 °C. Glucose and deoxy-xylose 5 phosphate (intermediates of TCA cycle) were found in higher amounts at temperature treatments of 15 and 25 °C, respectively. We conclude that a low-temperature treatment (15 °C) results in a stress-induced accumulation of a variety of pharmacologically important secondary metabolites.     

Metabolomics study of type 2 diabetes using ultra-performance LC-ESI/quadrupole-TOF high-definition MS coupled with pattern recognition methods

Type 2 diabetes (T2D), called the burden of the twenty-first century, is growing with an epidemic rate. Here, we explored the differences in metabolite concentrations between T2D patients and healthy volunteers. Metabolomics represents an emerging discipline concerned with comprehensive analysis of small molecule metabolites and provides a powerful approach to discover biomarkers in biological systems. The acquired data were analyzed by ultra-performance liquid chromatography–electrospray ionization/quadrupole time-of-flight high-definition mass spectrometry coupled with pattern recognition approach [principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA)] to identify potential disease-specific biomarkers. PCA showed satisfactory clustering between patients and healthy volunteers. Biomarkers reflected the biochemical events associated with early stages of T2D which were observed in PLS-DA loading plots. These urinary differential metabolites, such as adiponectin, acylcarnitines, citric acid, kynurenic acid, 3-indoxyl sulfate, urate, and glucose, were identified involving several key metabolic pathways such as taurine and hypotaurine metabolism; cysteine and methionine metabolism; valine, leucine, and isoleucine biosynthesis metabolism, etc. Our data suggest that robust metabolomics has the potential as a noninvasive strategy to evaluate the early diagnosis of T2D patients and provides new insight into pathophysiologic mechanisms and may enhance the understanding of its cause of disease.     

采用代谢组学分析技术分析工业啤酒发酵过程中风味物质生成规律

啤酒风味是保证啤酒品质的关键因素之一。运用代谢组学的方法,分析工业啤酒发酵过程中酵母胞内代谢物和啤酒风味物质的对应关系,从代谢水平上研究风味物质形成过程中的关键影响因素。在啤酒发酵过程中,同时检测风味物质的含量变化和酵母胞内代谢物的变化,对得到海量的、多维的代谢数据采用主成分分析（PCA）和偏最小二乘分析（PLS）的多元统计分析方法进行处理。由PCA分析结果可知：磷酸、海藻糖、琥珀酸、谷氨酸、天冬氨酸、丙氨酸对主成分贡献比较大,说明这些代谢物在不同发酵阶段含量变化显著。由PLS分析结果可知：对啤酒风味影响最大的物质主要为氨基酸,包括丝氨酸、缬氨酸、苏氨酸、赖氨酸、丙氨酸、亮氨酸和天冬酰胺等,这为啤酒中风味物质的调控提供了一定的理论指导。     

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl-l-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.     

Effect of benzothiadiazole on the metabolome of Arabidopsis thaliana

Benzothiadiazole (BTH) is a functional analog of the plant endogenous hormone-like compound, salicylic acid (SA), which is required for the induction of plant defense genes leading to systemic acquired resistance (SAR). Previous molecular and genetic studies have suggested that BTH itself might potentiate SAR resulting in the induction of several pathogenesis-related (PR) genes. However, the changes in the metabolome, which occur as a result of BTH-treatment, remain unclear. In this study, metabolic alterations in BTH-treated Arabidopsis thaliana were investigated using nuclear magnetic resonance (NMR) spectroscopy followed by multivariate data analyses such as principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA). Both PCA and PLS-DA show that increase of glucose, glutamine, inositol, malic acid, sucrose, and threonine as well as BTH and its degraded metabolites contribute to the clear discrimination of the metabolome of BTH-treated Arabidopsis from control plants. However, the levels of phenolic metabolites, which have generally been observed to be induced by other signaling molecules were significantly reduced in BTH-treated Arabidopsis. In addition to these changes due to BTH-treatment, it was also found that the EtOH used as a solvent in this treatment may per se act as an inducer of the accumulation of a flavonoid.     

Characterization of the Lipopolysaccharide and Structure of the O-Specific Polysaccharide of the Bacterium Pseudomonas syringae pv. atrofaciens IMV 948

Lipopolysaccharide (LPS) was isolated from the phytopathogenic bacterium Pseudomonas syringae pv. atrofaciens IMV 948 by mild extraction of the microbial cells with saline, and the properties, composition, and structure of the LPS were studied. The LPS showed low toxicity in D- galactosamine-sensitized mice and low biological activity in plants. Structural components of LPS--lipid A, core oligosaccharide, and O-specific polysaccharide (OPS)--were obtained by mild acid degradation and characterized. The lipid A contained fatty acids 3-HO-C10:0, C12:0, 2-HO-C12:0, 3-HO-C12:0, C16:0, C16:1, C18:0, and C18:1, as well as components of the hydrophilic moiety: GlcN, ethanolamine, phosphate, and phosphoethanolamine. The LPS core contained components typical of pseudomonads: glucose, rhamnose (Rha), L-glycero-D-manno-heptose, GlcN, GalN, 2-keto-3-deoxy-D-manno-octonic acid, alanine, and phosphate. The OPS consisted of L-Rha and D-GlcNAc in the ratio 4 : 1 and was structurally heterogeneous. The main pentasaccharide repeating unit of the OPS has the following structure: [structure see text]. Immunochemical studies showed that P. syringae pv. atrofaciens IMV 948 is serologically separate from other P. syringae strains, including those that have structurally similar OPS.