Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.     

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca²⁺ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.     

PROTEOMER: A workflow‐optimized laboratory information management system for 2‐D electrophoresis‐centered proteomics

In recent years proteomics became increasingly important to functional genomics. Although a large amount of data is generated by high throughput large‐scale techniques, a connection of these mostly heterogeneous data from different analytical platforms and of different experiments is limited. Data mining procedures and algorithms are often insufficient to extract meaningful results from large datasets and therefore limit the exploitation of the generated biological information. In our proteomic core facility, which almost exclusively focuses on 2‐DE/MS‐based proteomics, we developed a proteomic database custom tailored to our needs aiming at connecting MS protein identification information to 2‐DE derived protein expression profiles. The tools developed should not only enable an automatic evaluation of single experiments, but also link multiple 2‐DE experiments with MS‐data on different levels and thereby helping to create a comprehensive network of our proteomics data. Therefore the key feature of our “PROTEOMER” database is its high cross‐referencing capacity, enabling integration of a wide range of experimental data. To illustrate the workflow and utility of the system, two practical examples are provided to demonstrate that proper data cross‐referencing can transform information into biological knowledge.     

Mass Spectrometric Identification of 13C‐Labeled Metabolites During Anaerobic Propanoic Acid Oxidation

Biowaste digestion is a possibility to gain biogas as a renewable fuel source. However, the anaerobic food chain may be disrupted by, e.g., substrate overload or by inhibitors, leading to the accumulation of volatile fatty acids (VFAs), predominantly of propanoic acid (PA). VFA Accumulation may cause a rapid pH decrease, less biogas production, or even a total inhibition. To maintain high biogas productivity or to prevent a collapse of methanogenesis, metabolic properties of the degrading microorganisms must be elucidated, e.g., by investigation of the established pathways for degradation of VFAs. A Dani 3950 headspace system (HS), a Varian 431 gas chromatograph (GC), and a Varian 210 mass spectrometer (MS) have been combined to quantify and specifically identify metabolites of PA oxidation. The use of [1‐¹³C]‐labeled PA as a carbon source for microorganisms allows differentiation between the methyl‐malonyl‐CoA or the C(6)‐dismutation pathway, both resulting in AcOH production. Appearance of the ¹³C‐moiety either in the COO or Me group of AcO can easily be detected by MS. The methyl‐malonyl‐CoA pathway was successfully identified as the only pathway of PA degradation by organisms in a lab‐scale anaerobic digester. A similar approach can be applied to any degradation pathway involving VFAs.     

The Lan3‐2 glycoepitope of Hirudo medicinalis consists of β‐(1,4)‐linked mannopyranose

While glycosyltransferases are restrictively expressed in invertebrate model organisms, little is known of their glycan end products. One such restrictively expressed glycoepitope was localized to sensory and epithelial cells of leech and Caenorhabditis elegans using the Lan3‐2 monoclonal antibody. A biological function for the neural Lan3‐2 epitope was previously determined in the leech. Here we report on the chemical structure of this mannosidic epitope harvested from whole Hirudo medicinalis. Crude glycans were liberated from glycoproteins by hydrazinolysis. Re‐N‐acetylated glycans were subjected to immunoaffinity purification. The affinity‐purified glycans were fractioned by size chromatography into oligosaccharides and polysaccharides. Lan3‐2 oligosaccharide structure was characterized by gas chromatography of alditol acetates, methylation analysis, 500 MHz ¹H NMR spectroscopy, matrix‐assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem MS‐MS of permethylated derivatives. The predominant components of the Lan3‐2 oligosaccharide fraction were a series of linear β‐(1,4)‐linked mannose polymers. The homologous expression of the Lan3‐2 epitope in C. elegans will facilitate the exploration of its glycosylation pathway. Other invertebrates expressing the Lan3‐2 epitope are Planaria dugesia, Capitella sp. I and Lumbriculus variegatus. The glycoepitope was not detected in the diploblastic animals Hydra littoralis and Aptaisia sp. or in deuterostomes.     

Tyrosine phosphoproteome of hamster spermatozoa: Role of glycerol‐3‐phosphate dehydrogenase 2 in sperm capacitation

Capacitation confers on the spermatozoa the competence to fertilize the oocyte. At the molecular level, a cyclic adenosine monophosphate (cAMP) dependent protein tyrosine phosphorylation pathway operates in capacitated spermatozoa, thus resulting in tyrosine phosphorylation of specific proteins. Identification of these tyrosine‐phosphorylated proteins and their function with respect to hyperactivation and acrosome reaction, would unravel the molecular basis of capacitation. With this in view, 21 phosphotyrosine proteins have been identified in capacitated hamster spermatozoa out of which 11 did not identify with any known sperm protein. So, in the present study attempts have been made to ascertain the role of one of these eleven proteins namely glycerol‐3‐phosphate dehydrogenase 2 (GPD2) in hamster sperm capacitation. GPD2 is phosphorylated only in capacitated hamster spermatozoa and is noncanonically localized in the acrosome and principal piece in human, mouse, rat, and hamster spermatozoa, though in somatic cells it is localized in the mitochondria. This noncanonical localization may imply a role of GPD2 in acrosome reaction and hyperactivation. Further, enzymatic activity of GPD2 during capacitation correlates positively with hyperactivation and acrosome reaction thus demonstrating that GPD2 may be required for sperm capacitation.     

Mitsunobu Alkylation of Cancerostatic 5‐Fluorouridine with (2E)‐10‐Hydroxydec‐2‐enoic Acid,a Fatty Acid from Royal Jelly with Multiple Biological Activities

5‐Fluorouridine ( 1 ) – a nucleoside antimetabolite with strong cancerostatic properties – was protected i) at the 2′‐ and 3′‐OH groups with a heptan‐4‐ylidene residue and ii) at the 5′‐OH group with a (4‐methoxyphenyl)(diphenyl)methyl residue. This fully protected compound, 3 , was submitted to a Mitsunobu reaction with the N‐hydroxysuccinimide (NHS) ester, 5 , of (2E)‐10‐hydroxydec‐2‐enoic acid ( 4 ) which gave nucleolipid 6 . The latter was detritylated with Cl₂CHCOOH to yield the co‐drug 7 as NHS ester.     

Proteomic profiling of the prechylomicron transport vesicle involved in the assembly and secretion of apoB‐48‐containing chylomicrons in the intestinal enterocytes

Intracellular assembly of chylomicrons (CM) occurs in intestinal enterocytes through a series of complex vesicular interactions. CM are transported from the ER to the Golgi using a specialized vesicular compartment called the prechylomicron transport vesicle (PCTV). In this study, PCTVs were isolated from the enteric ER of the Syrian Golden hamster, and characterized using 2‐DE and MS. Proteomic profiles of PCTV‐associated proteins were developed with the intention of identifying proteins involved in the formation, transport, lipidation, and assembly of CM particles. Positively identified proteins included those involved in lipoprotein assembly, namely microsomal triglyceride transfer protein and apolipoprotein B‐48, as well as proteins involved in vesicular transport, such as Sar1 and vesicle‐associated membrane protein 7. Other groups of proteins found were chaperones, intracellular vesicular trafficking proteins, fatty acid‐binding proteins, and lipid‐related proteins. These findings have increased our understanding of the transport vesicle involved in the intracellular assembly and transport of CM and can provide insight into potential cellular factors responsible for dysregulation of intestinal CM production.     

2H‐Pyran‐2‐one and 2H‐Furan‐2‐one Derivatives from the Plant Endophytic Fungus Pestalotiopsis fici

Two new α‐pyrones (=2H‐pyran‐2‐ones), ficipyrones A and B ( 1 and 2 , resp.), and two new α‐furanones (=2H‐furan‐2‐ones), ficifuranones A and B ( 3 and 4 , resp.), together with three known metabolites, antibiotic F 0368 ( 5 ), hydroxyseiridin ( 6 ), and hydroxyisoseiridin ( 7 ), were isolated from solid cultures of the plant endophytic fungus Pestalotiopsis fici. Their structures were elucidated primarily by NMR spectroscopy, and the absolute configuration of 1 was deduced from the circular‐dichroism (CD) data. Compound 1 showed antifungal activity against the plant pathogen Gibberella zeae (CGMCC 3.2873) with an IC₅₀ value of 15.9 μM .     

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis‐related (PR) proteins. Endo‐β‐1,3‐glucanases (EGases) belong to the PR‐2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti‐P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR‐2 family from Vitis.     

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi‐functional platelets and cross‐species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high‐throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin‐stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across‐species evolutionary link that the orthologous proteins representing “core proteome”, and the “evolutionary proteome” is actually a relatively static proteome.     

Enantiopure 4‐oxazolin‐2‐ones and 4‐methylene‐2‐oxazolidinones as chiral building blocks in a divergent asymmetric synthesis of heterocycles

Enantiopure 3‐((R)‐ and 3‐((S)‐1‐phenylethyl)‐4‐oxazoline‐2‐ones were evaluated as chiral building blocks for the divergent construction of heterocycles with stereogenic quaternary centers. The N‐(R)‐ or N‐(S)‐1‐phenylethyl group of these compounds proved to be an efficient chiral auxiliary for the asymmetric induction of the 4‐ and 5‐positions of the 4‐oxazolin‐2‐one ring through thermal and MW‐promoted nucleophilic conjugated addition to Michael acceptors and alkyl halides. The resulting adducts were transformed via a cascade process into fused six‐membered carbo‐ and heterocycles. The structure of the reaction products depended on the electrophiles and reaction conditions used. Alternative isomeric 4‐methylene‐2‐oxazolidinones served as chiral precursors for a versatile and divergent approach to highly substituted cyclic carbamates. DFT quantum calculations showed that the formation of bicyclic pyranyl compounds was generated by a diastereoselective concerted hetero‐Diels‐Alder cycloaddition.     

Magnesium methoxide‐induced chemiluminescent decomposition of bicyclic dioxetanes bearing a 2′‐alkoxy‐2‐hydroxy‐1,1′‐binaphthyl‐7‐yl moiety

Bicyclic dioxetanes 2a–c bearing a 2′‐alkoxy‐2‐hydroxy‐1,1′‐binaphthyl‐7‐yl moiety were effectively synthesized and their base‐induced chemiluminescent decomposition was investigated by the use of alkaline metal (Na⁺ and K⁺) or Mg²⁺ alkoxide in MeOH. When 2a–c were treated with tetrabutylammonium fluoride (TBAF) in dimethyl sulfoxide (DMSO) as a reference system, they showed chemiluminescence as a flash of orange light (maximum wavelength λ_max^CL = 573–577 nm) with efficiency Φ^CL = 6–8 × 10^–2. On the other hand, for an alkaline metal (Na⁺ or K⁺) alkoxide/MeOH system, 2a–c decomposed slowly to emit a glow of chemiluminescence, the spectra of which were shifted slightly toward red from the TBAF/DMSO system, and Φ^CL (= 1.4–2.3 × 10^–3) was considerably decreased. In addition, Mg(OMe)₂ was found to play a characteristic role as a base for the chemiluminescent decomposition of 2a–c through coordination to the intermediary oxidoaryl‐substituted dioxetanes 13. Thus, Mg²⁺ increased Φ^CL to more than twice those with Na⁺ or K⁺, while it shifted λ_max^CL considerably toward blue (λ_max^CL = 550–566 nm). Copyright © 2012 John Wiley & Sons, Ltd.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis of dendrimer‐type chiral stationary phases based on the selector of (1S,2R)‐(+)‐2‐Amino‐1,2‐diphenylethanol derivate and their enantioseparation evaluation by HPLC

In our recent work, a series of dendritic chiral stationary phases (CSPs) were synthesized, in which the chiral selector was L‐2‐(p‐toluenesulfonamido)‐3‐phenylpropionyl chloride (selector I), and the CSP derived from three‐generation dendrimer showed the best separation ability. To further investigate the influence of the structures of dendrimer and chiral selector on enantioseparation ability, in this work, another series CSPs ( CSPs 1‐4 ) were prepared by immobilizing (1S,2R)‐1,2‐diphenyl‐2‐(3‐phenylureido)ethyl 4‐isocyanatophenylcarbamate (selector II) on one‐ to four‐generation dendrimers that were prepared in previous work. CSPs 1 and 4 demonstrated the equivalent enantioseparation ability. CSPs 2 and 3 showed the best and poorest enantioseparation ability respectively. Basically, these two series of CSPs exhibited the equivalent enantioseparation ability although the chiral selectors were different. Considering the enantioseparation ability of the CSP derived from aminated silica gel and selector II is much better than that of the one derived from aminated silica gel and selector I, it is believed that the dendrimer conformation essentially impacts enantioseparation. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Comparative proteomic profile of rat sciatic nerve and gastrocnemius muscle tissues in ageing by 2‐D DIGE

Ageing induces a progressive morphological change and functional decline in muscles and in nerves. Light and electron microscopy, 2‐D DIGE and MS, were applied to profile the qualitative and quantitative differences in the proteome and morphology of rat gastrocnemius muscle and sciatic nerve, in healthy 22‐month‐old rats. At muscle level, morphological changes are associated to fibre atrophy accompanied by myofibrillar loss and degeneration, disappearance of sarcomeres and sarcoplasmic reticulum dilatation, internal migration of nuclei, longitudinal fibre splitting, increment of subsarcolemmal mitochondria aggregates and increment of lipofuscin granules. Sciatic nerve shows myelin abnormalities like enfoldings, invaginations, onion bulbs, breakdowns and side axonal atrophy. Proteomic analysis identified changes correlated to morphological abnormalities in metabolic, contractile and cytoskeletal proteins, deregulation of iron homeostasis, change of Ca²⁺ balance and stress response proteins, accompanied by a deregulation of myelin membrane adhesion protein and proteins regulating the neuronal caliber. By comparing proteomic results from the two tissues, 16 protein isoforms showed the same up and down regulation trend suggesting that there are changes implying a general process which may act as a signal event of degeneration. Only β enolase and tropomyosin 1α were differentially expressed in the tissues.     

Synthesis of a novel fluorescent probe based on 7‐nitrobenzo‐2‐oxa‐1,3‐diazole skeleton for the rapid determination of vitamin B12 in pharmaceuticals

A new fluorescent probe, 4‐N,N‐di(2‐hydroxyethyl)imino‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (HINBD) was synthesized in a single step with reasonably good yield. The water‐soluble HINBD emits strongly in the visible region (λ_ex = 479 nm, λ_em = 545 nm) and is stable over a wide range of pH values. It was found that vitamin B₁₂ (VB₁₂) had the ability to quench the fluorescence of HINBD, and the quenched fluorescence intensity was proportional to the concentration of VB₁₂. A method for VB₁₂ determination based on the quenching fluorescence of HINBD was thus established. Interference effects of various substances, including sugars, vitamins, amino acids, inorganic cations and some organic substances have been studied. Under optimal conditions, the linear range is 0.0–2.4 × 10^–5 mol/L. The determination limit is 8.3 × 10^–8 mol/L. The method was applied to measure VB₁₂ in pharmaceutical preparations with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd.