Direct acting antiviral therapy rescues neutrophil dysfunction and reduces hemolysis in hepatitis C infection.

Chronic hepatitis C virus infection is characterized by multiple extra-hepatic manifestations. Innate immune dysfunction and hemolysis are symptoms which might be associated with each other. We investigated the impact of direct acting antivirals on neutrophil function and its connection to hemolysis. In this prospective study, 85 patients with or without cirrhosis and 21 healthy controls were included. Patients' blood samples were taken at baseline, at the end of therapy and at follow-up 12 weeks after end of therapy. Neutrophil phagocytosis, oxidative burst, and hemolysis parameters were studied. Multivariate analysis was performed to decipher the relationship between hemolysis and neutrophil function. Ex vivo cross-incubation experiments with neutrophils and serum fractions were done. Impaired neutrophil phagocytosis and mild hemolysis were observed in patients with and without cirrhosis. A proteome approach revealed different expression of hemolysis-related serum proteins in patients and controls. Direct acting antiviral therapy restored neutrophil function irrespective of severity of liver disease, achievement of sustained virologic response or type of drug and reduced hemolysis. Treatment with ribavirin delayed the improvement of neutrophil function. Statistical analysis revealed associations of haptoglobin with neutrophil phagocytic capacity. Neutrophil dysfunction could be transferred to healthy cells by incubation with patients' serum fractions (>30 kDa) ex vivo. Neutrophil dysfunction and hemolysis represent extrahepatic manifestations of chronic hepatitis C virus infection and simultaneously improve during direct acting antiviral therapy independently of therapy-related liver function recovery. Therefore, large-scale treatment would not only drive viral eradication but also improve patients' immune system and may reduce susceptibility to infections.

Determination of cathinone and cathine in Khat plant material by LC-MS/MS: Fresh vs. dried leaves.

The consumption of Khat leaves represents an ancient kind of drug abuse mainly observed in Eastern Africa and the Arab Peninsula among adult men. For this purpose, the leaves are directly collected from the shrub "Catha edulis" prior to extensive chewing process. Seizures in Europe are rare, since the leaves have to undergo quick transportation: After a short period of time, the harvested leaves decompose and suffer in decrease of concentration of the active ingredient cathinone, which makes long term transportation difficult. As an alternative, plant material can be dried to increase life period. In the past years, an increasing number of seizures were made by Austrian police, however, the content of cathinone and cathine in dry material is widely unknown. In this work, a seizure of fresh Khat leaves was compared with two seizures of dried material in terms of concentration of cathinone and cathine using LC-MS/MS analysis. For fresh leaves, a purity grade was found to be 0.115-0.158% for cathinone and 0.172-0.192% for cathine, respectively. In contrast, subsequent storage of dried Khat leaves over months led to a dramatic loss of cathinone: Analysis of two seizures revealed that concentration of cathinone dropped to 0.021-0.023%. These findings are intended to serve as a guideline for Justice authorities to estimate the content of the controlled ingredients of Khat leaves in future.

Myeloperoxidase-Derived 2-Chlorohexadecanal Is Generated in Mouse Heart during Endotoxemia and Induces Modification of Distinct Cardiomyocyte Protein Subsets In Vitro.

Sepsis is a major cause of mortality in critically ill patients and associated with cardiac dysfunction, a complication linked to immunological and metabolic aberrations. Cardiac neutrophil infiltration and subsequent release of myeloperoxidase (MPO) leads to the formation of the oxidant hypochlorous acid (HOCl) that is able to chemically modify plasmalogens (ether-phospholipids) abundantly present in the heart. This reaction gives rise to the formation of reactive lipid species including aldehydes and chlorinated fatty acids. During the present study, we tested whether endotoxemia increases MPO-dependent lipid oxidation/modification in the mouse heart. In hearts of lipopolysaccharide-injected mice, we observed significantly higher infiltration of MPO-positive cells, increased fatty acid content, and formation of 2-chlorohexadecanal (2-ClHDA), an MPO-derived plasmalogen modification product. Using murine HL-1 cardiomyocytes as in vitro model, we show that exogenously added HOCl attacks the cellular plasmalogen pool and gives rise to the formation of 2-ClHDA. Addition of 2-ClHDA to HL-1 cardiomyocytes resulted in conversion to 2-chlorohexadecanoic acid and 2-chlorohexadecanol, indicating fatty aldehyde dehydrogenase-mediated redox metabolism. However, a recovery of only 40% indicated the formation of non-extractable (protein) adducts. To identify protein targets, we used a clickable alkynyl analog, 2-chlorohexadec-15-yn-1-al (2-ClHDyA). After Huisgen 1,3-dipolar cycloaddition of 5-tetramethylrhodamine azide (N3-TAMRA) and two dimensional-gel electrophoresis (2D-GE), we were able to identify 51 proteins that form adducts with 2-ClHDyA. Gene ontology enrichment analyses revealed an overrepresentation of heat shock and chaperone, energy metabolism, and cytoskeletal proteins as major targets. Our observations in a murine endotoxemia model demonstrate formation of HOCl-modified lipids in the heart, while pathway analysis in vitro revealed that the chlorinated aldehyde targets specific protein subsets, which are central to cardiac function.

Bupleurum chinense Roots: a Bioactivity-Guided Approach toward Saponin-Type NF-κB Inhibitors.

The roots of Bupleurum chinense have a long history in traditional medicine to treat infectious diseases and inflammatory disorders. Two major compounds, saikosaponins A and D, were reported to exert potent anti-inflammatory activity by inhibiting NF-κB. In the present study, we isolated new saikosaponin analogues from the roots of B. chinese interfering with NF-κB activity in vitro. The methanol-soluble fraction of the dichloromethane extract of Radix Bupleuri was subjected to activity-guided isolation yielding 18 compounds, including triterpenoids and polyacetylenes. Their structures were determined by spectroscopic methods as saikogenin D (1), prosaikogenin D (2), saikosaponins B2 (3), W (4), B1 (5), Y (6), D (7), A (8), E (9), B4 (10), B3 (11), and T (12), saikodiyne A (13), D (14), E (15) and F (16), falcarindiol (17), and 1-linoleoyl-sn-glycero-3-phosphorylcholine (18). Among them, 4, 15, and 16 are new compounds, whereas 6, previously described as a semi-synthetic compound, is isolated from a natural source for the first time, and 13-17 are the first reports of polyacetylenes from this plant. Nine saponins/triterpenoids were tested for inhibition of NF-κB signaling in a cell-based NF-κB-dependent luciferase reporter gene model in vitro. Five of them (1, 2, 4, 6, and 8) showed strong (> 50%, at 30 µM) NF-κB inhibition, but also varying degrees of cytotoxicity, with compounds 1 and 4 (showing no significant cytotoxicity) presenting IC50 values of 14.0 µM and 14.1 µM in the cell-based assay, respectively.

Assessment of electrophile damage in a human brain endothelial cell line utilizing a clickable alkyne analog of 2-chlorohexadecanal.

Peripheral leukocytes aggravate brain damage by releasing cytotoxic mediators that compromise blood-brain barrier function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system. The reaction of HOCl with the endogenous plasmalogen pool of brain endothelial cells results in the generation of 2-chlorohexadecanal (2-ClHDA), a toxic, lipid-derived electrophile that induces blood-brain barrier dysfunction in vivo. Here, we synthesized an alkynyl-analog of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA) to identify potential protein targets in the human brain endothelial cell line hCMEC/D3. Similar to 2-ClHDA, 2-ClHDyA administration reduced cell viability/metabolic activity, induced processing of pro-caspase-3 and PARP, and led to endothelial barrier dysfunction at low micromolar concentrations. Protein-2-ClHDyA adducts were fluorescently labeled with tetramethylrhodamine azide (N3-TAMRA) by 1,3-dipolar cycloaddition in situ, which unveiled a preferential accumulation of 2-ClHDyA adducts in mitochondria, the Golgi, endoplasmic reticulum, and endosomes. Thirty-three proteins that are subject to 2-ClHDyA-modification in hCMEC/D3 cells were identified by mass spectrometry. Identified proteins include cytoskeletal components that are central to tight junction patterning, metabolic enzymes, induction of the oxidative stress response, and electrophile damage to the caveolar/endosomal Rab machinery. A subset of the targets was validated by a combination of N3-TAMRA click chemistry and specific antibodies by fluorescence microscopy. This novel alkyne analog is a valuable chemical tool to identify cellular organelles and protein targets of 2-ClHDA-mediated damage in settings where myeloperoxidase-derived oxidants may play a disease-propagating role.

Flavoalkaloids and Flavonoids from Astragalus monspessulanus.

A new flavonol tetraglycoside, quercetin-3-O-[α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-galactopyranosyl]-7-O-ß-D-glucopyranoside (1), and two new flavonol alkaloids, N-(8-methylquercetin-3-O-[α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-galactopyranosyl])-3-hydroxypiperidin-2-one (2) and N-(8-methylkaempferol-3-O-[α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-ß-D-galactopyranosyl])-3-hydroxypiperidin-2-one (3), were isolated from the aerial parts of Astragalus monspessulanus ssp. monspessulanus. Two rare flavonoids with an unusual 3-hydroxy-3-methylglutaric acid moiety, quercetin-3-O-α-L-rhamnopyranosyl-(1→2)-[6-O-(3-hydroxy-3-methylglutaryl)-ß-D-galactopyranoside (4) and kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-[6-O-(3-hydroxy-3-methylglutaryl)-ß-D-galactopyranoside (5), were isolated from the aerial parts of A. monspessulanus ssp. illyricus. In addition, the eight known flavonoids alangiflavoside (6), alcesefoliside (7), mauritianin (8), quercetin-3-ß-robinobioside (9), cosmosine (10), apigenin-4'-O-glucoside (11), trifolin (12), and rutin (13) were isolated from aerial parts of A. monspessulanus ssp. monspessulanus. Their structures were elucidated via NMR and HRESIMS data. In a model that tested t-BuOOH-induced oxidative stress on isolated rat hepatocytes, flavonoids 1-13 had statistically significant cytoprotective activity similar to that of silymarin, tested at 60 µg/mL. The most prominent effects were observed for flavonoids 1, 4, 7, and 12.

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6.

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/ß-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.

Polyyne hybrid compounds from Notopterygium incisum with peroxisome proliferator-activated receptor gamma agonistic effects.

In the search for peroxisome proliferator-activated receptor gamma (PPARγ) active constituents from the roots and rhizomes of Notopterygium incisum, 11 new polyacetylene derivatives (1-11) were isolated. Their structures were elucidated by NMR and HRESIMS as new polyyne hybrid molecules of falcarindiol with sesquiterpenoid or phenylpropanoid moieties, named notoethers A-H (1-8) and notoincisols A-C (9-11), respectively. Notoincisol B (10) and notoincisol C (11) represent two new carbon skeletons. When tested for PPARγ activation in a luciferase reporter assay with HEK-293 cells, notoethers A-C (1-3), notoincisol A (9), and notoincisol B (10) showed promising agonistic activity (EC50 values of 1.7 to 2.3 µM). In addition, notoincisol A (9) exhibited inhibitory activity on NO production of stimulated RAW 264.7 macrophages.

Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) deficiencies affect expression of lipolytic activities in mouse adipose tissues.

Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular degradation of triacylglycerols. It was the aim of this study to elucidate how the deficiency in one of these proteins affects the residual lipolytic proteome in adipose tissue. For this purpose, we compared the lipase patterns of brown and white adipose tissue from ATGL (-/-) and HSL (-/-) mice using differential activity-based gel electrophoresis. This method is based on activity-recognition probes possessing the same substrate analogous structure but carrying different fluorophores for specific detection of the enzyme patterns of two different tissues in one electrophoresis gel. We found that ATGL-deficiency in brown adipose tissue had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in brown adipose tissue. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in white adipose tissue. Enzyme activities of mouse tissues on acylglycerol substrates were analyzed as well, showing that ATGL-and HSL-deficiencies can be compensated for at least in part by other enzymes. The proteins that responded to ATGL-deficiency in brown adipose tissue were overexpressed and their activities on acylglycerols were analyzed. Among these enzymes, Es1, Es10, and Es31-like represent lipase candidates as they catalyze the hydrolysis of long-chain acylglycerols.

Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster α-Esterase-7.

Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such as xenobiotic detoxification, lipid metabolism and reproduction. Phylogenetically, many insect carboxylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene clusters such as the α-Esterase cluster. Much in contrast to the vital importance attributed to carboxylesterases in general, the in vivo function of individual α-Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fly Drosophila melanogaster fat body. One of the fat body carboxylesterases, α-Esterase-7, was selected for mutational analysis by gene targeting to generate a deletion mutant fly. Phenotypic characterization of α-Esterase-7 null mutants and transgenic flies, which overexpress a chimeric α-Esterase-7:EGFP gene, reveals important functions of α-Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The presented first deletion mutant of any α-Esterase in the model insect D. melanogaster generated by gene targeting not only provides experimental evidence for the endogenous functions of this gene family. It also offers an entry point for in vivo structure-function analyses of α-Esterase-7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species.

Absolute/relative bioavailability and metabolism of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (tetraenes) after intravenous and oral single doses to rats.

The present study assessed the absolute and relative bioavailabilities of dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides (tetraenes), the main bioactive constituents in Echinacea, administered as pure compounds or in the form of an Echinacea purpurea root extract preparation. Tetraenes were administered orally by gavage or intravenously in a dose of 0.75 mg/kg. The extract was administered orally in a dose of 158.6 mg/kg which corresponds to the same amount of tetraenes. Pharmacokinetic parameters of tetraenes were calculated by non-compartmental analysis using WinNonlin® 5.2 software. Mean dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamide dose-normalized plasma area under the concentration-time curve (AUC0-∞/dose) was 3.24 ± 0.32 min · ng/mL/µg and 0.95 ± 0.16 min · ng/mL/µg after iv and oral administrations, respectively, and 1.53 ± 0.18 min · ng/mL/µg after oral administration of the Echinacea root extract. The absolute oral bioavailability of dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides was 29.2 ± 2.3 %, which was increased to 47.1 ± 7.2 % (1.6-fold) by administration of the Echinacea extract. Administration of an Echinacea extract increased blood exposure with no impact on C(max), but prolonged the elimination half-life to 123.3 ± 15.7 min in comparison to 35.8 ± 6.5 min after administration of the pure dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides.

Development and validation of a LC-MS/MS method based on a new 96-well Hybrid-SPE™-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma.

A rapid and selective high-throughput HESI-LC-MS/MS method for determining eight cytochrome P450 probe drugs in one-step extraction and single run was developed and validated. The four specific probe substrates midazolam, dextromethorphan, tolbutamide, theophylline and their metabolites 1-hydroxymidazolam, dextrorphan, hydroxyl(methyl)tolbutamide, 1,3-dimethyluric acid, together with the deuterated internal standards, were extracted from rat plasma using a novel 96-well Hybrid-SPE™-precipitation technique. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11 min. The intra- and inter-day precisions for all eight substrates/metabolites were 1.62-12.81% and 2.09-13.02%, respectively, and the relative errors (accuracy) for the eight compounds ranged from -9.62% to 7.48% and -13.84% to 8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool for the evaluation of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2 and 2D6) activities with a cocktail approach in rats to clarify herb-drug interactions. The method can be used as a basic common validated high-throughput analytical assay for in vivo interaction studies.

15-Deoxy-Δ12,14-prostaglandin J2 induces Cox-2 expression in human osteosarcoma cells through MAPK and EGFR activation involving reactive oxygen species.

Prostaglandins (PGs), important modulators in bone biology, may also contribute to tumor formation and progression in human osteosarcoma. 15-Deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), a metabolite of PGD(2) and PPARγ-ligand, exerts a panel of biological activities via receptor-dependent and -independent mechanisms. As inducible cyclooxygenase-2 (Cox-2) is a candidate inflammatory marker in human osteosarcoma and a rate-limiting enzyme in PG biosynthesis, this study aimed at investigating intracellular redox status and signaling cascades leading to Cox-2 induction in human MG-63 osteosarcoma cells. 15d-PGJ(2) induced the accumulation of reactive oxygen species (ROS) that in turn may lead to upregulation of Cox-2 via two different routes in a PPARγ-independent manner. First, phosphorylation of p38 MAPK directly enhances Cox-2 expression by promoting mRNA stability. Second, 15d-PGJ(2) induces activation of epidermal growth factor receptors and downstream activation of Cox-2 via phosphorylation of p42/44 MAPK. Glutathione precursor molecules reversed enhanced ROS levels and Cox-2 expression. Functional activity of Cox-2 expression was tested by measurement of PGE(2) and PGF(2α). The synthetic compound 9,10-dihydro-15d-PGJ(2) lacking the α,ß-unsaturated carbonyl group in the cyclopentenone ring did not exhibit the cellular responses observed with 15d-PGJ(2). We conclude that the electrophilic carbon atom of 15d-PGJ(2) is responsible for alterations in intracellular redox status and Cox-2 expression.

Design, synthesis and antimycobacterial activities of 1-methyl-2-alkenyl-4(1H)-quinolones.

A series of 23 new 1-methyl-2-alkenyl-4(1H)quinolones have been synthesized and evaluated in vitro for their antimycobacterial activities against fast growing species of mycobacteria, such as Mycobacterium fortuitum, M. smegmatis and M. phlei. The compounds displayed good to excellent inhibition of the growth of the mycobacterial test strains with improved antimycobacterial activity compared to the hit compound, evocarpine. The most active compounds, which possessed chain length of 11-13 carbons at position-2 displayed potent inhibitory effects with an MIC value of 1.0mg/L. In a human diploid embryonic lung cell line, MRC-5 cytotoxicity assay, the alkaloids showed weak to moderate cytotoxic activity. Biological evaluation of these evocarpine analogues on the less pathogenic fast growing strains of mycobacteria showed an interesting antimycobacterial profile and provided significant insight into the structure-activity relationships.

Functional proteomic analysis of lipases and esterases in cultured human adipocytes.

This study reports on the analysis of the lipolytic proteome of cultured human fat cells. We used specific affinity tags to detect and identify the lipolytic and esterolytic enzymes in human subcutaneous (Sc) and visceral (Visc) adipocytes. For this purpose, differentiated fat cells were incubated with a fluorescent suicide inhibitor followed by protein separation using one- or two-dimensional gel electrophoresis. After detection by fluorescence laser scanning, the labeled proteins were tryptically digested and peptides were identified by mass spectrometry. In addition, a biotinylated probe was used for specific enzyme labeling with subsequent avidin affinity isolation of the tagged proteins. Finally, we determined the quantitative differences in protein expression levels between subcutaneous and visceral adipocytes using differential activity-based gel electrophoresis (DABGE). We found that the lipase/esterase patterns of both cell types are very similar, except for some proteins that were only found in Sc cells. Two novel enzyme candidates identified in this study were overexpressed and characterized using biologically relevant glycerolipid substrates in vitro. Both of them showed pronounced hydrolytic activities on hydrophobic acylglycerols and therefore may be considered lipases. The physiological functions of the novel lipolytic proteins in vivo are currently subject to investigation.

Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12 neurons in vitro.

Recent observations link myeloperoxidase (MPO) activation to neurodegeneration. In multiple sclerosis MPO is present in areas of active demyelination where the potent oxidant hypochlorous acid (HOCl), formed by MPO from H(2)O(2) and chloride ions, could oxidatively damage myelin-associated lipids. The purpose of this study was (i) to characterize reaction products of sphingomyelin (SM) formed in response to modification by HOCl, (ii) to define the impact of exogenously added SM and HOCl-modified SM (HOCl-SM) on viability parameters of a neuronal cell line (PC12), and (iii) to study alterations in the PC12 cell proteome in response to SM and HOCl-SM. MALDI-TOF-MS analyses revealed that HOCl, added as reagent or generated enzymatically, transforms SM into chlorinated species. On the cellular level HOCl-SM but not SM induced the formation of reactive oxygen species. HOCl-SM induced severely impaired cell viability, dissipation of the mitochondrial membrane potential, and activation of caspase-3 and DNA damage. Proteome analyses identified differential expression of specific subsets of proteins in response to SM and HOCl-SM. Our results demonstrate that HOCl modification of SM results in the generation of chlorinated lipid species with potent neurotoxic properties. Given the emerging connections between the MPO-H(2)O(2)-chloride axis and neurodegeneration, this chlorinating pathway might be implicated in neuropathogenesis.

The role of Cys108 in Trigonopsis variabilis d-amino acid oxidase examined through chemical oxidation studies and point mutations C108S and C108D.

Oxidative modification of Trigonopsis variabilis D-amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causing substantial loss of activity and thermostability of the enzyme. To simulate native and modified oxidase each as a microheterogeneity-resistant entity, we replaced Cys108 individually by a serine (C108S) and an aspartate (C108D), and characterized the purified variants with regard to their biochemical and kinetic properties, thermostability, and reactivity towards oxidation by hypochlorite. Tandem MS analysis of tryptic peptides derived from a hypochlorite-treated inactive preparation of recombinant wild-type oxidase showed that Cys108 was converted into cysteine sulfonic acid, mimicking the oxidative modification of native enzyme as isolated. Colorimetric titration of protein thiol groups revealed that in the presence of ammonium benzoate (0.12 mM), the two muteins were not oxidized at cysteines whereas in the wild-type enzyme, one thiol group was derivatized. Each site-directed replacement caused a conformational change in D-amino acid oxidase, detected with an assortment of probes, and resulted in a turnover number for the O2-dependent reaction with D-Met which in comparison with the corresponding wild-type value was decreased two- and threefold for C108S and C108D, respectively. Kinetic analysis of thermal denaturation at 50 degrees C was used to measure the relative contributions of partial unfolding and cofactor dissociation to the overall inactivation rate in each of the three enzymes. Unlike wild-type, C108S and C108D released the cofactor in a quasi-irreversible manner and were therefore not stabilized by external FAD against loss of activity. The results support a role of the anionic side chain of Cys108 in the fine-tuning of activity and stability of D-amino acid oxidase, explaining why C108S was a surprisingly poor mimic of the native enzyme.

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture.

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA(1), LPA(2), and LPA(3) on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.

Microwave-assisted high-throughput derivatization techniques utilizing silicon carbide microtiter platforms.

Parallel microwave-assisted gas chromatography (GC) derivatization protocols utilizing a silicon carbide (SiC)-based microtiter plate platform fitted with screw-capped GC vials were developed. For three selected standard derivatization protocols such as acetylation (exemplified for morphine), pentafluoropropionylation (for 6-monoacetylmorphine) and trimethylsilylation (for Delta(9)-tetrahydrocannabinol) complete derivatization was achieved within 5min at 100 degrees C in a dedicated multimode microwave instrument using online temperature monitoring. Microwave irradiation leads to rapid and homogeneous heating of the strongly microwave-absorbing SiC plate, with minimal deviations in the temperature recorded at different positions of the plate. The current platform allows the simultaneous derivatization of 80 reaction mixtures under strictly controlled temperature conditions. Similar results can also be obtained using a standard hotplate as heating source, although heating to the target temperature of 100 degrees C is slightly slower. The results demonstrate that parallel microwave derivatization procedures can significantly reduce the overall analysis time and increase sample throughput for GC-MS-based analytical methods.