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Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on hidden enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by unclustered protease genes required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and hidden proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5209 correlations between precursors and hidden proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of previously unreported class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of hidden lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis.

C/EBP homologous protein (CHOP) is a transcription factor that is elevated in adipose tissue across many models of diabetes and metabolic stress. Although increased CHOP levels are associated with the terminal response to endoplasmic reticulum stress and apoptosis, there is no evidence for CHOP mediated apoptosis in the adipose tissue during diabetes. CHOP protein levels increase in parallel with protein succination, a fumarate derived cysteine modification, in the adipocyte during metabolic stress. We investigated the factors contributing to sustained CHOP proteins levels in the adipocyte, with an emphasis on the regulation of CHOP protein turnover by metabolite-driven modification of Keap1 cysteines. CHOP protein stability was investigated in conditions of nutrient stress due to high glucose or elevated fumarate (fumarase knockdown model); where cysteine succination is specifically elevated. CHOP protein turnover is significantly reduced in models of elevated glucose and fumarate with a ~30% increase in CHOP stability (p > 0.01), in part due to decreased CHOP phosphorylation. Sustained CHOP levels occur in parallel with elevated heme-oxygenase-1, a production of increased Nrf2 transcriptional activity and Keap1 modification. While Keap1 is directly succinated in the presence of excess fumarate derived from genetic knockdown of fumarase (fumarate levels are elevated >20-fold), it is the oxidative modification of Keap1 that predominates in adipocytes matured in high glucose (fumarate increases 4–5 fold). Elevated fumarate indirectly regulates CHOP stability through the induction of oxidative stress. The antioxidant N-acetylcysteine (NAC) reduces fumarate levels, protein succination and CHOP levels in adipocytes matured in high glucose. Elevated CHOP does not contribute elevated apoptosis in adipocytes, but plays a redox-dependent role in decreasing the adipocyte secretion of interleukin-13, an anti-inflammatory chemokine. NAC treatment restores adipocyte IL-13 secretion, confirming the redox-dependent regulation of a potent anti-inflammatory eotaxin. This study demonstrates that physiological increases in the metabolite fumarate during high glucose exposure contributes to the presence of oxidative stress and sustained CHOP levels in the adipocyte during diabetes. The results reveal a novel metabolic link between mitochondrial metabolic stress and reduced anti-inflammatory adipocyte signaling as a consequence of reduced CHOP protein turnover.

Bacterial natural product biosynthetic genes, canonically clustered, have been increasingly found to rely on cryptic enzymes encoded elsewhere in the genome for completion of biosynthesis. The study and application of lanthipeptides are frequently hindered by cryptic proteases required for final maturation. Here, we establish a global correlation network bridging the gap between lanthipeptide precursors and cryptic proteases. Applying our analysis to 161,954 bacterial genomes, we establish 5,209 correlations between precursors and cryptic proteases, with 91 prioritized. We use network predictions and co-expression analysis to reveal a previously missing protease for the maturation of class I lanthipeptide paenilan. We further discover widely distributed bacterial M16B metallopeptidases of previously unclear biological function as a new family of lanthipeptide proteases. We show the involvement of a pair of bifunctional M16B proteases in the production of novel class III lanthipeptides with high substrate specificity. Together, these results demonstrate the strength of our correlational networking approach to the discovery of cryptic lanthipeptide proteases and potentially other missing enzymes for natural products biosynthesis.Graphical abstract

The sphingomyelin pathway is important in cell regulation and determining cellular fate. Inhibition of sphingosine kinase isoform 1 (SK1) within this pathway, leads to a buildup of sphingosine and ceramide, two molecules directly linked to cell apoptosis, while decreasing the intracellular concentration of sphingosine-1-phosphate (S1P), a molecule linked to cellular proliferation. Recently, an inhibitor capable of inhibiting SK1 in vitro was identified, but also shown to be ineffective in vivo. A set of compounds designed to assess the impact of synthetic modifications to the hydroxynaphthalene ring region of the template inhibitor with SK1 to obtain a compound with increased efficacy in vivo. Of these fifteen compounds, 4A was shown to have an IC50 = 6.55 μM with improved solubility and in vivo potential.

Emerging evidence suggests that gut microbiota may influence the response to chemotherapy. We sought to characterize the effects of 5 fluorouracil (5FU) chemotherapy on colon inflammation and functional measures in colorectal cancer (CRC) and to further determine whether gut microbiota can influence this response. 50 C57BL/6 were randomized into four groups; Control + Vehicle (n = 10), Control + 5FU (n = 10), AOM/DSS + Vehicle (n = 15), and AOM/DSS + 5FU (n = 15). CRC was induced chemically by a single 10 mg/kg injection of azoxymethane (AOM) followed by two cycles (2% and 1%) of dextran sodium sulfate (DSS). Mice were then treated with 3 cycles of vehicle or 5FU (cycle 1: 40 mg/kg, cycle 2 + 3: 20 mg/kg). Functional tests (grip strength and run-to-fatigue) were performed prior to 5FU treatment (baseline) and at the completion of the second cycle of 5FU. Following the third 5FU cycle, mice were euthanized and the colon was evaluated for expression of inflammatory genes using RT-qPCR and stool samples were profiled using 16S rRNA sequencing. A second experiment used fecal microbiota transplantation from 5FU treated mice to control mice (n = 10-15/group) to determine whether 5FU associated changes in the microbiota could influence functional measures and colon inflammation. 5FU reduced grip strength (p 0.05) and caused a trending decrease in run-to-fatigue performance in cancer mice (p = 0.06). Select intestinal inflammatory genes were significantly elevated with 5FU treatment and this was further exacerbated with cancer (p 0.05). Microbiota analysis revealed increased dissimilarity and alterations in bacterial taxonomy in 5FU and AOM/DSS-treated mice (p 0.05). Fecal transplant from 5FU treated mice reduced functional performance (p 0.05) and altered select colon inflammatory markers (p 0.05). This study provides evidence of an effect of 5FU on inflammatory responses and functional measures in a mouse model of CRC and suggests that gut microbes may play a role in some, but not all, 5FU related perturbations.

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.

Mauritius Island possesses unique plant biodiversity with a potential reservoir of biologically active compounds of pharmacological interest. In the current study, we investigated Mauritius endemic plant families Asteraceae, Ebenaceae, Sapotaceae, and Erythroxylaceae, for anti-cancer properties on T cell lymphoma and B16F10 Melanoma cells and immunomodulatory properties on primary T and B cells. The cytotoxicity of methanolic plant extracts at 1, 10, 25 µg/ml was determined. The most active plant species were evaluated for their apoptosis-inducing effects. The immunomodulatory properties of the plants were also studied, and preliminary phytochemical screening of selected plants was done by LC–MS analysis. Psiadia lithospermifolia (Lam.) Cordem (Asteraceae) at 25 µg/ml was the most cytotoxic on both EL4 and B16 cells and triggered apoptosis by the death receptor pathway, and at least in part, by the mitochondrial pathway. Most plant species from Asteraceae, Ebenaceae, Erythroxylaceae, and Sapotaceae inhibited the proliferation of activated T and B cells, although some promoted T cell proliferation. LC–MS profile of Asteraceae plants showed the presence of terpenes, terpenoids, fatty acids, and phenolic. Flavonoids and phenolic acid were also detected from Ebenaceae and Sapotaceae plants. Together, our study demonstrated that Mauritius endemic flora exhibit potential anti-cancer and anti-inflammatory properties worthy of further in-depth studies.

Background and Aims Cannabinoid receptor [CB] activation can attenuate inflammatory bowel disease [IBD] in experimental models and human cohorts. However, the roles of the microbiome, metabolome, and the respective contributions of haematopoietic and non-haematopoietic cells in the anti-colitic effects of cannabinoids have yet to be determined. Methods Female C57BL/6 mice were treated with either cannabidiol [CBD], Δ 9-tetrahydrocannabinol [THC], a combination of CBD and THC, or vehicle, in several models of chemically induced colitis. Clinical parameters of colitis were assessed by colonoscopy, histology, flow cytometry, and detection of serum biomarkers; single-cell RNA sequencing and qRT-PCR were used to evaluate the effects of cannabinoids on enterocytes. Immune cell transfer from CB2 knockout mice was used to evaluate the contribution of haematopoietic and non-haematopoietic cells to colitis protection. Results We found that THC prevented colitis and that CBD, at the dose tested, provided little benefit to the amelioration of colitis, nor when added synergistically with THC. THC increased colonic barrier integrity by stimulating mucus and tight junction and antimicrobial peptide production, and these effects were specific to the large intestine. THC increased colonic Gram-negative bacteria, but the anti-colitic effects of THC were independent of the microbiome. THC acted both on immune cells via CB2 and on enterocytes, to attenuate colitis. Conclusions Our findings demonstrate how cannabinoid receptor activation on both immune cells and colonocytes is critical to prevent colonic inflammation. These studies also suggest how cannabinoid receptor activation can be used as a preventive and therapeutic modality against colitis.

SummaryThe NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration, suggesting a mechanistic role in neurodegenerative decline. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle, is succinated in the NDUFS4 KO brain. Succination of DLST reduced KGDHC activity in the brainstem (BS) and olfactory bulb (OB) of KO mice. The defective production of KGDHC derived succinyl-CoA resulted in decreased mitochondrial substrate level phosphorylation, further aggravating the OXPHOS ATP deficit. Protein succinylation, an acylation modification that requires succinyl-CoA, was reduced in the KO mice. Our data demonstrate that the biochemical deficit extends beyond impaired Complex I assembly and OXPHOS deficiency, functionally impairing select components of the TCA cycle to drive metabolic perturbations in affected neurons.HighlightsCysteine succination by fumarate is increased in model of Complex I deficiencySuccination of DLST impairs KGDHC activity, limits SLP, and reduces lysine succinylationIrreversible succination drives neuropathology independent of the bioenergetic defect

Protein succination by fumarate increases in the adipose tissue of diabetic mice and in adipocytes matured in high glucose as a result of glucotoxicity-driven mitochondrial stress. The endoplasmic reticulum (ER) oxidoreductase protein disulfide isomerase (PDI) is succinated in adipocytes that are matured in high glucose, and in this study we investigated whether succination would alter PDI oxidoreductase activity, directly linking mitochondrial stress and ER stress.Protein succination and the ER stress marker C/EBP homologous protein (CHOP) were diminished after pharmaceutical targeting of mitochondrial stress with the chemical uncoupler niclosamide in adipocytes matured in high-glucose concentrations. PDI was succinated by fumarate on both CXXC-containing active sites, contributing to reduced enzymatic activity. Succinated PDI decreased reductase activity in adipocytes matured in high glucose, and in db/db epididymal adipose tissue, in association with increased levels of CHOP. PDI succination was increased in fumarase knockdown adipocytes, leading to reduced PDI oxidoreductase activity, increased CHOP levels, and pro-inflammatory cytokine secretion, confirming the specific role of elevated fumarate levels in contributing to ER stress. In addition, PDI succination and ER stress were decreased, and PDI reductase activity was restored when exposure to chronic high glucose was limited, highlighting the importance of calorie restriction in the improvement of adipocyte metabolic function.These experiments identify PDI succination as a novel biochemical mechanism linking altered mitochondrial metabolism to ER stress in the adipocyte during diabetes.The current study demonstrates that early biochemical changes in mitochondrial metabolism have important implications for the development of adipocyte stress. Antioxid. Redox Signal. 27, 1281-1296.

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3β in both neurons and non-neuronal cells. GSK-3β coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3β, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3β inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.

It is hypothesized that a high dietary n-6:n-3 (eg, 10-20:1) is partly responsible for the rise in obesity and related health ailments. However, no tightly controlled studies using high-fat diets differing in the n-6:n-3 have tested this hypothesis. The aim of the study was to determine the role that the dietary n-6:n-3 plays in non-alcoholic fatty-liver disease (NAFLD) and colitis development. We hypothesized that reducing the dietary n-6:n-3 would hinder the development of NAFLD and colitis. Male C57BL/6 J mice were fed high-fat diets, differing in the n-6:n-3 (1:1, 5:1, 10:1, 20:1), for 20 weeks. Gas chromatography-mass spectrometry was used to analyze the hepatic phospholipid arachidonic acid (AA):eicosapentaenoic acid and AA:docosahexaenoic acid. Hepatic metabolism, inflammatory signaling, macrophage polarization, gene expression of inflammatory mediators, oxidative and endoplasmic reticulum stress, and oxidative capacity were assessed as well as colonic inflammatory signaling, and gene expression of inflammatory mediators and tight-junction proteins. Although reducing the dietary n-6:n-3 lowered the hepatic phospholipid AA:eicosapentaenoic acid and AA:docosahexaenoic acid in a dose-dependent manner and mildly influenced inflammatory signaling, it did not significantly attenuate NAFLD development. Furthermore, the onset of NAFLD was not paired to colitis development or changes in tight-junction protein gene expression. In conclusion, reducing the dietary n-6:n-3 did not attenuate NAFLD progression; nor is it likely that colitis, or gut permeability, plays a role in NAFLD initiation in this model.

This manuscript investigates how incorporation of benzophenone, a well-known triplet sensitizer, within a bis-urea macrocycle, which self-assembles into a columnar host, influences its photophysical properties and affects the reactivity of bound guest molecules. We further report the generation of a remarkably stable organic radical. As expected, UV irradiation of the host suspended in oxygenated solvents efficiently generates singlet oxygen similar to the parent benzophenone. In addition, this host can bind guests such as 2-methyl-2-butene and cumene to form stable solid host-guest complexes. Subsequent UV irradiation of these complexes facilitated the selective oxidation of 2-methyl-2-butene into the allylic alcohol, 3-methyl-2-buten-1-ol, at 90% selectivity as well as the selective reaction of cumene to the tertiary alcohol, α,α'-dimethyl benzyl alcohol, at 63% selectivity. However, these products usually arise through radical pathways and are not observed in the presence of benzophenone in solution. In contrast, typical reactions with benzophenone result in the formation of the reactive singlet oxygen that reacts with alkenes to form endoperoxides, diooxetanes, or hydroperoxides, which are not observed in our system. Our results suggest that the confinement, the formation of a stable radical species, and the singlet oxygen photoproduction are responsible for the selective oxidation processes. A greater understanding of the mechanism of this selective oxidation could lead to development of greener oxidants.

A group of coagulase negative staphylococcal strains isolated from indoor air of occupied school rooms were the subject of this study. Conventional MALDI TOF MS profiling of cellular extracts and physiological tests (including API STAPH) provided incomplete identification of the set of strains. After separation of a 100 kDa band using 1D gel electrophoresis, profiling of peptides (released with tryptic digestion) using MALDI TOF MS allowed improved bacterial speciation in addition to determination of the identity of the protein of origin (aconitate hydratase). This was performed by Mascot search, empirical observation and computer-generated cross-correlation analysis of environmental isolates versus reference strains. The species studied included some with sequenced genomes and others with un-sequenced genomes. Peptide sequences were confirmed to originate from aconitate hydratase using MALDI TOF-TOF MS-MS analysis of a diverse set of m/z values representing variable and conserved sequences. The methodological approach described here might have widespread application in speciation of environmental isolates of diverse origin and in identification of their expressed proteins.

The intake of excess nutrients surpasses the energy requirements of the adipocyte and leads to increased mitochondrial stress. We have previously shown that this is associated with the accumulation of the mitochondrial metabolite fumarate. Fumarate can react with protein thiols to form the chemical modification S-(2-succino) cysteine (2SC), also termed protein succination. Succination is significantly increased in the adipose tissue of type 2 diabetic mouse models, and in adipocytes matured in high glucose, resulting in impaired protein function. We consistently observe that the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is elevated in parallel with increased fumarate and protein succination, independent of other markers of endoplasmic reticulum stress. We propose that fumarate may indirectly promote CHOP stability through the succination of Keap1 thiols, as Keap1 regulates CHOP ubiquitination and turnover. Fumarase knockdown (FHKD) or control 3T3-L1 adipocytes were matured in normal (5 mM) or high (25-30 mM) glucose. Protein succination, CHOP stability and activation of Keap1/Nrf2 in adipocyte lysates and adipose tissue (db/db mice) were analyzed. Keap1 succination sites were identified by LC-MS/MS. Fumarate, glutathione, and reactive oxygen species were measured in control and FHKD adipocytes. We discovered that while Keap1 is directly succinated in the presence of excess fumarate derived from fumarase knockdown cells, it is the oxidative modification of Keap1 that predominates in adipocytes matured in high glucose. We also determined that succination indirectly regulates CHOP stability through the induction of oxidative stress in the FHKD model. N-acetylcysteine reduced fumarate levels, protein succination and CHOP levels in adipocytes matured in high glucose. The results shown here demonstrate that elevated fumarate can induce oxidative stress, and that the oxidation of Keap1 thiols contributes to reduced CHOP turnover in the adipocyte during diabetes. This study emphasizes the combined roles of both metabolite derived stress and oxidative stress in the modulation of adipocyte proteins during diabetes.

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 > or =5-fold during adipogenesis in medium containing 30 mm glucose, producing a > or =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(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 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.

Systemin is a wound-signaling peptide that mediates defenses of tomato plants against herbivorous insects. Perception of systemin by the membrane-bound receptor SR160 results in activation of MAPKs, synthesis of jasmonic acid (JA), and expression of defense genes. To test the function of MAPKs in the response to systemin, we used virus-induced gene silencing (VIGS) in plants that overexpress the systemin precursor prosystemin ( 35S::prosys plants). These transgenic plants accumulate high levels of defense proteins and exhibit increased resistance to herbivorous insects. Cosilencing of the MAPKs MPK1 and MPK2 reduced MPK1/2 kinase activity, JA biosynthesis, and expression of JA-dependent defense genes. Application of methyl-JA restored the full defense response. These data show that MPK1 and MPK2 are essential components of the systemin signaling pathway and most likely function upstream of JA biosynthesis. MPK1 and MPK2 are 95% identical at the amino acid level. Specific VIGS of only MPK1 or MPK2 resulted in the same reduction of defense gene expression as cosilencing of MPK1 and MPK2 , indicating that gene dosage effects may be important for MPK signaling. In addition, VIGS of the closely related MPK3 also reduced systemin-induced defense responses. The function of MPK1/2 and orthologs in pathogen-induced defenses is well established. Here we show that cosilencing of MPK1 and MPK2 compromised prosystemin-mediated resistance to Manduca sexta (Lepidoptera) herbivory, demonstrating that MPK1 and MPK2 are also required for successful defenses against herbivorous insects.

We thank Ms. Jennifer Bethard, Medical University of South Carolina for the mass spectrometric analysis of the 2D gel spots. We would like to thank Professor John Baynes, USC for helpful discussion. We thank Dr. Boris Kantor, USC Viral Vector Core for production of the lentiviral vectors. This work was supported by grants from the National Institutes of Health (R03 HD077187, R01 NS092938, R37 DK19971 and P20 GM109091), the American Diabetes Association (1-11-JF-13), and a University of South Carolina Research Foundation ASPIRE-I award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-Translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltagedependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys77 and Cys48 were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.

Selenocyanate (SeCN−) is a major contaminant in the effluents from some oil refineries, power plants, and in mine drainage water. In this study, we determined the potential of Indian mustard (Brassica juncea) and muskgrass (a macroalga,Chara canescens) for SeCN− phytoremediation in upland and wetland situations, respectively. The tolerance of Indian mustard to toxic levels of SeCN− was similar to or higher than other toxic forms of Se. Indian mustard treated with 20 μm SeCN− removed 30% (w/v) of the Se supplied in 5 d, accumulating 554 and 86 μg of Se g−1 dry weight in roots and shoots, respectively. Under similar conditions, muskgrass removed approximately 9% (w/v) of the Se supplied as SeCN− and accumulated 27 μg of Se g−1 dry weight. A biochemical pathway for SeCN− degradation was proposed for Indian mustard. Indian mustard and muskgrass efficiently degraded SeCN− as none of the Se accumulated by either organism remained in this form. Indian mustard accumulated predominantly organic Se, whereas muskgrass contained Se mainly as selenite and organic Se forms. Indian mustard produced volatile Se from SeCN− in the form of less toxic dimethylselenide. Se volatilization by Indian mustard accounted for only 0.7% (w/v) of the SeCN− removed, likely because the biochemical steps in the production of dimethylselenide from organic Se were rate limiting. Indian mustard is promising for the phytoremediation of SeCN−-contaminated soil and water because of its remarkable abilities to phytoextract SeCN−and degrade all the accumulated SeCN− to other Se forms.

Single nucleotide polymorphisms (SNPs) and mutations were genotyped for both homozygous and heterozygous PCR products of p53, a tumor suppressor gene, and K-ras, an oncogene, using electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and ESI-quadrupole MS analysis. Mass accuracy was adequate for both instruments to detect genetic changes in homozygous PCR products, including the most difficult to distinguish (adenine [A] --> thymine [T] transversion). However, for the detection of A --> T shifts (9.0 Da difference) in heterozygous PCR products, the increased resolution of ESI-Q-TOFMS proved essential. Although, greater mass differences in heterozygotes (e.g. cytosine [C] T or guanine [G] A) can be discriminated using ESI-quadrupole MS analysis.

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and β-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db / db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 μM) inhibited polymerization up to 35% and 59% respectively. Using MS we identified Cys347α, Cys376α, Cys12β and Cys303β as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteine residues increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose compared with normal glucose also had reduced reactivity with the anti-α-tubulin antibody; suggesting that succination may interfere with tubulin–protein interactions. DMF reacted rapidly with 11 of the 20 cysteine residues in the αβ-tubulin dimer, decreased the number of free thiols and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggest that inhibition of tubulin polymerization is an important undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics. Abbreviations: C2SC, cysteine succination by fumarate; CID, collision-induced dissociation; CTT, C-terminal tail of tubulin; DMF, dimethylfumarate; DMEM, Dulbecco’s modified Eagle’s medium; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); DTPA, diethylenetriaminepenta-acetic acid; GI, gastrointestinal; 4-HNE, 4-hydroxynonenal; MAP, microtubule-associated protein; NEM, N-ethylmaleimide; PE, pyridylethylation; PTM, post-translational modification; RIPA, radioimmunoprecipitation assay; 2SC, S-2-(succino)cysteine; TFA, trifluoroacetic acid; TOF, time-of-flight

Aims To examine the effect of manipulating the omega-6:omega-3 (1∶1, 5∶1, 10∶1, and 20∶1) utilizing only α-linolenic and linoleic acid within a clinically-relevant high-fat diet (HFD) composed of up to seven sources of fat and designed to be similar to the standard American diet (MUFA∶PUFA of 2∶1, 12% and 40% of calories from saturated and total fat, respectively) on body composition, macrophage polarization, inflammation, and metabolic dysfunction in mice. Methods Diets were administered for 20 weeks. Body composition and metabolism (HOMA index and lipid profile) were examined monthly. GC-MS was utilized to determine the eicosapentaenoic acid (EPA):arachidonic acid (AA) and the docosahexaenoic acid (DHA):AA in AT phospholipids. Adipose tissue (AT) mRNA expression of chemokines (MCP-1, Fetuin-A, CXCL14), marker genes for M1 and M2 macrophages (CD11c and CD206, respectively) and inflammatory markers (TNF-α, IL-6, IL-1β, TLR-2, TLR-4, IL-10, GPR120) were measured along with activation of NFκB, JNK, and STAT-3. Macrophage infiltration into AT was examined using F4/80 immunohistochemistry. Results Any therapeutic benefit produced by reducing the omega-6:omega-3 was evident only when comparing the 1∶1 to 20∶1 HFD; the 1∶1 HFD resulted in a lower TC:HDL-C and decreased AT CXCL14 gene expression and AT macrophage infiltration, which was linked to a higher EPA:AA and DHA:AA in AT phospholipids. However, despite these effects, and independent of the omega-6:omega-3, all HFDs, in general, led to similar levels of adiposity, insulin resistance, and AT inflammation. Conclusion Reducing the omega-6:omega-3 using α-linolenic acid is not an effective therapy for attenuating obesity and type II diabetes mellitus development.

Despite the global prevalence of marine sedimentary habitats, the potential for chemically mediated ecological interactions within these assemblages remains poorly known. Using GC/MS, we examined methanol extracts of worm, mollusc, and crustacean macroinfauna from 2 local intertidal sandflat communities for volatile organohalogens, a common, often bioactive class of secondary metabolites from marine organisms. Halogenated compounds were newly identified from 11 common polychaete species in the families Capitellidae, Chaetopteridae, Cirratulidae, Glyceridae, Pectinariidae and Spionidae. Tentative structures of these halometabolites are proposed based on GC/MS data. The represented chemical classes included aromatic mono- and dibrominated hydroxyphenylpropanoids, nitrogen-containing bromoalkylpyrroles, as well as brominated and chlorinated hydrocarbons and sulfides. Among the molluscs, only Terebra dislocata (Prosobranchia) contained an organohalogen compound (2,6-dibromophenol), which is likely derived from its diet of hemichordate worms. Volatile haloorganics were not found in amphipod and nemertean taxa. Possession of these compounds was not a function of any particular phylum, size categorization, or trophic mode, although hemichordates and all Capitellid and Spionid polychaetes collected at these sites invariably contained halocompounds. Most (40 of ca 54 taxa) of the numerically dominant macroinfaunal taxa at these 2 sites were examined in this survey; of these, 43% contained halometabolites, including the broadly distributed species Mediomastus ambiseta, Heteromastus filiformis, Streblospio benedicti, Tharyx marioni, and Saccoglossus kowalevskii, Thus, the potential for widespread occurrence of halogenated compounds among infauna is great. The presence of organohalogens in many common and abundant polychaete species suggests a general hiological significance for these metabolites, a result of potential interest to marine ecologists, toxicologists and environmental chemists.

Organochlorine pesticides and polychlorinated biphenyls were determined in high volume air samples collected in Green Bay from the University of Wisconsin (1989), and from shipboard over the four lower Great Lakes (1990). Average concentrations of Σchlordane, DDT, DDE, toxaphene and ΣPCBs in Green Bay were 35, 8.7, 15, 59 and 330 pg m−3, respectively. Air concentrations of the pesticides were positively correlated to each other (R2=0.896–0.997), and analysis of back trajectory data from Green Bay showed that higher atmospheric concentrations of organochlorine insecticides were associated with air masses originating in southern USA. Average concentrations from the Great Lakes were: Σchlordane, 187 pg m−3; DDT, 38 pg m−3; pg m−3; DDE, 59 pg m−3; toxaphene, 33 pg m−3; and ΣPCBs, 385 pg m−3. Higher air concentrations of PCBs were found in areas of industrial and urban development. There was no strong positive correlation of concentration values between any pesticides, with the exception of the chlordane isomers, or between any pesticide and ΣPCB from the Great Lakes cruise. Results from 5-day air mass back trajectory data suggest that, at the time of the cruise, atmospheric sources of organochlorines were not long-range transport from southern USA, but local or regional volatilization.

A rapid extraction method has been developed for the quantitation of (Z,E)-propanethial S-oxide, the lachrymatory factor (LF), and other flavor chemicals in onion (Allium cepa) using gas chromatogr...

Chlorinated bornanes (CHBs, e.g. toxaphene) and other organochlorine (OC) pesticides were measured in air and surface water at Resolute Bay, N.W.T., during August 1992 to determine summertime levels in the Arctic and the net direction of air-sea gas exchange. Mean concentrations in air (pg/m3) were: hexachlorocyclohexanes (α-HCH + γ-HCH) = 124, CHBs = 6.9, endosulfan I = 4.0, chlordanes + nonachlors = 2.8 andp,p′-DDT < 0.3. OCs in seawater (pg/l) were: HCHs = 5100, CHBs = 48, chlordanes + nonachlors = 14 andp,p′-DDE = 1.0. One sample from Amituk Lake on Cornwallis Island yielded levels of CHBs 3 times higher and HCHs 3 times lower than respective levels in Resolute Bay. Chlordanes in the two waters were approximately the same. Chromatographic profiles of CHBs in air and water were skewed toward the lower molecular weight components of toxaphene. The octa- and nonachlorobornanes (designated as T2 and T12), which strongly accumulate in fish, marine mammals and human milk, were also found in air and water. Atmospheric concentrations of γ-HCH andcis-chlordane in surface seawater were close to Henry's Law equilibrium. CHBs were undersaturated, indicating a potential for air-to-sea transfer. Alpha-HCH andtrans-chlordane were oversaturated, implying volatilization. Differences among these OCs might be explained in part by temporal variations in their atmospheric concentrations.

Organochlorine contaminants were measured in water, fish, and Baikal seal from Lake Baikal, Siberia. Organochlorine levels in water were comparable to those reported from the upper Great Lakes, with the exception of chlordanes, which were a factor of 2 lower. Dissolved toxaphene and ΣDDT (sum of 4,4'-DDE, 4,4'-DDD, 2,4'-DDT, and 4,4'-DDT) averaged 64±37 pg/L and 87±37 pg/L, respectively. Total polychlorinated biphenyl (ΣPCB) concentrations in biota ranged from 1.2 to 26 mg/kg of lipid in the omul (an endemic salmonid) and the Baikal seal, respectively. Tetra- and pentachlorobiphenyl congeners accounted for a greater proportion of ΣPCB in omul and hexa-, hepta-, and octachlorobiphenyl congeners in pelagic sculpins and seal

Water and biological samples were obtained from Lake Baikal in June 1991 and analyzed for toxaphene and other organochlorines. Dissolved toxaphene concentration ranged from 34 pg/L to 143 pg/L and were similar in magnitude to ∑DDT (47 pg/L; sum of 2,4′-DDT, 4,4′-DDT, and 4,4′-DDE) and ∑chlordanes (28 pg/L; sum of cis- and trans-chlordane and cis- and trans-nonachlor). Toxaphene in biota was 1.9 mg/kg-lipid in the pelagic sculpin Comephorus dybowskii , 1.1 mg/kg-lipid in the omul ( Coregonus autumnalis migratorious ) and 2.3 mg/kg-lipid in Baikal seal (Phoca siberica) blubber. Toxaphene chromatographic patterns, determined by gas chromatography electron capture negative ion-mass spectrometry, were transformed in the seal relative to either of the fish, which is likely due to the seal's greater ability to metabolize some toxaphene congeners. The major peak in the Baikal seal toxaphene chromatogram was an 8-chlorinated bornane unlike that found by other investigators for narwhal or beluga whale blubber, but similar to ringed seals from the Canadian Arctic.

Complex mixtures of chlorinated bornanes and bornenes (CHBs), typically referred to as toxaphene or polychlorinated camphenes, were measured in the air and surface water of Lake Baikal, Russia during June, 1991. The mean CHB concentration was 16 ± 4 pg m −3 in air and 64 ± 37 pg/L in water. Levels in air were similar to previous measurements in Arctic regions, and the chromatographic pattern of CHB congeners was extremely “weathered” compared to a toxaphene standard suggesting long-range atmospheric transport rather that a nearby source. The congener pattern in the water samples was similar to that in the air indicating the importance of atmospheric deposition processes to Lake Baikal. Estimates of gas flux of CHBs across the air-water interface predict that this process could cause a substantial contribution to the lakes' overall pollutant budget.

Chlorinated pesticides in air were measured from the Greenpeace ship Gondwana between New Zealand and Ross Island, January–March, 1990. Geometric mean concentrations (pg m −3 ) were: α and γ -hexachlorocyclohexanes=4.0 and 3.8, hexachlorobenzene=60, heptachlor epoxide=0.52, chlordanes+nonachlors=1.8, p,p′-DDE+p,p′-DDT=0.81. A trend of decreasing pesticide concentration at higher latitudes was noted. The low levels of DDTs found in this study and also by Larsson et al . (1992) during 1988–1990 on Ross Island indicates that atmospheric DDT over the Southern Ocean has dropped markedly in the last decade. Recent concentrations of DDTs in Antarctic air are similar to those in the Arctic.

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 increasedor =5-fold during adipogenesis in medium containing 30 mm glucose, producing aor =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(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 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.

S-(2-Succinyl)cysteine (2SC) has been identified as a chemical modification in plasma proteins, in the non-mercaptalbumin fraction of human plasma albumin, in human skin collagen, and in rat skeletal muscle proteins and urine. 2SC increases in human skin collagen with age and is increased in muscle protein of diabetic vs. control rats. The concentration of 2SC in skin collagen and muscle protein correlated strongly with that of the advanced glycation/lipoxidation end-product (AGE/ALE), N(epsilon)-(carboxymethyl)lysine (CML). 2SC is formed by a Michael addition reaction of cysteine sulfhydryl groups with fumarate at physiological pH. Fumarate, but not succinate, inactivates the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase in vitro, in concert with formation of 2SC. 2SC is the first example of spontaneous chemical modification of protein by a metabolic intermediate in the Krebs cycle. These observations identify fumarate as an endogenous electrophile and suggest a role for fumarate in regulation of metabolism.

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 alpha-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 b5 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 b5 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.

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., Author Summary Poplar is considered as the model tree species for the production of lignocellulosic biomass destined for biofuel production. The plant growth promoting endophytic bacterium Enterobacter sp. 638 can improve the growth of poplar on marginal soils by as much as 40%. This prompted us to sequence the genome of this strain and, via comparative genomics, identify functions essential for the successful colonization and endophytic association with its poplar host. Analysis of the genome sequence, combined with metabolite analysis and quantitative PCR, pointed to a remarkable interaction between Enterobacter sp. 638 and its poplar host with the endophyte responsible for the production of a phytohormone, and a precursor for another that poplar is unable to synthesize, and where the production of the plant growth promoting compounds depended on the presence of plant synthesized compounds, such as sucrose, in the growth medium. Our results provide the basis to better understanding the synergistic interactions between poplar and Enterobacter sp. 638. This information can be further exploited to improve establishment and sustainable production of poplar on marginal, non-agricultural soils using endophytic bacteria such as Enterobacter sp. 638 as growth promoting agents.

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.

A senior-level undergraduate laboratory experiment that demonstrates the use of solid-phase microextraction (SPME) and capillary gas chromatography-mass spectrometry (GC-MS) was developed for the identification of volatile compounds in consumer products. SPME minimizes sample preparation and concentrates volatile analytes in a solvent-free manner.

The chemistry of Maillard or browning reactions of glycated proteins is being studied in model systems in vitro in order to characterize potential reaction pathways and products in biological systems. In previous work with the Amadori rearrangement product N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in proteins, we showed that fFL was oxidatively cleaved between C-2 and C-3 of the carbohydrate chain to yield N epsilon-carboxymethyllysine (CML) and D-erythronic acid. We then detected CML in proteins glycated in vitro, as well as in human lens proteins and collagen in vivo (Ahmed, M. U., Thorpe, S. R., and Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894). This work provided an explanation for the origin of CML in human urine and evidence for non-browning pathways of the Maillard reaction in vivo. In this report we describe the identification of a second set of products resulting from oxidative cleavage of fFL between C-3 and C-4 of the sugar chain, i.e. 3-(N epsilon-lysino)-lactic acid (LL) and D-glyceric acid. The formation of LL from fFL was increased at slightly acid pH, representing about 30% of the yield of CML at pH 6.4, compared with 4% at pH 7.4 in phosphate buffer. By gas chromatography-mass spectroscopy, LL was detected in proteins glycated in vitro and then identified as a natural product in human lens proteins and urine. Our results indicate that oxidative degradation of Amadori adducts to proteins occurs in vivo, leading to formation and excretion of CML and LL. These non-browning pathways for reaction of Amadori compounds may be physiologically relevant mechanisms for averting potentially damaging consequences of the Maillard reaction.

Structures are proposed for three new flavonoids. In common with other major flavonoids of D. aurantiacus, all three have a C10-monoterpene side chain. Two of the flavonoids are monomethoxylated derivatives of the two most abundant resin flavonoids while the third is distinct in having one less phenolic group.

Capillary gas chromatography using fused-silica columns followed by electron impact or chemical ionization mass spectrometry was used to profile and identify neutral and amino sugars present in several legionellae and other bacteria. A modified alditol acetate derivatization method was employed to produce volatile carbohydrate derivatives. Muramic acid, a component of bacterial peptidoglycan, was detected in all legionellae examined. Heptose, a component of bacterial lipopolysaccharide, was identified in Escherichia coli organisms and in several purified Gram-negative bacterial lipopolysaccharides but not in the legionellae examined. Two amino dideoxyhexoses were found to be present in several of the Legionellae examined. The potential of gas chromatography-mass spectrometry for the direct chemical characterization of microorganisms is discussed.

Fused-silica capillary gas chromatography—mass spectrometry was employed to investigate the production of several bacterial pyrolysis products, including acetamide, propionamide, and furfuryl alcohol. A variety of model compounds, selected for their similarity with bacterial structures, were pyrolyzed and amounts of these pyrolysis products were quantitatively determined. Model compounds included several analogues of muramyl dipeptide differing in chain length at the lactyl—peptide bridge as well as several neutral and amino sugars, amino acids, and nucleic acids. Bacterial cell walls and whole microbial cells were also analyzed. Measurement of the amounts of a few chemically identified pyrolysis products provides some chemotaxonomic differentiation of microorganisms, as demonstrated by the pyrolysis of a group of Gram-positive and Gram-negative bacteria (including samples of Legionella pneumophila ), and fungi.

The abundant species of infaunal soft-bodied organisms from two geographically separated sites with very different predatory regimes were examined for the occurrence of volatile brominated compounds. Of the 10 species, three contained substantial amounts of brominated compounds while two others contained trace amounts. A greater proportion of species from the Western Atlantic site with large, sediment-disturbing predators and much lower infaunal densities contained these compounds. The occurrence of the compounds did not follow phylogenetic lineages or food sources. The toxicity of such compounds and potential for predator deterrence may be major determinants of their occurrence.

Two gas chromatographic-mass spectrometric methods, one based on chemical derivatisation and the other on analytical pyrolysis, were employed for the characterisation of a diverse group of bacteria. The muramic acid content of some Gram-positive and Gram-negative bacteria (including legionellae) was determined by aldononitrile acetate derivativisation and capillary GC-MS analysis. Muramic acid levels were generally higher in Gram-positive than in Gram-negative bacteria. When applied to the same set of bacteria, pyrolysis GC-MS and non-linear mapping were also able to distinguish Gram-negative from Gram-positive bacteria. The potential of GC-MS techniques for the direct chemical characterisation of bacteria is discussed.