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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.

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.

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.

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.

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.

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...

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.

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.

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.

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).

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.

Catalyst-free growth of boron nanoribbons was observed by pyrolysis of diborane at 630−750 °C and ∼200 mTorr in a quartz tube furnace. Nanodiffraction analysis indicates the nanoribbons are single crystal α-tetragonal boron. TEM images show the nanoribbon is covered by a 1−2 nm thick amorphous layer. Elemental analysis by EELS, EDX, and XPS shows the nanoribbons consist of boron with small amounts of oxygen and carbon. Infrared and Raman spectra are also reported.

The extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. Here, the four disulfide bonds of AVR4 were identified as Cys-11-41, Cys-21-27, Cys-35-80, and Cys-57-72 by partial reduction with Tris-(2-carboxyethyl)-phosphine hydrochloride, subsequent cyanylation, and base-catalyzed chain cleavage. The resulting peptide fragments were analyzed by mass spectrometry. Sequence homology and the disulfide bond pattern revealed that AVR4 contains an invertebrate (inv) chitin-binding domain (ChBD). Binding of AVR4 to chitin was confirmed experimentally. The three disulfide bonds encompassing the inv ChBD motif are also required for protein stability of AVR4. Independent disruption of each of the three conserved disulfide bonds in AVR4 resulted in a protease-sensitive protein, whereas the fourth disulfide bond appeared not to be required for protein stability. Most strains of C. fulvum virulent on Cf-4 tomato contain Cys to Tyr substitutions in AVR4 involving two (Cys-11-41, Cys-35-80) of the three disulfide bonds present in the inv ChBD motif. These natural Cys to Tyr mutant AVR4 proteins did retain their chitin binding ability and when bound to chitin were less sensitive to proteases. Thus, the widely applied tomato Cf-4 resistance gene is circumvented by C. fulvum by amino acid substitutions affecting two disulfide bonds in AVR4 resulting in the absence of the corresponding AVR4 isoforms in apoplastic fluid. However, these natural isoforms of AVR4 appear to have retained their intrinsic function, i.e. binding to chitin present in the cell wall of C. fulvum, most likely to protect it against the deleterious effects of plant chitinases.

The allowed conrotatory cyclobutene ring-opening has a distinctly nonplanar carbon skeleton. Classic experiments by Brauman and Archie, and by Freedman et al., placed the allowed/forbidden gap at greater than 15 kcal/mol. Wolfgang Roth proposed that a system forced to planarity might have a smaller preference for the conrotatory mode than unconstrained systems. Such systems have now been studied theoretically and experimentally, and results that confirm Roth's postulate are presented here. The experiments were performed in Bochum, and the calculations were carried out in Osaka and Los Angeles. As the cyclobutene ring-opening transition structure approaches planarity, the energy gap between allowed conrotatory and the forbidden disrotatory pathways decreases. For the ring-opening of a cyclobutene fused to norbornene, the energy gap between the forbidden and the allowed transition state is only 4.1 kcal/mol by CASSCF and 8.0 kcal/mol by CAS-MP2 as compared to 13.4 and 19.2 kcal/mol, respectively, for the parent cyclobutene. Experimental studies of 3,4-dimethylcyclobutenes fused to various ring systems are reported, and a trend is found toward a reduced allowed/forbidden gap as the planarity of the cyclobutene is enforced.

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.Cell Death and Differentiation advance online publication, 22 August 2014; doi:10.1038/cdd.2014.110.

Keratocytes of the corneal stroma secrete a unique population of proteoglycan molecules considered essential for corneal transparency. In healing corneal wounds, keratocytes exhibit a myofibroblastic phenotype in response to transforming growth factor beta (TGF-beta), characterized by expression of alpha-smooth muscle actin. This study examined proteoglycan and collagen expression by keratocytes in vitro during the TGF-beta-induced keratocyte-myofibroblast transition. TGF-beta-treated primary bovine keratocytes developed myofibroblastic features, including actin stress fibers anchored to paxillin-containing focal adhesions, cell-associated fibronectin, alpha(5) integrin, and alpha-smooth muscle actin. Collagen I and III protein and mRNA increased in response to TGF-beta. Secretion of [(35)S]sulfate-labeled keratan sulfate proteoglycans decreased markedly in response to TGF-beta. Dermatan sulfate proteoglycans, however, increased in size and abundance. Protein and mRNA transcripts for normal stromal proteoglycans (lumican, keratocan, mimecan, and decorin) all decreased in response to TGF-beta, but protein expression and mRNA for biglycan, a proteoglycan present in fibrotic tissue, was markedly up-regulated. These results show that TGF-beta in vitro induces a proteoglycan expression pattern similar to that of corneal scars in vivo. This altered proteoglycan expression occurred coordinately with transdifferentiation of keratocytes to the myofibroblastic phenotype, implicating these cells as the source of fibrotic tissue in nontransparent corneal scars.

The concerted formation of the orthogonal trimethylene-type diradicals 3, 6, and 12 has been demonstrated for the three homofulvenes 2, 4, and 7 and shown to proceed preferentially in a disrotatory manner. The same type of orthogonal diradicals are formed, but in a nonconcerted fashion, from the bicyclic methylenecyclopropanes 26, 30, 33, and 40, through the planar diradicals 28, 31, 35, and 50, respectively. The different reaction modes are viewed as being caused by the different coupling probabilities of the underlying vibrations. The orthogonal or planar geometries of the diradicals were derived from stereochemical and kinetic arguments. Additional thermodynamic characterization was achieved for three diradicals [3c, (E)-19, and 41] by oxygen-trapping experiments.

Characteristics of low density, high stiffness, good creep resistance and high strength at a wide range of temperatures make titanium aluminide a potentially important material in respect of weight savings in high performance components operating at high temperatures. Some work has already been carried out to examine properties of the machining of this alloy using mechanical stock-removal techniques such as, turning. Such methods are found to have limitations in terms of surface integrity defects and the formation of surface hardened layer. In this paper the electrochemical machining (ECM) characteristics of titanium aluminide are examined. Conditions under which reproducible ECM is viable for this material have been established and parameterised in terms of machining parameters generated from chronoamperometric analyses for both chloride and perchlorate electrolyte systems. Stable dissolution has been shown to be limited by surface passivation, in case of the chloride system, and choking in case of the perchlorate system. In case of the perchlorate surface there is evidence of an increased amount of cross-colony attack when compared to surfaces machined using chloride. Chronoamperometric analysis has indicated that this form of attack can be correlated to differences in the valency (2.4 for the perchlorate system and 3.0 for the chloride system) and the overpotential (3.8 V for the perchlorate system and 4.3 V for the chloride system). These differences have been related to measured differences in the surface dissolution characteristics of the microstructural phases of this material. Surface hardness of the ECM TiAl has been shown to have decreased by 46% with respect to conventionally machined surfaces.

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

Whereas 1,5-dimethylsemibullvalene (1b) equilibrates with 1,5-dimethylcyclooctatetraene (2b), the semibullvalene-2,6-dicarbonitriles 13 rearrange irreversibly to afford mixtures of the isomeric cyclooctatetraene-1,5-dicarbonitriles 14 and 15. Arrhenius and Eyring parameters of these thermal isomerisation reactions have been determined for the gas phase (1b 2b) and [D6]benzene solutions (1b 2b, 13 14 + 15). Furthermore, the activation parameters of the known rearrangement of octamethylcyclooctatetraene (3) to octamethylsemibullvalene (4) have been determined. – The data for these compounds, together with those for related compounds previously reported in the literature, show that substituents not only influence the relative stabilities of the semibullvalene and cyclooctatetraene systems but also the height of the energy barrier for their interconversion. Those substituents that lower the barrier toward the degenerate Cope rearrangement of semibullvalenes simultaneously accelerate their rearrangement to cyclooctatetraenes thus limiting the thermal stability of the former.

Purpose: Cryptorchidism is the most common congenital abnormality of the genitalia. However, its exact etiology remains to be defined. Homeobox (HOX) containing genes have a key role in the morphogenesis of segmental structures along the primary body axis, including the urogenital mesenchyma. In male mice with a targeted deletion of the HOXA10 gene cryptorchidism manifests in the absence of other major defects. Because to our knowledge this gene has never been examined for alterations in humans, we evaluated whether mutations of HOXA10 are associated with cryptorchidism in humans.Materials and Methods: Genomic deoxyribonucleic acid (DNA) was extracted from human blood or tissue samples from 16 noncryptorchid control subjects and 45 cryptorchid boys. To screen for mutations exons 1 and 2 of the HOXA10 gene were amplified individually by polymerase chain reaction using 6 overlapping oligonucleotide primer pairs. Single strand conformational polymorphism (SSCP) analysis of the amplified radiolabeled ...

Activation parameters have been determined in the gas phase for the geometrical isomerization of the double bonds in two phenyl-substituted methylenecyclopropanes (2 and 8). Comparison with the analogous values for three sterically non-restricted styrene derivatives (15, 16 and 17) shows that the enthalpies of activation for the methylenecyclopropanes are lower by 3.7 kcal mol−1. This value, which is an upper limit to the amount of strain energy released by pyramidalization of the ring carbon atom in the orthogonal diradical transition state when undergoing geometrical isomerization, is considerably smaller than the value of 12-14 kcal mol−1 by which the strain energy of methylenecyclopropane is larger relative to that of cyclopropane. Our kinetics experiments thus show that the angle strain, associated with incorporating a trigonal carbon atom into the three-membered ring, is not responsible for the majority of the additional strain energy of methylenecyclopropane, relative to that of cyclopropane.

The effects of acid precipitation and ozone on the composition of the ectomycorrhizal community of red spruce saplings were evaluated. In 1986, saplings were excavated from a site in Maine that had been clear-cut in 1979. Saplings were then potted in native soil and transported to Ithaca, New York. With the exception of an ambient control treatment, trees were grown in open-top chambers. Saplings were exposed to five levels of ozone and three levels of acid precipitation beginning in July 1987. Ectomycorrhizae were sampled in 1988 and 1991 after one and four years of treatment, respectively. Although the percentage of root tips infected by ectomycorrhizal fungi was not affected by treatments, a shift in the composition of the ectomycorrhizal community occurred in response to acid precipitation treatments for both sampling years. Among the seven ectomycorrhizal morphotypes identified, the percent composition of one morphotype increased and another decreased in response to higher rain acidity. Alone, ozone treatments did not influence ectomycorrhizal composition; however, a significant interactive response to ozone and acid precipitation was observed in the organic soil horizon in 1988. Such shifts in the composition of the ectomycorrhizal community may indicate that the experimental trees were stressed by pollution treatments.

Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3' noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5'-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency.

Bovine cornea contains three unique keratan sulfate proteoglycans (KSPGs), of which two (lumican and keratocan) have been characterized using molecular cloning. The gene for the third protein (KSPG25) has not been identified. This study examined the relationship between the KSPG25 protein and the gene for osteoglycin, a 12-kDa bone glycoprotein. The N-terminal amino acid sequence of KSPG25 occurs in osteoglycin cDNA cloned from bovine cornea. The osteoglycin amino acid sequence makes up the C-terminal 47% of the deduced sequence of the KSPG25 protein. Antibodies to osteoglycin reacted with intact corneal KSPG, with KSPG25 protein, and with a 36-kDa protein, distinct from lumican and keratocan. KSPG25-related proteins, not modified with keratan sulfate, were also detected in several connective tissues. Northern blot analysis showed mRNA transcripts of 2.4, 2.5, and 2.6 kilobases in numerous tissues with the 2.4-kilobase transcript enriched in ocular tissues. Ribonuclease protection analysis detected several protected KSPG25 mRNA fragments, suggesting alternate splicing of KSPG25 transcripts. We conclude that the full-length translation product of the gene producing osteoglycin is a corneal keratan sulfate proteoglycan, also present in many non-corneal tissues without keratan sulfate chains. The multiple size protein products of this gene appear to result from in situ proteolytic processing and/or alternative splicing of mRNA. The name mimecan is proposed for this gene and its products.

From the O2-dependence of the trapping rate of 1,1-difluoro-2,3-diphenylcyclopropane in supercritical CO2 in the temperature range 110–180°C and the rates of its geometrical isomerization and racemization of the trans-isomer, the energy profile for the geometrical isomerization is derived. Assuming that 7 is a common intermediate, the temperature dependence of the ratio of the rates of isomerization/racemization leads to a lowering of 1.2 kcal · mol−1 for the activation energy for the disrotatory cyclization of the intermediate diradical compared to the conrotatory mode. The energy barriers associated with cyclization of the diradical amount to 2.1 and 0.9 kcal · mol−1, respectively.

In the course of a lytic infection the Salmonella phage P22 occasionally encapsulates bacterial DNA instead of phage DNA. Thus, phage lysates include two classes of viral particles. Phage particles carrying bacterial DNA are referred to as transducing particles and deliver this DNA to a host as efficiently as particles carrying phage DNA. Once injected, the transduced DNA can either recombine with the recipient chromosome to form a “complete” transductant, or it can establish itself as an expressible, nonreplicating genetic element and form an “abortive” transductant. In this work, we describe a P22-phage mutant with reduced ability to form abortive transductants. The mutation responsible for this phenotype, called tdx-1, was found as one of two mutations contributing to the high-transducing phenotype of the P22-mutant HT12/4. In addition, the tdx-1 mutation is lethal when combined with an erf-am mutation. The tdx-1 mutation has been mapped to a region of the P22 genome that encodes several injected proteins and may involve more than one mutant locus. The phenotypes of the tdx-1 mutation suggest that the Tdx protein(s) normally assist in the circularization of the P22 genome and also contribute to the formation of DNA circles thought to be required for abortive transduction.

Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG (galactose-acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.