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Enterobacter sp. 638 is an endophytic plant growth promoting gamma-proteobacterium that was isolated from the stem of poplar (Populus trichocarpa×deltoides cv. H11-11), a potentially important biofuel feed stock plant. The Enterobacter sp. 638 genome sequence reveals the presence of a 4,518,712 bp chromosome and a 157,749 bp plasmid (pENT638-1). Genome annotation and comparative genomics allowed the identification of an extended set of genes specific to the plant niche adaptation of this bacterium. This includes genes that code for putative proteins involved in survival in the rhizosphere (to cope with oxidative stress or uptake of nutrients released by plant roots), root adhesion (pili, adhesion, hemagglutinin, cellulose biosynthesis), colonization/establishment inside the plant (chemiotaxis, flagella, cellobiose phosphorylase), plant protection against fungal and bacterial infections (siderophore production and synthesis of the antimicrobial compounds 4-hydroxybenzoate and 2-phenylethanol), and improved poplar growth and development through the production of the phytohormones indole acetic acid, acetoin, and 2,3-butanediol. Metabolite analysis confirmed by quantitative RT–PCR showed that, the production of acetoin and 2,3-butanediol is induced by the presence of sucrose in the growth medium. Interestingly, both the genetic determinants required for sucrose metabolism and the synthesis of acetoin and 2,3-butanediol are clustered on a genomic island. These findings point to a close interaction between Enterobacter sp. 638 and its poplar host, where the availability of sucrose, a major plant sugar, affects the synthesis of plant growth promoting phytohormones by the endophytic bacterium. The availability of the genome sequence, combined with metabolome and transcriptome analysis, will provide a better understanding of the synergistic interactions between poplar and its growth promoting endophyte Enterobacter sp. 638. This information can be further exploited to improve establishment and sustainable production of poplar as an energy feedstock on marginal, non-agricultural soils using endophytic bacteria as growth promoting agents.

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Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased >5-fold during adipogenesis in medium containing 30 mM glucose, producing a >10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N ⑀ -(carboxymethyl)lysine and N ⑀ -(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one-and two-dimensional electrophoresis and ϳ60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.The adipocyte is increasingly recognized as a dynamic cell that readily adapts to the changing nutritional status of the body. In a state of over-nutrition, the adipocyte responds by synthesizing and storing triglycerides, a process that may eventually lead to obesity and insulin resistance and then to diabetes.Chemical modification of proteins by advanced glycoxidation and lipoxidation end products (AGE/ALEs) 3 is increased in diabetes and is strongly implicated in the development of diabetic complications (1-3). However, relatively little is known about the chemical modification of proteins in adipocytes or adipose tissue or the possible role of chemical modifications of proteins in the regulation of adipocyte metabolism during adipogenesis or diabetogenesis.In general, AGE/ALEs are derivatives of lysine and arginine residues, formed by reaction of the amino or guanidino groups on protein with electrophilic intermediates in carbohydrate and lipid autoxidation or metabolism (4). In contrast to extracellular proteins, the lower pK a sulfhydryl group of cysteine residues is a more likely target for modification by electrophiles on intracellular proteins. We recently described S-(2-succinyl)cysteine (2SC) as a novel chemical modification of cysteine residues in proteins, formed by a...

2-Hydroxysphingolipids are a subset of sphingolipids containing 2-hydroxy fatty acids. The 2-hydroxylation occurs during de novo ceramide synthesis and is catalyzed by fatty acid 2-hydroxylase (also known as fatty acid ␣-hydroxylase). In mammals, 2-hydroxysphingolipids are present abundantly in brain because the major myelin lipids galactosylceramides and sulfatides contain 2-hydroxy fatty acids. Here we report identification and characterization of a human gene that encodes a fatty acid 2-hydroxylase. Data base searches revealed a human homologue of the yeast ceramide 2-hydroxylase gene (FAH1), which we named FA2H. The FA2H gene encodes a 372-amino acid protein with 36% identity and 46% similarity to yeast Fah1p. The amino acid sequence indicates that FA2H protein contains an N-terminal cytochrome b 5 domain and four potential transmembrane domains. FA2H also contains the iron-binding histidine motif conserved among membrane-bound desaturases/ hydroxylases. COS7 cells expressing human FA2H contained 3-20-fold higher levels of 2-hydroxyceramides (C16, C18, C24, and C24:1) and 2-hydroxy fatty acids compared with control cells. Microsomal fractions prepared from transfected COS7 cells showed tetracosanoic acid 2-hydroxylase activities in an NADPH-and NADPH: cytochrome P-450 reductase-dependent manner. FA2H lacking the N-terminal cytochrome b 5 domain had little activity, indicating that this domain is a functional component of this enzyme. Northern blot analysis showed that the FA2H gene is highly expressed in brain and colon tissues. These results demonstrate that the human FA2H gene encodes a fatty acid 2-hydroxylase. FA2H is likely involved in the formation of myelin 2-hydroxy galactosylceramides and -sulfatides.Sphingolipids are a large class of lipids found in all eukaryotic cells and are involved in a variety of cellular processes. The structural diversity of sphingolipids stems from over 300 known distinct head groups as well as modifications of the hydrophobic ceramide moiety. One of the common modifications of the ceramide moiety is 2-hydroxylation of the N-acyl chain. Sphingolipids with 2-hydroxy fatty acid are found in most organisms including plants, yeast, worms, vertebrate animals, and some bacterial species.In mammals, 2-hydroxy fatty acid-containing sphingolipids are uniquely abundant in nervous and epidermal tissues. In mammalian central and peripheral nervous systems, galactosylceramides and sulfatides (3-sulfate ester of galactosylceramide) are major lipid components of myelin (1, 2). These glycosphingolipids contain a high proportion (ϳ50%) of 2-hydroxy fatty acid (3) and are critical components of myelin (4, 5). In mammalian epidermal tissues, there are several unique, very long chain ceramides with 2-hydroxy fatty acids, which are critical for the permeability barrier function of epidermis (6, 7).Several biophysical studies (8 -10) demonstrated that the 2-hydroxyl group in sphingolipids has a profound effect in the lipid organization within membranes because of its hydrogenbonding capability....

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of glucose adducts to protein in vitro and has been detected in human tissue proteins and urine [Ahmed, M. U., Thorpe, S. R., & Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894; Dunn, J. A., Patrick, J. S., Thorpe, S. R., & Baynes, J. W. (1989) Biochemistry 28, 9464-9468]. In the present study we show that CML is also formed in reactions between ascorbate and lysine residues in model compounds and protein in vitro. The formation of CML from ascorbate and lysine proceeds spontaneously at physiological pH and temperature under air. Kinetic studies indicate that oxidation of ascorbic acid to dehydroascorbate is required. Threose and N epsilon-threuloselysine, the Amadori adduct of threose to lysine, were identified in the ascorbate reaction mixtures, suggesting that CML was formed by oxidative cleavage of N epsilon-threuloselysine. Support for this mechanism was obtained by identifying CML as a product of reaction between threose and lysine and by analysis of the relative rates of formation of threuloselysine and CML in reactions of ascorbate or threose with lysine. The detection of CML as a product of reaction of ascorbate and threose with lysine suggests that other sugars, in addition to glucose, may be sources of CML in proteins in vivo. The proposed mechanism for formation of CML from ascorbate is an example of autoxidative glycosylation of protein and suggests that CML may also be an indicator of autoxidative glycosylation of proteins in vivo.

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Residues of the insecticide toxaphene (polychlorinated camphenes, PCCs) and other organochlorines (0 0) were determined in air, snow, seawater, zooplankton, and benthic amphipods collected from an ice island in the Canadian Arctic. The simultaneous determination of OCs in the atmospheric, hydrologic, and biologic compartments provided evidence of an atmospheric link to polar food chains. PCCs were identified and quantified using capillary gas chromatographynegative ion mass spectrometry. The order of OC abundance in arctic air was: hexachlorocyclohexanes (HCHs) > hexachlorobenzene > PCCs > polychlorinated biphenyls (PCBs) > chlordanes > DDTs. In seawater, PCCs were exceeded only by the HCHs. Concentrations of PCBs and PCCs in two samples of benthic amphipods were the highest of the OCs detected.