ABSTRACT
Cell walls are crucial for the integrity and function of all land plants and are of central importance in human health, livestock production, and as a source of renewable bioenergy. Many enzymes that mediate the biosynthesis of cell wall polysaccharides are encoded by members of the large cellulose synthase (CesA) gene superfamily. Here, we analyzed 29 sequenced genomes and 17 transcriptomes to revise the phylogeny of the CesA gene superfamily in angiosperms. Our results identify ancestral gene clusters that predate the monocot-eudicot divergence and reveal several novel evolutionary observations, including the expansion of the Poaceae-specific cellulose synthase-like CslF family to the graminids and restiids and the characterization of a previously unreported eudicot lineage, CslM, that forms a reciprocally monophyletic eudicot-monocot grouping with the CslJ clade. The CslM lineage is widely distributed in eudicots, and the CslJ clade, which was thought previously to be restricted to the Poales, is widely distributed in monocots. Our analyses show that some members of the CslJ lineage, but not the newly identified CslM genes, are capable of directing (1,3;1,4)-ß-glucan biosynthesis, which, contrary to current dogma, is not restricted to Poaceae.
Subject(s)
Cell Wall/metabolism , Glucosyltransferases/genetics , Phylogeny , Plant Proteins/genetics , Evolution, Molecular , Glucosyltransferases/metabolism , Magnoliopsida/enzymology , Magnoliopsida/genetics , Multigene Family , Plant Proteins/metabolism , Plants, Genetically Modified , Poaceae/enzymology , Poaceae/genetics , Nicotiana/genetics , Nicotiana/metabolism , beta-Glucans/metabolismABSTRACT
Metabolism is a key health risk factor following exposures to pro-carcinogenic polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[def,p]chrysene (DBC), an IARC classified 2A probable human carcinogen. Human exposure to PAHs occurs primarily from the diet in nonsmokers. However, little data is available on the metabolism and pharmacokinetics in humans of high molecular weight PAHs (≥4 aromatic rings), including DBC. We previously determined the pharmacokinetics of DBC in human volunteers orally administered a microdose (29 ng; 5 nCi) of [14C]-DBC by accelerator mass spectrometry (AMS) analysis of total [14C] in plasma and urine. In the current study, we utilized a novel "moving wire" interface between ultraperformance liquid chromatography (UPLC) and AMS to detect and quantify parent DBC and its major metabolites. The major [14C] product identified in plasma was unmetabolized [14C]-DBC itself (Cmax = 18.5 ±15.9 fg/mL, Tmax= 2.1 ± 1.0 h), whereas the major metabolite was identified as [14C]-(+/-)-DBC-11,12-diol (Cmax= 2.5 ±1.3 fg/mL, Tmax= 1.8 h). Several minor species of [14C]-DBC metabolites were also detected for which no reference standards were available. Free and conjugated metabolites were detected in urine with [14C]-(+/-)-DBC-11,12,13,14-tetraol isomers identified as the major metabolites, 56.3% of which were conjugated (Cmax= 35.8 ± 23.0 pg/pool, Tmax = 6-12 h pool). [14C]-DBC-11,12-diol, of which 97.5% was conjugated, was also identified in urine (Cmax = 29.4 ± 11.6 pg/pool, Tmax = 6-12 h pool). Parent [14C]-DBC was not detected in urine. This is the first data set to assess metabolite profiles and associated pharmacokinetics of a carcinogenic PAH in human volunteers at an environmentally relevant dose, providing the data necessary for translation of high dose animal models to humans for translation of environmental health risk assessment.
Subject(s)
Benzopyrenes/metabolism , Benzopyrenes/pharmacokinetics , Adult , Aged , Benzopyrenes/analysis , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Female , Healthy Volunteers , Humans , Male , Mass Spectrometry , Middle Aged , Molecular Structure , Young AdultABSTRACT
BACKGROUND: The ability to increase cellulose content and improve the stem strength of cereals could have beneficial applications in stem lodging and producing crops with higher cellulose content for biofuel feedstocks. Here, such potential is explored in the commercially important crop barley through the manipulation of cellulose synthase genes (CesA). RESULTS: Barley plants transformed with primary cell wall (PCW) and secondary cell wall (SCW) barley cellulose synthase (HvCesA) cDNAs driven by the CaMV 35S promoter, were analysed for growth and morphology, transcript levels, cellulose content, stem strength, tissue morphology and crystalline cellulose distribution. Transcript levels of the PCW HvCesA transgenes were much lower than expected and silencing of both the endogenous CesA genes and introduced transgenes was often observed. These plants showed no aberrant phenotypes. Although attempts to over-express the SCW HvCesA genes also resulted in silencing of the transgenes and endogenous SCW HvCesA genes, aberrant phenotypes were sometimes observed. These included brittle nodes and, with the 35S:HvCesA4 construct, a more severe dwarfing phenotype, where xylem cells were irregular in shape and partially collapsed. Reductions in cellulose content were also observed in the dwarf plants and transmission electron microscopy showed a significant decrease in cell wall thickness. However, there were no increases in overall crystalline cellulose content or stem strength in the CesA over-expression transgenic plants, despite the use of a powerful constitutive promoter. CONCLUSIONS: The results indicate that the cellulose biosynthetic pathway is tightly regulated, that individual CesA proteins may play different roles in the synthase complex, and that the sensitivity to CesA gene manipulation observed here suggests that in planta engineering of cellulose levels is likely to require more sophisticated strategies.
Subject(s)
Cell Wall/metabolism , Cellulose/metabolism , Gene Silencing , Hordeum/cytology , Hordeum/genetics , Transcription, Genetic , Cell Wall/ultrastructure , Fluorescent Antibody Technique , Gene Expression Regulation, Plant , Genes, Plant , Glucosyltransferases/genetics , Lignin/metabolism , Organ Specificity , Phenotype , Plant Stems/metabolism , Plants, Genetically Modified , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Setaria viridis has emerged as a model species for the larger C4 grasses. Here the cellulose synthase (CesA) superfamily has been defined, with an emphasis on the amounts and distribution of (1,3;1,4)-ß-glucan, a cell wall polysaccharide that is characteristic of the grasses and is of considerable value for human health. METHODS: Orthologous relationship of the CesA and Poales-specific cellulose synthase-like (Csl) genes among Setaria italica (Si), Sorghum bicolor (Sb), Oryza sativa (Os), Brachypodium distachyon (Bradi) and Hordeum vulgare (Hv) were compared using bioinformatics analysis. Transcription profiling of Csl gene families, which are involved in (1,3;1,4)-ß-glucan synthesis, was performed using real-time quantitative PCR (Q-PCR). The amount of (1,3;1,4)-ß-glucan was measured using a modified Megazyme assay. The fine structures of the (1,3;1,4)-ß-glucan, as denoted by the ratio of cellotriosyl to cellotetraosyl residues (DP3:DP4 ratio) was assessed by chromatography (HPLC and HPAEC-PAD). The distribution and deposition of the MLG was examined using the specific antibody BG-1 and captured using fluorescence and transmission electron microscopy (TEM). RESULTS: The cellulose synthase gene superfamily contains 13 CesA and 35 Csl genes in Setaria. Transcript profiling of CslF, CslH and CslJ gene families across a vegetative tissue series indicated that SvCslF6 transcripts were the most abundant relative to all other Csl transcripts. The amounts of (1,3;1,4)-ß-glucan in Setaria vegetative tissues ranged from 0.2% to 2.9% w/w with much smaller amounts in developing grain (0.003% to 0.013% w/w). In general, the amount of (1,3;1,4)-ß-glucan was greater in younger than in older tissues. The DP3:DP4 ratios varied between tissue types and across developmental stages, and ranged from 2.4 to 3.0:1. The DP3:DP4 ratios in developing grain ranged from 2.5 to 2.8:1. Micrographs revealing the distribution of (1,3;1,4)-ß-glucan in walls of different cell types and the data were consistent with the quantitative (1,3;1,4)-ß-glucan assays. CONCLUSION: The characteristics of the cellulose synthase gene superfamily and the accumulation and distribution of (1,3;1,4)-ß-glucans in Setaria are similar to those in other C4 grasses, including sorghum. This suggests that Setaria is a suitable model plant for cell wall polysaccharide biology in C4 grasses.
Subject(s)
Cell Wall/metabolism , Glucosyltransferases/genetics , Polysaccharides/genetics , Setaria Plant/genetics , beta-Glucans/metabolism , Glucosyltransferases/metabolism , Phylogeny , Polysaccharides/metabolism , Setaria Plant/cytology , Setaria Plant/metabolismABSTRACT
Heterodera avenae (cereal cyst nematode, CCN) infects the roots of barley (Hordeum vulgare) forming syncytial feeding sites. In resistant host plants, relatively few females develop to maturity. Little is known about the physiological and biochemical changes induced during CCN infection. Responses to CCN infection were investigated in resistant (Rha2) and susceptible barley cultivars through histological, compositional and transcriptional analysis. Two phases were identified that influence CCN viability, including feeding site establishment and subsequent cyst maturation. Syncytial development progressed faster in the resistant cultivar Chebec than in the susceptible cultivar Skiff, and was accompanied by changes in cell wall polysaccharide abundance, particularly (1,3;1,4)-ß-glucan. Transcriptional profiling identified several glycosyl transferase genes, including CELLULOSE SYNTHASE-LIKE F10 (HvCslF10), which may contribute to differences in polysaccharide abundance between resistant and susceptible cultivars. In barley, Rha2-mediated CCN resistance drives rapid deterioration of CCN feeding sites, specific changes in cell wall-related transcript abundance and changes in cell wall composition. During H. avenae infection, (1,3;1,4)-ß-glucan may influence CCN feeding site development by limiting solute flow, similar to (1,3)-ß-glucan during dicot cyst nematode infections. Dynamic transcriptional changes in uncharacterized HvCslF genes, possibly involved in (1,3;1,4)-ß-glucan synthesis, suggest a role for these genes in the CCN infection process.
Subject(s)
Disease Resistance/genetics , Hordeum/genetics , Hordeum/parasitology , Plant Diseases/parasitology , Plant Proteins/genetics , Tylenchoidea/physiology , beta-Glucans/metabolism , Animals , Cell Wall , Cluster Analysis , Crosses, Genetic , Disease Susceptibility , Fluorescence , Gene Expression Regulation, Plant , Genes, Plant , Giant Cells/physiology , Herbivory/physiology , Hordeum/immunology , Monosaccharides/analysis , Plant Diseases/genetics , Plant Proteins/metabolism , Plant Roots/metabolism , Plant Roots/parasitology , Polysaccharides/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
In cereals, the presence of soluble polysaccharides including (1,3;1,4)-ß-glucan has downstream implications for human health, animal feed and biofuel applications. Sorghum bicolor (L.) Moench is a versatile crop, but there are limited reports regarding the content of such soluble polysaccharides. Here, the amount of (1,3;1,4)-ß-glucan present in sorghum tissues was measured using a Megazyme assay. Very low amounts were present in the grain, ranging from 0.16%-0.27% (w/w), while there was a greater quantity in vegetative tissues at 0.12-1.71% (w/w). The fine structure of (1,3;1,4)-ß-glucan, as denoted by the ratio of cellotriosyl and cellotetraosyl residues, was assessed by high performance liquid chromatography (HPLC) and ranged from 2.6-3:1 in the grain, while ratios in vegetative tissues were lower at 2.1-2.6:1. The distribution of (1,3;1,4)-ß-glucan was examined using a specific antibody and observed with fluorescence and transmission electron microscopy. Micrographs showed a variable distribution of (1,3;1,4)-ß-glucan influenced by temporal and spatial factors. The sorghum orthologs of genes implicated in the synthesis of (1,3;1,4)-ß-glucan in other cereals, such as the Cellulose synthase-like (Csl) F and H gene families were defined. Transcript profiling of these genes across sorghum tissues was carried out using real-time quantitative polymerase chain reaction, indicating that, as in other cereals, CslF6 transcripts dominated.
Subject(s)
Biosynthetic Pathways/genetics , Genes, Plant , Multigene Family , Sorghum/genetics , beta-Glucans/metabolism , Gene Expression Profiling , Gene Expression Regulation, Plant , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Plant Roots/metabolism , Plant Stems/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Seeds/metabolism , Seeds/ultrastructure , Sorghum/cytologyABSTRACT
In plants, cell walls are one of the first lines of defence for protecting cells from successful invasion by fungal pathogens and are a major factor in basal host resistance. For the plant cell to block penetration attempts, it must adapt its cell wall to withstand the physical and chemical forces applied by the fungus. Papillae that have been effective in preventing penetration by pathogens are traditionally believed to contain callose as the main polysaccharide component. Here, we have re-examined the composition of papillae of barley (Hordeum vulgare) attacked by the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) using a range of antibodies and carbohydrate-binding modules that are targeted to cell wall polysaccharides. The data show that barley papillae induced during infection with Bgh contain, in addition to callose, significant concentrations of cellulose and arabinoxylan. Higher concentrations of callose, arabinoxylan and cellulose are found in effective papillae, compared with ineffective papillae. The papillae have a layered structure, with the inner core consisting of callose and arabinoxylan and the outer layer containing arabinoxylan and cellulose. The association of arabinoxylan and cellulose with penetration resistance suggests new targets for the improvement of papilla composition and enhanced disease resistance.
Subject(s)
Ascomycota/physiology , Cellulose/metabolism , Glucans/metabolism , Hordeum/microbiology , Plant Leaves/metabolism , Xylans/metabolism , Hordeum/metabolism , Plant Diseases , Plant Leaves/cytology , Plant Leaves/microbiologyABSTRACT
Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC-MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2-7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with Kms ranging from 7 to 160 µM and turnover numbers of 30-40 min(-1). The product formed was identified by LC-MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1.
Subject(s)
Oxygenases/metabolism , Thiones/metabolism , Thiourea/metabolism , Animals , Cell Line , Humans , Insecta , Mice , Oxidation-Reduction , Oxygenases/chemistry , Oxygenases/genetics , Thiones/chemistry , Thiourea/chemistryABSTRACT
To explain the low levels of starch, high levels of (1,3;1,4)-ß-glucan, and thick cell walls in grains of Brachypodium distachyon L. relative to those in other Pooideae, aspects of grain development were compared between B. distachyon and barley (Hordeum vulgare L.). Cell proliferation, cell expansion, and endoreduplication were reduced in B. distachyon relative to barley and, consistent with these changes, transcriptional downregulation of the cell-cycle genes CDKB1 and cyclin A3 was observed. Similarly, reduced transcription of starch synthase I and starch-branching enzyme I was observed as well as reduced activity of starch synthase and ADP-glucose pyrophosphorylase, which are consistent with the lowered starch content in B. distachyon grains. No change was detected in transcription of the major gene involved in (1,3;1,4)-ß-glucan synthesis, cellulose synthase-like F6. These results suggest that, while low starch content results from a reduced capacity for starch synthesis, the unusually thick cell walls in B. distachyon endosperm probably result from continuing (1,3;1,4)-ß-glucan deposition in endosperm cells that fail to expand. This raises the possibility that endosperm expansion is linked to starch deposition.
Subject(s)
Brachypodium/metabolism , Cell Wall/metabolism , Seeds/growth & development , Starch/metabolism , Brachypodium/genetics , Brachypodium/growth & development , Cell Wall/genetics , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Proteins/metabolism , Poaceae/genetics , Poaceae/growth & development , Poaceae/metabolism , Seeds/genetics , Seeds/metabolismABSTRACT
Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-ß-d-glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)-ß-d-glucans in the Poaceae is mediated, in part at least, by the cellulose synthase-like CslF family of genes. Over-expression of the barley CslF6 gene under the control of an endosperm-specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)-ß-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-ß-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-ß-d-glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)-ß-d-glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)-ß-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-ß-d-glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)-ß-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-ß-d-glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.
Subject(s)
Cell Wall/enzymology , Glucosyltransferases/genetics , Hordeum/enzymology , Plant Proteins/genetics , Plants, Genetically Modified/enzymology , beta-Glucans/metabolism , Cell Wall/genetics , Cell Wall/ultrastructure , Dietary Fiber/metabolism , Genetic Engineering , Glucosyltransferases/metabolism , Hordeum/genetics , Hordeum/ultrastructure , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Plant Proteins/metabolism , Plant Proteins/physiology , RNA, Messenger/metabolism , Seeds/genetics , Seeds/metabolism , Seeds/ultrastructureABSTRACT
Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA >30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes "superinduction" of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (>300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.
Subject(s)
Liver/enzymology , Oxygenases/biosynthesis , Polycyclic Aromatic Hydrocarbons/toxicity , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Benzo(a)pyrene/toxicity , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Liver/drug effects , Liver Neoplasms, Experimental/enzymology , Male , Methylcholanthrene/toxicity , Mice , Mice, Inbred C57BL , Oxygenases/genetics , Polychlorinated Dibenzodioxins/toxicity , RNA, Messenger/metabolism , Receptors, Aryl Hydrocarbon/metabolism , XenobioticsABSTRACT
Catalytically active human flavin-containing monooxygenase isoform 2 (FMO2.1) is encoded by an allele detected only in individuals of African or Hispanic origin. Genotyping and haplotyping studies indicate that S195L and N413K occasionally occur secondary to the functional FMO2*1 allele encoding reference protein Gln472. Sulfoxygenation under a range of conditions reveals the role these alterations may play in individuals expressing active FMO2 and provides insight into FMO structure. Expressed S195L lost rather than gained activity as pH was increased or when cholate was present. The activity of S195L was mostly eliminated after heating at 45 degrees C for 5 min in the absence of NADPH, but activity was preserved if NADPH was present. By contrast, Gln472 was less sensitive to heat, a response not affected by NADPH. A major consequence of the S195L mutation was a mean 12-fold increase in K(m) for NADPH compared with Gln472. Modeling an S213L substitution, the equivalent site, in the structural model of FMO from the Methylophaga bacterium leads to disruption of interactions with NADP(+). N413K had the same pattern of activity as Gln472 in response to pH, cholate, and magnesium, but product formation was always elevated by comparison. N413K also lost more activity when heated than Gln472; however, NADPH attenuated this loss. The major effects of N413K were increases in velocity and k(cat) compared with Gln472. Although these allelic variants are expected to occur infrequently as mutations to the FMO2*1 allele, they contribute to our overall understanding of mammalian FMO structure and function.
Subject(s)
Oxygenases/metabolism , Polymorphism, Single Nucleotide , Sulfoxides/metabolism , Cholates/metabolism , Databases, Protein , Enzyme Stability , Genotype , Hot Temperature , Humans , Hydrogen-Ion Concentration , Isoenzymes , Kinetics , Magnesium/metabolism , Models, Molecular , NADP/metabolism , Oxidation-Reduction , Oxygenases/chemistry , Oxygenases/genetics , Phenotype , Protein Conformation , Protein Denaturation , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
The significance of active versus inactive flavin-containing monooxygenase 2 (FMO2) for human drug and xenobiotic metabolism and sensitivity is unknown, but the underlying ethnic polymorphism is well documented. We used quantitative real-time PCR to measure message levels of Fmo1, Fmo2, Fmo3 and Fmo5 in lung and liver from eight strains of 8 week old female mice to determine if a strain could be identified that predominately expressed Fmo2 in lung, recapitulating the human FMO expression profile and being the ideal strain for Fmo2 knockout studies. We also characterized enzyme activity of baculovirus expressed mouse Fmo1, Fmo2 and Fmo3 to identify a substrate or incubation conditions capable of discriminating Fmo2 from Fmo mixtures. Fmo transcript expression patterns were similar for all strains. In lung, 59% of total FMO message was Fmo2, but Fmo1 levels were also high, averaging 34%, whereas Fmo3 and Fmo5 levels were 2 and 5%, respectively. In liver, Fmo1, Fmo2, Fmo3 and Fmo5 contributed 16, 1, 7 and 76% respectively, of detected message. Peak activity varied by isoform and was pH- and substrate-dependent. Fmo3 oxidation of methyl p-tolyl sulfide was negligible at pH 9.5, but Fmo3 oxidation of methimazole was comparable to Fmo1 and Fmo2. Heating microsomes at 50 degrees C for 10min eliminated most Fmo1 and Fmo3 activity, while 94% of Fmo2 activity remained. Measurement of activity in heated and unheated lung and liver microsomes verified relative transcript abundance. Our results show that dual Fmo1/2 knockouts will be required to model the human lung FMO profile.
Subject(s)
Isoenzymes/genetics , Liver/enzymology , Lung/enzymology , Oxygenases/genetics , RNA, Messenger/analysis , Animals , Female , Kinetics , Mice , Mice, Inbred Strains , Oxygenases/metabolismABSTRACT
Tuberculosis (TB) results from infection with Mycobacterium tuberculosis and remains endemic throughout the world with one-third of the world's population infected. The prevalence of multi-drug resistant strains necessitates the use of more toxic second-line drugs such as ethionamide (ETA), a pro-drug requiring bioactivation to exert toxicity. M. tuberculosis possesses a flavin monooxygenase (EtaA) that oxygenates ETA first to the sulfoxide and then to 2-ethyl-4-amidopyridine, presumably through a second oxygenation involving sulfinic acid. ETA is also a substrate for mammalian flavin-containing monooxygenases (FMOs). We examined activity of expressed human and mouse FMOs toward ETA, as well as liver and lung microsomes. All FMOs converted ETA to the S-oxide (ETASO), the first step in bioactivation. Compared to M. tuberculosis, the second S-oxygenation to the sulfinic acid is slow. Mouse liver and lung microsomes, as well as human lung microsomes from an individual expressing active FMO, oxygenated ETA in the same manner as expressed FMOs, confirming this reaction functions in the major target organs for therapeutics (lung) and toxicity (liver). Inhibition by thiourea, and lack of inhibition by SKF-525A, confirm ETASO formation is primarily via FMO, particularly in lung. ETASO production was attenuated in a concentration-dependent manner by glutathione. FMO3 in human liver may contribute to the toxicity and/or affect efficacy of ETA administration. Additionally, there may be therapeutic implications of efficacy and toxicity in human lung based on the FMO2 genetic polymorphism, though further studies are needed to confirm that suggestion.
Subject(s)
Antitubercular Agents/metabolism , Ethionamide/metabolism , Lung/metabolism , Microsomes, Liver/metabolism , Oxygenases/metabolism , Animals , Antitubercular Agents/pharmacokinetics , Chromatography, High Pressure Liquid , Ethionamide/pharmacokinetics , Glutathione/metabolism , Humans , In Vitro Techniques , Lung/cytology , Lung/drug effects , Lung/enzymology , Mass Spectrometry , Mice , Microsomes/drug effects , Microsomes/enzymology , Microsomes/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/enzymologyABSTRACT
Global grape production could generate up to 13 Mt/yr of wasted biomass. The compositions of Cabernet Sauvignon (red marc) and Sauvignon Blanc (white marc) were analyzed with a view to using marc as raw material for biofuel production. On a dry weight basis, 31-54% w/w of the grape marc consisted of carbohydrate, of which 47-80% was soluble in aqueous media. Ethanol insoluble residues consisted mainly of polyphenols, pectic polysaccharides, heteroxylans and cellulose. Acid and thermal pre-treatments were investigated for their effects on subsequent cellulose saccharification. A 0.5M sulfuric acid pre-treatment yielded a 10% increase in the amount of liberated glucose after enzymatic saccharification. The theoretical amount of bioethanol that could be produced by fermentation of grape marc was up to 400 L/t. However, bioethanol from only soluble carbohydrates could yield 270 L/t, leaving a polyphenol enriched fraction that may be used in animal feed or as fertilizer.
Subject(s)
Cellulose/chemistry , Ethanol/chemistry , Glucose/chemistry , Polysaccharides/chemistry , Vitis/chemistry , Animal Feed , Biofuels , Biomass , Fermentation/physiology , Polyphenols/chemistry , Sulfuric Acids/chemistryABSTRACT
Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like--rich in moisture, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47-50% w/w) and non-cellulosic polysaccharides (16-22% w/w), and whole leaves were low in lignin (9-13% w/w). Of the dry mass of whole Agave leaves, 85-95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41-48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition.
Subject(s)
Agave/chemistry , Cellulose/chemistry , Plant Leaves/chemistry , Renewable Energy , Agave/metabolism , Biomass , Fermentation , Hydrolysis , Lignin/chemistry , Plant Leaves/metabolism , Polysaccharides/chemistryABSTRACT
Phorate and disulfoton are organophosphate insecticides containing three oxidizable sulfurs, including a thioether. Previous studies have shown that only the thioether is oxygenated by flavin-containing monooxygenase (FMO) and the sole product is the sulfoxide with no oxygenation to the sulfone. The major FMO in lung of most mammals, including non-human primates, is FMO2. The FMO2*2 allele, found in all Caucasians and Asians genotyped to date, codes for a truncated, non-functional, protein (FMO2.2A). Twenty-six percent of individuals of African descent and 5% of Hispanics have the FMO2*1 allele, coding for full-length, functional protein (FMO2.1). We have here demonstrated that the thioether-containing organophosphate insecticides, phorate and disulfoton, are substrates for expressed human FMO2.1 with Km of 57 and 32 microM, respectively. LC/MS confirmed the addition of oxygen and formation of a single polar metabolite for each chemical. MS/MS analysis confirmed the metabolites to be the respective sulfoxides. Co-incubations with glutathione did not reduce yield, suggesting they are not highly electrophilic. As the sulfoxide of phorate is a markedly less effective acetylcholinesterase inhibitor than the cytochrome P450 metabolites (oxon, oxon sulfoxide or oxon sulfone), humans possessing the FMO2*1 allele may be more resistant to organophosphate-mediated toxicity when pulmonary metabolism is an important route of exposure or disposition.
Subject(s)
Disulfoton/metabolism , Insecticides/metabolism , Lung/enzymology , Oxygenases/metabolism , Phorate/metabolism , Animals , Humans , Mice , Microsomes/metabolism , Oxidation-Reduction , RabbitsABSTRACT
We studied the estrogenic effects of model chemicals in one-year-old juvenile rainbow trout. Methoxychlor (20 mg/kg), diethylstilbestrol (15 mg/kg), 4-tert-octylphenol (25 and 50 mg/kg), and biochanin A (25 and 50 mg/kg) were injected intraperitoneally on days 1, 4, and 7. Fish were sacrificed on day 9 and examined for multiple biomarkers. All of the test chemicals caused increases in plasma vitellogenin levels, a biomarker of estrogenicity. Treatment with the xenoestrogens decreased hepatic lauric acid hydroxylase activity and, as shown by Western blots, also generally reduced expression of hepatic cytochrome P450s 2K1 (CYP2K1), 2M1 (CYP2M1), and 3A27 (CYP3A27) at the protein level. Both doses of biochanin A also significantly induced P4501A (CYPIA) and greatly increased hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity. These findings suggest that methoxychlor, diethylstilbestrol, 4-tert-octylphenol, and biochanin A were all estrogenic and mimicked 17beta-estradiol (E2) in repressing the expression of cytochrome P450 isoforms (CYP2KI, CYP2M1, and CYP3A27) in the rainbow trout liver. Additionally, biochanin A was found to induce CYPIA in this fish species.
Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Microsomes, Liver/metabolism , Oncorhynchus mykiss/metabolism , Water Pollutants, Chemical/pharmacology , Animals , Blotting, Northern , Blotting, Western , Cytochrome P-450 CYP4A , Cytochrome P-450 Enzyme Inhibitors , Diethylstilbestrol/pharmacology , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Estradiol/pharmacology , Female , Genistein/pharmacology , Injections, Intraperitoneal , Isoenzymes , Male , Methoxychlor/pharmacology , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Mixed Function Oxygenases/antagonists & inhibitors , Phenols/pharmacology , Vitellogenins/bloodABSTRACT
Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30-50% of O2 consumed as H2O2 upon addition of NADPH. Addition of substrate had little effect on H2O2 production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H2O2. FMO2 S195L exhibited higher "leakage", producing much greater amounts of H2O2, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H2O2 generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H2O2 release. Catalase did not reduce levels of H2O2 with either FMO2.1 or FMO3 but inhibited H2O2 generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP(+) binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H2O2.