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Transthyretin amyloidosis is an increasingly recognized cause of restrictive cardiomyopathy related to amyloid fibril deposition in cardiac tissues. As treatment therapies have emerged for transthyretin amyloidosis (ATTR), so has interest in using biomarkers to identify disease prior to advanced presentation. Lower levels of transthyretin and retinol binding protein-4 have been demonstrated in patients with pathogenic mutations of transthyretin either with or without clinical disease. Levels associate with the severity of mutations as well as response to treatment with transthyretin stabilizers or small interfering RNA molecules which silence transthyretin production. Transthyretin stability is the rate limiting step of amyloid fibril formation and directly measuring transthyretin kinetic stability has the potential to identify patients as risk as well as therapeutic response to treatment regardless of pathogenic or wild-type genetics. In addition, non-antibody protein-based peptide probes have been developed that directedly measure misfolded transthyretin oligomers due to transthyretin breakdown. Although promising, both TTR kinetic and protein peptide probes remain in early stages of clinical investigation. Transthyretin, retinol binding protein-4, transthyretin kinetic stability, and protein-based peptide probes have potential as biomarkers to facilitate an earlier ATTR diagnosis for patients with pathogenic transthyretin mutations.

Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its composition may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently investigated in the context of oral health and disease. Furthermore, its potential as an alternative matrix for analytical purposes has also been recognized in other disciplines like archeology, food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, on the other hand, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for determination of lipid compositions of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for analytical purposes has been performed in this work. An untargeted lipidomics workflow utilizing a ultra-high-performance liquid chromatography (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theoretical fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amounts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepared by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-analysis. It was discovered that most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was observed in the cultivated biofilms. On the other hand, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate were shown to be specifically increased in plaque samples. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-02364-2) contains supplementary material, which is available to authorized users.

Xylem sap of angiosperm species has been found to include low concentrations of polar lipids and nanoparticles, including surfactant-coated nanobubbles. Although the nanoparticles have been suggested to consist of polar lipids, no attempt has been made to determine if nanoparticle and lipid concentrations are related. Here, we examined concentrations of nanoparticles and lipids in xylem sap and contamination control samples of six temperate angiosperm species with a NanoSight device and based on mass spectrometry. We found (1) that the concentration of nanoparticles and lipids were both diluted when an increasing amount of sap was extracted, (2) that their concentrations were significantly correlated in three species, (3) that their concentrations were affected by vessel anatomy, and (4) that concentrations of nanoparticles and lipids were very low in contamination-control samples. Moreover, there was little seasonal difference, no freezing-thawing effect on nanoparticles, and little seasonal variation in lipid composition. These findings indicate that lipids and nanoparticles are related to each other, and largely do not pass interconduit pit membranes. Further research is needed to examine the formation and stability of nanoparticles in xylem sap in relation to lipid composition, and the complicated interactions among the gas, liquid, and solid phases in xylem conduits.

BACKGROUND Ammonia concentrations typically increase during mammalian cell cultures, mainly due to glutamine and other amino acid consumption. An early ammonia stress indicator is a metabolic shift with respect to alanine. To determine the underlying mechanisms of this metabolic shift, a Chinese hamster ovary (CHO) cell line with two distinct ages (standard and young) was cultured in parallel fed-batch bioreactors with 0 mM or 10 mM ammonia added at 12 h. Reduced viable cell densities were observed for the stressed cells, while viability was not significantly affected. The stressed cultures had higher alanine, lactate, and glutamate accumulation. Interestingly, the ammonia concentrations were similar by Day 8.5 for all cultures. We hypothesized the ammonia was converted to alanine as a coping mechanism. Interestingly, no significant differences were observed for metabolite profiles due to cell age. Glycosylation analysis showed the ammonia stress reduced galactosylation, sialylation, and fucosylation. Transcriptome analysis of the standard-aged cultures indicated the ammonia stress had a limited impact on the transcriptome, where few of the significant changes were directly related metabolite or glycosylation reactions. These results indicate that mechanisms used to alleviate ammonia stress are most likely controlled post-transcriptionally, and this is where future research should focus.

Eosinophils play an important role in mediating itch and inflammation in dermatitis. The role of the eosinophil granule protein eosinophil peroxidase (EPX) in mediating inflammation and itch was tested in a dermatitis mouse model. Mice were sensitized to trimellitic anhydride (TMA) and subsequently challenged chronically on the ear to establish dermatitis. Loss of EPX (in EPX (-/-) mice) or blocking EPX with the drug resorcinol significantly reduced dermatitis in mice exposed to TMA. Resorcinol also reduced levels of thymic stromal lymphopoietin protein (TSLP) in skin. Further studies showed that EPX increased different cytokines in keratinocytes in cell culture via two distinct mechanisms. EPX induced TSLP expression requires lysophosphatidic acid signaling while EPX induced expression of TNF-α, CSF2, CSF3, and IL1α required IL-1 signaling. We also showed that blocking IL-1 reduced inflammation in skin following TMA exposure in mice. Thus, EPX is an important mediator of inflammation and itch, that are mediated via at least two pathways. This suggests that both EPX and its’ signaling pathways may provide novel therapeutic strategies in dermatitis.

Genome-wide association studies identified the BIN1 locus as a leading modulator of genetic risk in Alzheimer’s disease (AD). One limitation in understanding BIN1’s contribution to AD is its unknown function in the brain. AD-associated BIN1 variants are generally noncoding and likely change expression. Here, we determined the effects of increasing expression of the major neuronal isoform of human BIN1 in cultured hippocampal neurons. Higher BIN1 induced network hyperexcitability on multielectrode arrays, increased frequency of synaptic transmission, and elevated calcium transients, indicating that increasing BIN1 drives greater neuronal activity. In exploring the mechanism of these effects on neuronal physiology, we found that BIN1 interacted with L-type voltage-gated calcium channels (LVGCCs) and that BIN1–LVGCCs interactions were modulated by Tau in vitro and in vivo. Finally, Tau reduction prevented BIN1-induced network hyperexcitability. These data shed light on BIN1’s neuronal function and suggest that it may contribute to Tau-dependent hyperexcitability in AD.

In response to elevated temperatures, plants alter the activities of enzymes that affect lipid composition. While it has long been known that plant leaf membrane lipids become less unsaturated in response to heat, other changes, including polygalactosylation of galactolipids, head group acylation of galactolipids, increases in phosphatidic acid and triacylglycerols, and formation of sterol glucosides and acyl sterol glucosides, have been observed more recently. In this work, by measuring lipid levels with mass spectrometry, we confirm the previously observed changes in Arabidopsis thaliana leaf lipids under three heat stress regimens. Additionally, in response to heat, increased oxidation of the fatty acyl chains of leaf galactolipids, sulfoquinovosyldiacylglycerols, and phosphatidylglycerols, and incorporation of oxidized acyl chains into acylated monogalactosyldiacylglycerols are shown. We also observed increased levels of digalactosylmonoacylglycerols and monogalactosylmonoacylglycerols. The hypothesis that a defect in sterol glycosylation would adversely affect regrowth of plants after a severe heat stress regimen was tested, but differences between wild-type and sterol glycosylation-defective plants were not detected.

Infrared (IR) optical materials have enabled a broad range of optical sensing and measurement applications in the mid-wave and long-wave IR. Many IR transmitting glasses are based on covalently-bonded selenides and tellurides, such as As2Se3 and GeTe2, which typically have relatively low glass transition temperatures (Tg) on the order of 200 to 350 °C. Many applications have working temperatures above the Tg of these materials, which compels the development of new IR materials. This work studies the underlying short-range order (SRO) structure and glass formability of a new family of ionically-bonded sulfide glasses, xBaS + yLa2S3 + (1 − x − y)GeS2, to develop high Tg optical materials with a broad IR transmission range. These sulfide glasses were produced by melting sulfide materials inside evacuated and sealed carbon-coated silica ampoules at 1150 °C for 12 h and quenching to room temperature to form glass. Glass samples were then characterized by IR and Raman spectroscopies and differential thermal analysis (DTA). It was found that by increasing the modifier concentration, the predominantly Ge4 SRO units, the superscript defines the number of bridging sulfur (BS) ions in the tetrahedral network found in GeS2 glasses, are ultimately converted to Ge0 units at >40 mol% network modifier content through the generation of non-bridging sulfur (NBS) ions. These molecular ionic units still form a glassy network, with some of the highest reported Tg values to date for a pure sulfide glass. This suggests that this composition has strong ionic bonds between negatively-charged tetrahedral SRO units and the positively-charged modifier cations. While the glass network is depolymerized in the high modifier content glasses though the formation of a high concentration of molecular ionic Ge0 SRO groups, they are, nevertheless, homogeneous glassy materials that exhibit the largest Tg and ΔT (difference between crystallization temperature, Tc, and Tg) values of glasses in this system, making them the optimal glasses for high Tg IR optical components, including, potentially, refractory IR optical fibers.

Aim: Phospholipid fatty acid methyl ester (FAME) analysis offers a simple option additionally to 16S rRNA sequencing to characterize microbial communities and to monitor changes. A method was established for the characterization of dental plaque via FAME profiles. Methodology: Fatty acids were determined as FAMEs (direct, acidic transesterification) and analyzed by GC–MS using an optimized temperature gradient. The transesterification reaction was optimized using a fractional factorial central composite face-centered design. Results: Optimal conditions for the transesterification in methanol/toluene: hydrochloric acid concentration 2% (w/v), reaction time 40 min, temperature 110 °C. Method validation showed satisfactory accuracy, precision and linearity. Conclusion: The method provides a useful tool to characterize plaque via FAME profiles and was successfully applied to samples from ten subjects demonstrating its applicability.

Background Lipidomics plays an important role in understanding plant adaptation to different stresses and improving our knowledge of the genes underlying lipid metabolism. Lipidomics involves lipid extraction, sample preparation, mass spectrometry analysis, and data interpretation. One of the practical challenges for large-scale lipidomics studies on plant leaves is the requirement of an efficient and rapid extraction method. Results A single-extraction method with a polar solvent mixture gives results comparable to a widely used, multi-extraction method when tested on both Arabidopsis thaliana and Sorghum bicolor leaf tissue. This single-extraction method uses a mixture of 30 parts chloroform, 25 parts isopropanol, 41.5 parts methanol, and 3.5 parts water (v/v/v/v) and a 24-h extraction time. Neither inclusion of ammonium acetate nor inclusion of acetic acid increased extraction efficiency. Conclusions The extract produced by this method can be used for analysis by mass spectrometry without a solvent evaporation step. The amount of lipid extracted, including phosphatidic acid, is comparable to widely used, more labor-intensive methods. The single-extraction protocol is less laborious, reducing the potential for human error. Electronic supplementary material The online version of this article (10.1186/s13007-018-0282-y) contains supplementary material, which is available to authorized users.

While most missense suppressors have very narrow specificities and only suppress the allele against which they were isolated, the sumA missense suppressor from Salmonella enterica serovar Typhimurium is a promiscuous or broad-acting missense suppressor that suppresses numerous missense mutants. The sumA missense suppressor was identified as a glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid. In addition to rescuing missense mutants caused by glycine to aspartic acid changes as expected, sumA could also rescue a number of other missense mutants as well by changing a neighboring (contacting) aspartic acid to glycine, which compensated for the other amino acid change. Thus the ability of sumA to rescue numerous missense mutants was due in part to the large number of glycine codons in genes that can be mutated to an aspartic acid codon and in part to the general tolerability and/or preference for glycine amino acids in proteins. Because the glyV tRNA Gly3(GAU/C) missense suppressor has also been extensively characterized in Escherichia coli as the mutA mutator, we demonstrated that all gain-of-function mutants isolated in a glyV tRNA Gly3(GAU/C) missense suppressor are transferable to a wild-type background and thus the increased mutation rates, which occur in glyV tRNA Gly3(GAU/C) missense suppressors, are not due to the suppression of these mutants.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

The microtubule-associated protein Tau is strongly implicated in Alzheimer's disease (AD) and aggregates into neurofibrillary tangles in AD. Genetic reduction of Tau is protective in several animal models of AD and cell culture models of amyloid-{beta} (A{beta}) toxicity, making it an exciting therapeutic target for treating AD. A variety of evidence indicates that Tau's interactions with Fyn kinase and other SH3 domain-containing proteins, which bind to PxxP motifs in Tau's proline-rich domain, may contribute to AD deficits and A{beta} toxicity. Thus, we sought to determine if inhibiting Tau-SH3 interactions ameliorates A{beta} toxicity. We developed a peptide inhibitor of Tau-SH3 interactions and a proximity ligation assay (PLA)-based target engagement assay. Then, we used membrane trafficking and neurite degeneration assays to determine if inhibiting Tau-SH3 interactions ameliorated A{beta} oligomer (A{beta}o)-induced toxicity in primary hippocampal neurons from rats. We verified that Tau reduction ameliorated A{beta}o toxicity in neurons. Using PLA, we identified a peptide inhibitor that reduced Tau-SH3 interactions in HEK-293 cells and primary neurons. This peptide reduced Tau phosphorylation by Fyn without affecting Fyn's kinase activity state. In primary neurons, endogenous Tau-Fyn interaction was present primarily in neurites and was reduced by the peptide inhibitor, from which we inferred target engagement. Reducing Tau-SH3 interactions in neurons ameliorated A{beta}o toxicity by multiple outcome measures, namely A{beta}o-induced membrane trafficking abnormalities and neurite degeneration. Our results indicate that Tau-SH3 interactions are critical for A{beta}o toxicity and that inhibiting them is a promising therapeutic target for AD.

The epidermis is the first line of defense against ultraviolet (UV) light from the sun. Keratinocytes and melanocytes respond to UV exposure by eliciting a tanning response dependent in part on paracrine signaling, but how keratinocyte:melanocyte communication is regulated during this response remains understudied. Here, we uncover a surprising new function for the keratinocyte-specific cell-cell adhesion molecule desmoglein 1 (Dsg1) in regulating keratinocyte:melanocyte paracrine signaling to promote the tanning response in the absence of UV exposure. Melanocytes within Dsg1-silenced human skin equivalents exhibited increased pigmentation and altered dendrite morphology, phenotypes which were confirmed in 2D culture using conditioned media from Dsg1-silenced keratinocytes. Dsg1-silenced keratinocytes increased melanocyte-stimulating hormone precursor (Pomc) and cytokine mRNA. Melanocytes cultured in media conditioned by Dsg1-silenced keratinocytes increased Mitf and Tyrp1 mRNA, TYRP1 protein, and melanin production and secretion. Melanocytes in Dsg1-silenced skin equivalents mislocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Together with our previous report that UV reduces Dsg1 expression, these data support a role for Dsg1 in controlling keratinocyte:melanocyte paracrine communication and raise the possibility that a Dsg1-deficient niche contributes to pagetoid behavior, such as occurs in early melanoma development.

Lipids have been observed attached to lumen-facing surfaces of mature xylem conduits of several plant species, but there has been little research on their functions or effects on water transport, and only one lipidomic study of the xylem apoplast. Therefore, we conducted lipidomic analyses of xylem sap from woody stems of seven plants representing six major angiosperm clades, including basal magnoliids, monocots, and eudicots, to characterize and quantify phospholipids, galactolipids, and sulfolipids in sap using mass spectrometry. Locations of lipids in vessels of Laurus nobilis were imaged using TEM and confocal microscopy. Xylem sap contained the galactolipids di- and mono-galactosyldiacylglycerol (DGDG and MGDG), as well as all common plant phospholipids, but only traces of sulfolipids, with total lipid concentrations in extracted sap ranging from 0.18 to 0.63 nmol / mL across all seven species. Contamination of extracted sap from lipids in cut living cells was found to be negligible. Lipid composition of sap was compared to wood in two species and was largely similar, suggesting that sap lipids, including galactolipids, originate from cell content of living vessels. Seasonal changes in lipid composition of sap were observed for one species. Lipid layers coated all lumen-facing vessel surfaces of Laurus nobilis, and lipids were highly concentrated in inter-vessel pits. The findings suggest that apoplastic, amphiphilic xylem lipids are a universal feature of angiosperms. The findings require a reinterpretation of the cohesion-tension theory of water transport to account for the effects of apoplastic lipids on dynamic surface tension and hydraulic conductance in xylem.

Until today, the oral delivery of peptide drugs is hampered due to their instability in the gastrointestinal tract and low mucosal penetration. To overcome these hurdles, PLA (polylactide acid)-nanoparticles were coated with a cyclic, polyarginine-rich, cell penetrating peptide (cyclic R9-CPP). These surface-modified nanoparticles showed a size and polydispersity index comparable to standard PLA-nanoparticles. The zeta potential showed a significant increase indicating successful CPP-coupling to the surface of the nanoparticles. Cryo-EM micrographs confirmed the appropriate size and morphology of the modified nanoparticles. A high encapsulation efficiency of liraglutide could be achieved. In vitro tests using Caco-2 cells showed high viability indicating the tolerability of this novel formulation. A strongly enhanced mucosal binding and penetration was demonstrated by a Caco-2 binding and uptake assay. In Wistar rats, the novel nanoparticles showed a substantial, 4.5-fold increase in the oral bioavailability of liraglutide revealing great potential for the oral delivery of peptide drugs.

Understanding how wheat (Triticum aestivum L.) plants under high temperature (HT) regulate lipid composition is critical to developing climate-resilient varieties. We measured 165 glycerolipids and sterol derivatives under optimum and high day and night temperatures in wheat leaves using electrospray ionization-tandem mass spectrometry. Levels of polar lipid fatty acyl chain unsaturation were lower in both heat-tolerant genotype Ventnor and susceptible genotype Karl 92 under HT, compared with optimum temperature. The lower unsaturation was predominantly because of lower levels of 18:3 acyl chains and higher levels of 18:1 and 16:0 acyl chains. Levels of 18:3-containing triacylglycerols increased threefold/more under HT, consistent with their possible role in sequestering fatty acids during membrane lipid remodelling. Phospholipids containing odd-numbered or oxidized acyl chains accumulated in leaves under HT. Sterol glycosides (SG) and 16:0-acylated sterol glycosides (ASG) were higher under HT than optimum temperatures. Ventnor had lower amounts of phospholipids with oxidized acyl chains under HT and higher amounts of SG and 16:0-ASG than Karl 92. Taken together, the data demonstrate that wheat leaf lipid composition is altered by HT, in which some lipids are particularly responsive to HT, and that two wheat genotypes, chosen for their differing physiological responses to HT, differ in lipid profile under HT.

Coronary artery disease is a growing clinical problem in HIV-infected subjects. The increased risk of coronary events in this population has been linked to low levels of HDL, but the effects of HIV infection and anti-retroviral treatment (ART) on HDL structure and function remain unknown. Here, we aimed to determine the composition and function of HDL particles isolated from ART-naive and ART-positive HIV-infected patients.Proteomic profiling revealed decreased levels of paraoxonase (PON) 1 and PON 3 in HDL from HIV patients relative to HDL from uninfected controls (p0.0001), and PON activity of HDL from control group (0.13 ± 0.01 U/μl) was significantly higher than PON activity of HDL from HIV-infected untreated subjects (0.12 ± 0.01 U/μl, p = 0.0035), subjects treated with non-nucleoside reverse transcriptase inhibitor (NNRTI)-based therapy (0.11 ± 0.01 U/μl, p0.0001), subjects treated with protease inhibitor (PI)-based therapy with detectable viral load (0.11 ± 0.01 U/μl, p0.0001), and PI-treated patients with undetectable viral load (0.12 ± 0.01 U/μl, p = 0.0164). Lipidomic profiling uncovered a negative correlation between CD4 T cell counts and particle sphingomyelin, lyso-phosphatidylcholine and ether-linked phosphatidylserine content in the ART-naive (R(2) = 0.2611, p0.05; R(2) = 0.2722, p0.05; and R(2) = 0.3977, p0.05, respectively) but not treated HIV-infected subjects. Functional analysis demonstrated a negative correlation between cholesterol efflux capacity of HDL and viral load in the ART-naive HIV-infected group (R(2) = 0.26, p = 0.026).Taken together, these results indicate that HIV infection associates with a number of both protein and lipid compositional changes in HDL particles. Moreover, HIV infection affects cholesterol efflux function of HDL, thus contributing to an increased risk of atherosclerosis in this patient population.

A direct-infusion electrospray ionization triple-quadrupole mass spectrometry method with multiple reaction monitoring (MRM) was employed to measure 264 lipid analytes extracted from leaves of Arabidopsis thaliana subjected to mechanical wounding. The method provided precise measurements with an average coefficient of variation of 6.1%. Lipid classes analyzed comprised galactolipids and phospholipids (including monoacyl molecular species, molecular species with oxidized acyl chains, phosphatidic acids (PAs)), tri- and tetra-galactosyldiacylglycerols (TrGDGs and TeGDGs), head-group-acylated galactolipids, and head-group-acylated phosphatidylglycerol (acPG), sulfoquinovosyldiacylglycerols (SQDGs), sphingolipids, di- and tri-acylglycerols (DAGs and TAGs), and sterol derivatives. Of the 264 lipid analytes, 254 changed significantly in response to wounding. In general, levels of structural lipids decreased, whereas monoacyl molecular species, galactolipids and phosphatidylglycerols (PGs) with oxidized fatty acyl chains, PAs, TrGDGs, TeGDGs, TAGs, head-group-acylated galactolipids, acPG, and some sterol derivatives increased, many transiently. The observed changes are consistent with activation of lipid oxidizing, hydrolyzing, glycosylating, and acylating activities in the wounding response. Correlation analysis of the levels of lipid analytes across individual control and treated plants was used to construct a lipid dendrogram and to define clusters and sub-clusters of lipid analytes, each composed of a group of lipids which occurred in a coordinated manner. Current knowledge of metabolism supports the notion that observed sub-clusters comprise lipids generated by a common enzyme and/or metabolically downstream of a common enzyme. This work demonstrates that co-occurrence analysis, based on correlation of lipid levels among plants, is a powerful approach to defining lipids generated in vivo by a common enzymatic pathway.

The availability of nitrogen (N) to plants has a profound impact on carbohydrate and protein metabolism, but little is known about its effect on membrane lipid species. This study examines the changes in galactolipid and phospholipid species in soybean as affected by the availability of N, either supplied to soil or obtained through Bradyrhizobium japonicum nodulation. When N was limited in soil, the content of galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacyglycerol (DGDG), decreased drastically in leaves, while a smaller decrease of DGDG was observed in roots. In both leaves and roots, the overall content of different phospholipid classes was largely unchanged by N limitation, although some individual phospholipid molecular species did display significant changes. Nodulation with Bradyrhizobium of soybean grown in N-deficient soil resulted in a large increase in levels of plastidic lipid classes, MGDG, DGDG, and phosphatidylglycerol, along with smaller increases in non-plastidic phospholipids in leaves. Nodulation also led to higher levels of phospholipids in roots without changes in root levels of MGDG and DGDG. Overall, N availability alters lipid content more in leaves than roots and more in galactolipids than phospholipids. Increased N availability leads to increased galactolipid accumulation in leaves, regardless of whether N is supplied from the soil or symbiotic fixation.

Dietary omega-3 fatty acids (n-3 PUFA), notably eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), impart health benefits in humans and animals. In horses, dietary n-3 PUFAs elevate EPA and DHA and may promote anti-inflammatory effects. No reports document effects of dietary n-3 PUFA on fatty acyl components of circulating and cellular phospholipids in horses nor whether responses to dietary n-3 PUFA are similar among horse breeds. Ten Quarter Horse and 10 American Miniature Horse mares were assigned to n-3 PUFA (64.4 mg· kg body weight [BW] −1 ·d −1 ) or control diet for 56 days. Blood was sampled at 0, 28, and 56 days. Apparent phospholipid molecular species from several classes (phosphatidylcholine [PC]; "ether-linked" phosphatidylcholine [i.e., alk(en)yl, acyl glycerophosphocholine] [ePC]; phosphatidylethanolamine [PE]; phosphatidylinositol [PI]; and phosphatidylserine [PS]) were determined in plasma and peripheral blood mononuclear cells (PBMC) by mass spectrometry. Statistical analysis showed that six phospholipid species had diet × day interactions ( P P

The release of fatty acids from membrane lipids has been implicated in various metabolic and physiological processes, but in many cases, the enzymes involved and their functions in plants remain unclear. Patatin-related phospholipase As (pPLAs) constitute a major family of acyl-hydrolyzing enzymes in plants. Here, we show that pPLAIIIδ promotes the production of triacylglycerols with 20- and 22-carbon fatty acids in Arabidopsis (Arabidopsis thaliana). Of the four pPLAIIIs (α, β, γ, δ), only pPLAIIIδ gene knockout results in a decrease in seed oil content, and pPLAIIIδ is most highly expressed in developing embryos. The overexpression of pPLAIIIδ increases the content of triacylglycerol and 20- and 22-carbon fatty acids in seeds with a corresponding decrease in 18-carbon fatty acids. Several genes in the glycerolipid biosynthetic pathways are up-regulated in pPLAIIIδ-overexpressing siliques. pPLAIIIδ hydrolyzes phosphatidylcholine and also acyl-coenzyme A to release fatty acids. pPLAIIIδ-overexpressing plants have a lower level, whereas pPLAIIIδ knockout plants have a higher level, of acyl-coenzyme A than the wild type. Whereas seed yield decreases in transgenic plants that ubiquitously overexpress pPLAIIIδ, seed-specific overexpression of pPLAIIIδ increases seed oil content without any detrimental effect on overall seed yield. These results indicate that pPLAIIIδ-mediated phospholipid turnover plays a role in fatty acid remodeling and glycerolipid production.

The patatin-related phospholipase A (pPLA) hydrolyzes membrane glycerolipids to produce monoacyl compounds and free fatty acids. Phospholipids are cleaved by pPLAIIα at the sn-1 and sn-2 positions, and galactolipids, including those containing oxophytodienoic acids, can also serve as substrates. Ablation of pPLAIIα decreased lysophosphatidylcholine and lysophosphatidylethanolamine levels, but increased free linolenic acid. pPLAIIα-deficient plants displayed a higher level of jasmonic acid and methyl jasmonate, as well as the oxylipin-biosynthetic intermediates 13-hydroperoxylinolenic acid and 12-oxophytodienoic acid than wild-type (WT) plants. The expression of genes involved in oxylipin production was also higher in the pPLAIIα-deficient mutant than in WT plants. The mutant plants lost water more quickly than WT plants. The stomata of WT and mutant plants responded similarly to abscisic acid. In response to desiccation, the mutant and WT leaves produced abscisic acid at the same rate, but, after 4 h of desiccation, the jasmonic acid level was much higher in mutant than WT leaves. These results indicate that pPLAIIα negatively regulates oxylipin production and suggest a role in the removal of oxidatively modified fatty acids from membranes.

Candida albicans-associated bloodstream infections are linked to the ability of this yeast to form biofilms. In this study, we used lipidomics to compare the lipid profiles of C. albicans biofilms and planktonic cells, in early and mature developmental phases. Our results showed that significant differences exist in lipid composition in both developmental phases. Biofilms contained higher levels of phospholipid and sphingolipids than planktonic cells (nmol per g biomass, PP≤0.05). The ratio of phosphatidylcholine to phosphatidylethanolamine was lower in biofilms compared to planktonic cells in both early (1.17 vs 2.52, P≤0.001) and late (2.34 vs 3.81, P≤0.001) developmental phases. The unsaturation index of phospholipids decreased with time, with this effect being particularly strong for biofilms. Inhibition of the biosynthetic pathway for sphingolipid [mannosyl diinositolphosphoryl ceramide, M(IP)2C] by myriocin or aureobasidin A, and disruption of the gene encoding inositolphosphotransferase (Ipt1p), abrogated the ability of C. albicans to form biofilms. The differences in lipid profiles between biofilms and planktonic Candida cells may have important implications for the biology and antifungal resistance of biofilms.

Conventional wisdom holds that microbes support their growth in vertebrate hosts by exploiting a large variety of nutrients. We show here that use of a specific nutrient (ethanolamine) confers a marked growth advantage on Salmonella enterica serovar Typhimurium ( S. Typhimurium) in the lumen of the inflamed intestine. In the anaerobic environment of the gut, ethanolamine supports little or no growth by fermentation. However, S. Typhimurium is able to use this carbon source by inducing the gut to produce a respiratory electron acceptor (tetrathionate), which supports anaerobic growth on ethanolamine. The gut normally converts ambient hydrogen sulfide to thiosulfate, which it then oxidizes further to tetrathionate during inflammation. Evidence is provided that S. Typhimurium's growth advantage in an inflamed gut is because of its ability to respire ethanolamine, which is released from host tissue, but is not utilizable by competing bacteria. By inducing intestinal inflammation, S. Typhimurium sidesteps nutritional competition and gains the ability to use an abundant simple substrate, ethanolamine, which is provided by the host.

Changes in phospholipid composition and consequent loss of membrane integrity are correlated with loss of seed viability. Furthermore, phospholipid compositional changes affect the composition of the triacylglycerols, i.e. the storage lipids. Phospholipase D (PLD) catalyzes the hydrolysis of phospholipids to phosphatidic acid, and PLDα is an abundant PLD isoform. Although wild type seeds stored for 33 months were non-viable, 30 to 50% of PLDα-knockdown (PLD-KD) soybean seeds stored for 33 months germinated. Wild type and PLD-KD seeds increased in lysophospholipid levels and in triacylglycerol fatty acid unsaturation during aging, but the levels of lysophospholipids increased more in wild type than in PLD-KD seeds. The loss of viability of wild type seeds was correlated with alterations in ultrastructure, including detachment of the plasma membrane from the cell wall complex and disorganization of oil bodies. The data demonstrate that, during natural aging, PLDα affects the soybean phospholipid profile and the triacylglycerol profile. Suppression of PLD activity in soybean seed has potential for improving seed quality during long-term storage.

King, J. N., Hotz, R., Reagan, E. L., Roth, D. R., Seewald, W., Lees, P. Safety of oral robenacoxib in the cat. J. vet. Pharmacol. Therap. 35, 290–300. The safety of robenacoxib, a nonsteroidal anti-inflammatory drug with high selectivity for inhibition of the cyclooxygenase (COX)-2 isoform of COX, was investigated in the cat in two randomized, blinded, placebo-controlled, parallel-group studies. Robenacoxib was administered orally to healthy young domestic short-hair cats at dosages of 0 (placebo), 5 and 10 mg/kg once daily for 28 days (study 1) and at 0 (placebo), 2, 6 and 10 mg/kg twice daily for 42 days (study 2). The recommended minimum dosage for robenacoxib tablets in cats is 1 mg/kg once daily (range 1–2.4 mg/kg). Relative to placebo treatment, no toxicologically significant effects of robenacoxib were recorded in either study, based on general observations of health, haematological and clinical chemistry variables and urinalyses in life, and by post mortem organ weight, gross pathology and histopathology assessments. Pharmacokinetic–pharmacodynamic simulations indicated that all dosages of robenacoxib were associated with marked inhibition of COX-2 at peak effect (median Imax 97.8–99.4% inhibition) with lesser inhibition of COX-1 (median Imax 26.8–58.3% inhibition). Inhibition of the COXs was short lasting, with >10% median inhibition persisting for 4.0 h for COX-2 and 1.5 h for COX-1. These levels of inhibition of COX-1 and COX-2 twice daily with robenacoxib were not associated with any detectable toxicity, suggesting that, as previously described in dogs, the high safety index of robenacoxib in cats may be related to a combination of its high COX-2 selectivity and short residence time in the central compartment.

LipidomeDB Data Calculation Environment (DCE) is a web application to quantify complex lipids by processing data acquired after direct infusion of a lipid-containing biological extract, to which a cocktail of internal standards has been added, into an electrospray source of a triple quadrupole mass spectrometer. LipidomeDB DCE is located on the public Internet at http://lipidome.bcf.ku.edu:9000/Lipidomics . LipidomeDB DCE supports targeted analyses; analyte information can be entered, or pre-formulated lists of typical plant or animal polar lipid analytes can be selected. LipidomeDB DCE performs isotopic deconvolution and quantification in comparison to internal standard spectral peaks. Multiple precursor or neutral loss spectra from up to 35 samples may be processed simultaneously with data input as Excel files and output as tables viewable on the web and exportable in Excel. The pre-formulated compound lists and web access, used with direct-infusion mass spectrometry, provide a simple approach to lipidomic analysis, particularly for new users.

By employing electrospray ionization tandem mass spectrometry (ESI-MS/MS), the phospholipidomes of eight hemiascomycetous human pathogenic Candida species have been characterized. Over 200 phospholipid molecular species were identified and quantified. There were no large differences among Candida species in phosphoglyceride class composition; however, differences in phosphoglycerides components (i.e., fatty acyl chains) were identified. In contrast, differences in sphingolipid class composition as well as in molecular species were quite evident. The phospholipid compositions of C. albicans, C. glabrata, C. parapsilosis, C. kefyr, C. tropicalis, C. dubliniensis, C. krusei, and C. utilis could be further discriminated by principal component analysis. Notwithstanding that a single strain of each species was analyzed, our data do point to a typical molecular species imprint of Candida strains.

Oat (Avena sativa L.) kernels appear to contain much higher polar lipid concentrations than other plant tissues. We have extracted, identified, and quantified polar lipids from 18 oat genotypes grown in replicated plots in three environments to determine genotypic or environmental variation in these lipids. Validation experiments indicated a solid phase silica gel extraction step elution provided excellent and clean separation of extracted lipids into neutral lipid, glycolipid, and phospholipid fractions. Analysis of phospholipids by HPLC (normal phase, diol column) indicated phosphatidylethanolamine, phosphatidylcholine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and lyso- forms but very little genotypic or environmental variation. Di, tri and tetragalactosyl-diacylglycerols were quantified in the glycolipids, along with their mono-, di-, and triacyl estolides. Most of these exhibited significant genotypic variation. Molecular species analysis of the glycolipids in the Morton...

Summary 1. The St. Johns River Water Management District removed over 5.4 million kg of gizzard shad (Dorosoma cepedianum) from Lake Apopka, FL during 1993–2005, as a means of reducing lake phosphorus and phytoplankton concentrations and improving water clarity. Other steps included reduction of external nutrient inputs and operation of a treatment wetland. We measured nutrient excretion by Lake Apopka gizzard shad to quantify the nutrient effect of this biomanipulation. 2. Both N and P excretion were significantly affected by fish body mass and temperature. Larger fish had lower mass-specific rates of excretion than smaller fish. 3. High water temperature increased P excretion to a much greater extent than N, resulting in a low N : P of nutrient excretion in midsummer. The N : P of excretion was lower than has been observed in other systems, probably because of higher water temperature. 4. Removal of gizzard shad >200 g prevented the annual release of 45 800 kg N year−1 (3.46 kg N ha−1 year−1) and 7700 kg P year−1 (0.62 kg P ha−1 year−1) on average. The actual impact on the P cycle varied substantially from year to year (range 7900–78 800 kg N year−1; 1200–14 800 kg P year−1), primarily because of fluctuations in the catch. 5. On an annual basis, the P directly removed in fish tissues was similar to that removed by the treatment wetland. The P excretion prevented by the removal of fish was approximately 20% of the reduction in external P loading achieved during 1993–2005. 6. In the short term, most of the P demand of planktonic primary producers is met through recycling of P, which greatly exceeds external P loading. Depending on population biomass, phosphorus excretion by the resident gizzard shad population was similar in magnitude to the P release by diffusive flux from the sediments.

Catalytic material-assisted growth of alkaline-earth metal hexaboride (MB6, M = Sr, Ba) 1D nanostructures was achieved by pyrolysis of diborane (B2H6) over alkaline-earth metal oxide (MO) powders at elevated temperature (∼840−870 °C) and low pressure (∼165 mTorr). The as-synthesized MB6 1D nanostructures were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy. Results show that the MB6 nanostructures are single crystalline with the preferred growth direction along [001]. The SrB6 nanowires are ∼20−100 nm in diameter and ∼2−5 μm in length. The BaB6 nanostructures have a rectangular cross section (width ∼20−300 nm) and are ∼5−20 μm in length. The growth of these MB6 nanostructures can be attributed to a nontraditional vapor−solid−liquid (VLS)-like growth mechanism. The as-synthesized MB6 1D nanostructures are promising n-type low-dimensional thermoelectric materials.

Oat kernels were extracted with methanol, and glycolipid-enriched fractions were prepared using silica solid phase extraction. Using direct infusion electrospray ionization (ESI) tandem mass spectrometry (MS), high performance liquid chromatography (HPLC)-ESI-MS, and HPLC-atmospheric pressure chemical ionization (APCI)-MS, we confirmed previous reports that digalactosyldiacylglycerol (DGDG) was the most abundant glycolipid in oat kernels and confirmed a previous report of the presence of a DGDG mono-estolide in oat kernels. In the current study we also identified several additional natural galactolipid estolides: two new DGDG estolides (di- and tri-estolides), two trigalactosyldiacylglycerol (TriGDG) estolides (mono- and di-estolides), and one tetragalactosyldiacylglycerol (TetraGDG) estolide (mono-estolide). The levels of total galactolipid estolides in oat kernels were estimated to be about 29% of the total glycolipid fraction. To our knowledge, this report is the first evidence of natural di- and tri-estolides of polar lipids.

Objective: The hypothesis of this two-part study is that adhesive systems for bonding orthodontic brackets (ie, two self-etch primers [Transbond and M-Bond] and a conventional phosphoric acid etch [Rely-a-Bond]) would show a difference with respect to rebonded enamel surface morphology and chemical composition. Materials and Methods: This study examined the enamel surface before and after debonding with scanning electron microscopy and the enamel surface chemical composition for the elements Ca, P, O, F, Si, and C using x-ray photoelectron spectroscopy. Results: The etching of the two self-etch groups is less aggressive and less uniform than that of phosphoric acid. The change in the concentration of C indicated that the separation of the bracket from the enamel surface is at the resin-enamel interface for the phosphoric acid–etched adhesive and a mixed mode involving the enamel-resin-bracket interfaces for the self-etching systems. F release appears to occur for Transbond but not for M-Bond. Conclusions: The results confirm the original hypothesis that differences in adhesive systems are manifested in less aggressive etches and less adhesive left on the enamel surface for the self-etching adhesive systems.

Systemic acquired resistance (SAR) is an inducible defense mechanism that is activated throughout the plant, subsequent to localized inoculation with a pathogen. The establishment of SAR requires translocation of an unknown signal from the pathogen-inoculated leaf to the distal organs, where salicylic acid-dependent defenses are activated. We demonstrate here that petiole exudates (PeXs) collected from Arabidopsis leaves inoculated with an avirulent (Avr) Pseudomonas syringae strain promote resistance when applied to Arabidopsis, tomato (Lycopersicum esculentum) and wheat (Triticum aestivum). Arabidopsis FATTY ACID DESATURASE7 (FAD7), SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 (SFD1) and SFD2 genes are required for accumulation of the SAR-inducing activity. In contrast to Avr PeX from wild-type plants, Avr PeXs from fad7, sfd1 and sfd2 mutants were unable to activate SAR when applied to wild-type plants. However, the SAR-inducing activity was reconstituted by mixing Avr PeXs collected from fad7 and sfd1 with Avr PeX from the SAR-deficient dir1 mutant. Since FAD7, SFD1 and SFD2 are involved in plastid glycerolipid biosynthesis and SAR is also compromised in the Arabidopsis monogalactosyldiacylglycerol synthase1 mutant we suggest that a plastid glycerolipid-dependent factor is required in Avr PeX along with the DIR1-encoded lipid transfer protein for long-distance signaling in SAR. FAD7-synthesized lipids provide fatty acids for synthesis of jasmonic acid (JA). However, co-infiltration of JA and methylJA with Avr PeX from fad7 and sfd1 did not reconstitute the SAR-inducing activity. In addition, JA did not co-purify with the SAR-inducing activity confirming that JA is not the mobile signal in SAR.

Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine, but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma’s, but only 3% of the host’s, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine, but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host’s immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen.

Seed aging decreases the quality of seed and grain and results in agricultural and economic losses. Alterations that impair cellular structures and metabolism are implicated in seed deterioration, but the molecular and biochemical bases for seed aging are not well understood. Ablation of the gene for a membrane lipid-hydrolyzing phospholipase D (PLDalpha1) in Arabidopsis enhanced seed germination and oil stability after storage or exposure of seeds to adverse conditions. The PLDalpha1-deficient seeds exhibited a smaller loss of unsaturated fatty acids and lower accumulation of lipid peroxides than did wild-type seeds. However, PLDalpha1-knockdown seeds were more tolerant of aging than were PLDalpha1-knockout seeds. The results demonstrate the PLDalpha1 plays an important role in seed deterioration and aging in Arabidopsis. A high level of PLDalpha1 is detrimental to seed quality, and attenuation of PLDalpha1 expression has the potential to improve oil stability, seed quality and seed longevity.

Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for approximately 1 min per sample.

Direct-infusion electrospray ionization triple quadrupole mass spectrometry method with multiple reaction monitoring (MRM) has been employed to measure Arabidopsis thaliana galactolipids and phosph...

Changes in gene copy number are among the most frequent mutational events in all genomes and were among the mutations for which a physical basis was first known. Yet mechanisms of gene duplication remain uncertain because formation rates are difficult to measure and mechanisms may vary with position in a genome. Duplications are compared here to deletions, which seem formally similar but can arise at very different rates by distinct mechanisms. Methods of assessing duplication rates and dependencies are described with several proposed formation mechanisms. Emphasis is placed on duplications formed in extensively studied experimental situations. Duplications studied in microbes are compared with those observed in metazoan cells, specifically those in genomes of cancer cells. Duplications, and especially their derived amplifications, are suggested to form by multistep processes often under positive selection for increased copy number.

In an attempt to develop a low-k interlayer dielectric, adamantane-diphenyldiethynyl moiety containing oligomer is prepared. Oligomerization of 1,3,5,7-tetrakis[3/4-ethynylphenyl]adamantane (4) is accomplished by a Glaser–Hay oxidative coupling with 1,3,5-triethynylbenzene and phenylacetylene end-capping agent. The CHCl3 soluble oligomer is then thermally treated by step-curing at 200, 300, 380, and 450 °C for 30 min at each temperature under nitrogen flow to render a shiny void-free black polymer. TGA analysis indicates that the polymer is stable under nitrogen up to 500 °C with a marginal decomposition up to 800 °C. Solid-state 13C NMR, Raman scattering, and FTIR are used to characterize the structure of the polymer. The polymer consists of amorphous carbon networks with the adamantane moieties and nanosized graphitic regions (clusters), which are generated from the thermal crosslinking of the diphenyldiethynyl units. It shows a remarkably low linear coefficient of thermal expansion (∼25 ppm/°C), presumably due to the presence of the disordered graphitic structure. Its high density (∼1.21 g/cm3), refractive index (∼1.80 at 632 nm), and Young's modulus (∼17.0 GPa) are also consistent with the interpretation. This study reveals important details about the effect of microscopic structure on the macroscopic properties of the highly crosslinked polymer. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 6909–6925, 2006

Lipid profiling is a targeted metabolomics platform that provides a comprehensive analysis of lipid species with high sensitivity. Profiling based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides quantitative data and is adaptable to high throughput analyses. Here we report the profiling of 140 apparent molecular species of polar glycerolipids in Arabidopsis leaves, flower stalks, flowers, siliques, roots, and seeds. Considerable differences in lipid species occur among these organs, providing insights into the different lipid metabolic activities in a specific organ. In addition, comparative profiling between wild-type and a knockout mutant pldalpha1 (locus ID: AT3G15730) provides insight into the metabolic function of phospholipase D (PLD) in different organs. PLDalpha1 contributes significantly to phosphatidic acid (PA) levels in roots, seeds, flowers, and flower stalks, but little to basal PA levels in siliques and leaves. In seeds of the pldalpha1 mutant plants, levels of PA, lysophosphatidylcholine, and lysophosphatidylethanolamine were significantly lower than those of wild-type seeds, suggesting a role for PLDalpha1 in membrane lipid degradation in seeds.

Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastid-localized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI) collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-of-flight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidylglycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and 16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA- and/or dnOPDA-containing MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023 nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200- to 1,000-fold. In contrast, levels of OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each containing a single oxylipin chain, rose 2- to 9-fold. The rapid accumulation of high levels of galactolipid species containing OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic oxylipin biosynthesis.

Forbidden Reactions, II. – The Disrotatory Cyclobutene Ringopening The energy profiles for the con- and disrotatory opening of benzocyclobutene, methylenecyclobutene, and cyclobutene derivatives were established by NO and oxygen trapping. The enthalpy for the transition states for the forbidden reactions proved to be identical with the heat of formation of the orthogonal diradicals derived by geometrical isomerization of the dienes formed in these reactions. These diradicals describe very well the activation barriers observed for the disrotatory opening of bicyclic cyclobutenes ([3.2.0] and [2.2.0] systems), but not for bicyclo[2.1.0]pent-2-ene, indicating here a truly forbidden reaction.

On the Energy Well of Diradicals, VI1 . – The Energy Profile for the NO2-Addition to Conjugated Double Bonds: A Reaction with Negative Activation Energy From the NO and temperature dependence of the reaction rates of 1,3-butadiene, styrene, and methylenebenzocyclobutene with NO2 in the presence of NO, the energy profiles for the formation of the primary adducts are derived, which explain the inverse temperature dependence for the disappearance of the substrate at low NO concentrations and allow to calculate the bond strength of the C–NO2 bond (57.9 ± 1.3 kcal · mol−1).

On the Energy Well of Diradicals, VIII1 . – Energy Profile of the Degenerate Methylenecyclobutane Rearrangement From the temperature and oxygen dependence of the trapping rates of the diradicals 4 and 6, recombination enthalpies of 4.7 and 1.8 kcal · mol−1 are derived. These enthalpies agree well with estimates from our reaction force field (5.5 and 2.0 kcal · mol−1). The increasing stereoselectivity in the methylenecyclobutane rearrangement 1a 1b with electronically less stabilizing substituents (R = Ph, CH3) is related to the decreasing recombination enthalpy of the intermediate diradical 2. This finding can be rationalized from the changing curve of the electronic energy during the recombination.

Salmonella enterica can obtain pyridine from exogenous nicotinamide mononucleotide (NMN) by three routes. In route 1, nicotinamide is removed from NMN in the periplasm and enters the cell as the free base. In route 2, described here, phosphate is removed from NMN in the periplasm by acid phosphatase (AphA), and the produced nicotinamide ribonucleoside (NmR) enters the cell via the PnuC transporter. Internal NmR is then converted back to NMN by the NmR kinase activity of NadR. Route 3 is seen only in pnuC * transporter mutants, which import NMN intact and can therefore grow on lower levels of NMN. Internal NMN produced by either route 2 or route 3 is deamidated to nicotinic acid mononucleotide and converted to NAD by the biosynthetic enzymes NadD and NadE.

Adenosylcobalamin (Ado-B 12 ) is both the cofactor and inducer of ethanolamine ammonia lyase (EA-lyase), a catabolic enzyme for ethanolamine. De novo synthesis of Ado-B 12 by Salmonella enterica occurs only under anaerobic conditions. Therefore, aerobic growth on ethanolamine requires import of Ado-B 12 or a precursor (CN-B 12 or OH-B 12 ) that can be adenosylated internally. Several known enzymes adenosylate corrinoids. The CobA enzyme transfers adenosine from ATP to a biosynthetic intermediate in de novo B 12 synthesis and to imported CN-B 12 , OH-B 12 , or Cbi (a B 12 precursor). The PduO adenosyl transferase is encoded in an operon ( pdu ) for cobalamin-dependent propanediol degradation and is induced by propanediol. Evidence is presented here that a third transferase (EutT) is encoded within the operon for ethanolamine utilization ( eut ). Surprisingly, these three transferases share no apparent sequence similarity. CobA produces sufficient Ado-B 12 to initiate eut operon induction and to serve as a cofactor for EA-lyase when B 12 levels are high. Once the eut operon is induced, the EutT transferase supplies more Ado-B 12 during the period of high demand. Another protein encoded in the operon (EutA) protects EA-lyase from inhibition by CN-B 12 but does so without adenosylation of this corrinoid.

The eutH gene is part of an operon that allows Salmonella enterica to use ethanolamine as a sole source of nitrogen, carbon, and energy. Although the sequence of EutH suggests a role in transport, eutH mutants use ethanolamine normally under standard conditions (pH 7.0). These mutants fail to use ethanolamine at a low pH. Evidence is presented that protonated ethanolamine (Eth 0 ) does not enter cells, while uncharged ethanolamine (Eth 0 ) diffuses freely across the membrane. The external concentration of Eth 0 varies with the pH (pK = 9.5). At pH 7.0, the standard ethanolamine concentration (41 mM) provides enough Eth 0 for an influx rate that can support growth with or without EutH. When a lowered pH and/or ethanolamine concentration reduced the Eth 0 concentration below 25 μM, EutH was needed to facilitate diffusion. EutH + cells grew normally at Eth 0 concentrations above 3 μM, close to the Km (9 μM) of the first degradative enzyme, ethanolamine ammonia lyase. It is suggested that EutH facilitates diffusion of Eth 0 . As predicted for a transporter, EutH contributed to the toxicity of ethanolamine seen under some conditions; furthermore, fusion of EutH to fluorescent Yfp protein provided evidence that EutH is a membrane protein.