Development, characterization, and application of flowing atmospheric-pressure afterglow ionization for mass spectrometric analysis of ambient organic aerosols

Addressing current limitations of state-of-the-art instrumentation in aerosol research, the aim of this work was to explore and assess the applicability of a novel soft ionization technique, namely flowing atmospheric-pressure afterglow (FAPA), for the mass spectrometric analysis of airborne particulate organic matter. Among other soft ionization methods, the FAPA ionization technique was developed in the last decade during the advent of ambient desorption/ionization mass spectrometry (ADI–MS). Based on a helium glow discharge plasma at atmospheric-pressure, excited helium species and primary reagent ions are generated which exit the discharge region through a capillary electrode, forming the so-called afterglow region where desorption and ionization of the analytes occurs. Commonly, fragmentation of the analytes during ionization is reported to occur only to a minimum extent, predominantly resulting in the formation of quasimolecular ions, i.e. [M+H]+ and [M–H]– in the positive and the negative ion mode, respectively. Thus, identification and detection of signals and their corresponding compounds is facilitated in the acquired mass spectra. The focus of the first part of this study lies on the application, characterization and assessment of FAPA–MS in the offline mode, i.e. desorption and ionization of the analytes from surfaces. Experiments in both positive and negative ion mode revealed ionization patterns for a variety of compound classes comprising alkanes, alcohols, aldehydes, ketones, carboxylic acids, organic peroxides, and alkaloids. Besides the always emphasized detection of quasimolecular ions, a broad range of signals for adducts and losses was found. Additionally, the capabilities and limitations of the technique were studied in three proof-of-principle applications. In general, the method showed to be best suited for polar analytes with high volatilities and low molecular weights, ideally containing nitrogen- and/or oxygen functionalities. However, for compounds with low vapor pressures, containing long carbon chains and/or high molecular weights, desorption and ionization is in direct competition with oxidation of the analytes, leading to the formation of adducts and oxidation products which impede a clear signal assignment in the acquired mass spectra. Nonetheless, FAPA–MS showed to be capable of detecting and identifying common limonene oxidation products in secondary OA (SOA) particles on a filter sample and, thus, is considered a suitable method for offline analysis of OA particles. In the second as well as the subsequent parts, FAPA–MS was applied online, i.e. for real time analysis of OA particles suspended in air. Therefore, the acronym AeroFAPA–MS (i.e. Aerosol FAPA–MS) was chosen to refer to this method. After optimization and characterization, the method was used to measure a range of model compounds and to evaluate typical ionization patterns in the positive and the negative ion mode. In addition, results from laboratory studies as well as from a field campaign in Central Europe (F–BEACh 2014) are presented and discussed. During the F–BEACh campaign AeroFAPA–MS was used in combination with complementary MS techniques, giving a comprehensive characterization of the sampled OA particles. For example, several common SOA marker compounds were identified in real time by MSn experiments, indicating that photochemically aged SOA particles were present during the campaign period. Moreover, AeroFAPA–MS was capable of detecting highly oxidized sulfur-containing compounds in the particle phase, presenting the first real-time measurements of this compound class. Further comparisons with data from other aerosol and gas-phase measurements suggest that both particulate sulfate as well as highly oxidized peroxyradicals in the gas phase might play a role during formation of these species. Besides applying AeroFAPA–MS for the analysis of aerosol particles, desorption processes of particles in the afterglow region were investigated in order to gain a more detailed understanding of the method. While during the previous measurements aerosol particles were pre-evaporated prior to AeroFAPA–MS analysis, in this part no external heat source was applied. Particle size distribution measurements before and after the AeroFAPA source revealed that only an interfacial layer of OA particles is desorbed and, thus, chemically characterized. For particles with initial diameters of 112 nm, desorption radii of 2.5–36.6 nm were found at discharge currents of 15–55 mA from these measurements. In addition, the method was applied for the analysis of laboratory-generated core-shell particles in a proof-of-principle study. As expected, predominantly compounds residing in the shell of the particles were desorbed and ionized with increasing probing depths, suggesting that AeroFAPA–MS might represent a promising technique for depth profiling of OA particles in future studies.

Xenobiotic metabolism: a view through the metabolometer

The combination of advanced ultraperformance liquid chromatography coupled with mass spectrometry, chemometrics, and genetically modified mice provide an attractive raft of technologies with which to examine the metabolism of xenobiotics. Here, a reexamination of the metabolism of the food mutagen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), the suspect carcinogen areca alkaloids (arecoline, arecaidine, and arecoline 1-oxide), the hormone supplement melatonin, and the metabolism of the experimental cancer therapeutic agent aminoflavone is presented. In all cases, the metabolic maps of the xenobiotics were considerably enlarged, providing new insights into their toxicology. The inclusion of transgenic mice permitted unequivocal attribution of individual and often novel metabolic pathways to particular enzymes. Last, a future perspective for xenobiotic metabolomics is discussed and its impact on the metabolome is described. The studies reviewed here are not specific to the mouse and can be adapted to study xenobiotic metabolism in any animal species, including humans. The view through the metabolometer is unique and visualizes a metabolic space that contains both established and unknown metabolites of a xenobiotic, thereby enhancing knowledge of their modes of toxic action.

Molecular structure of antibodies against small ligands

Antibodies which bind bioactive ligands can serve as a template for the generation of a second antibody which may react with the physiological receptor. This phenomenon of molecular mimicry by antibodies has been described in a variety of systems. In order to understand the chemical and molecular mechanisms involved in these interactions, monoclonal antibodies directed against two pharmacologically active alkaloids, morphine and nicotine, were carefully studied using experimental and theoretical molecular modeling techniques. The molecular characterization of these antibodies involved binding studies with ligand analogs and determination of the variable region amino acid sequence. A three-dimensional model of the anti-morphine binding site was constructed using computational and graphics display techniques. The antibody response in BALB/c mice to morphine appears relatively restricted, in that all of the antibodies examined in this study contained a $\lambda$ light chain, which is normally found in only 5% of mouse immunoglobulins. This study represents the first use of theoretical and experimental modeling techniques to describe the antigen binding site of a mouse Fv region containing a $\lambda$ light chain. The binding site model indicates that a charged glutamic acid residue and aromatic side chains are key features in ionic and hydrophobic interactions with the ligand morphine. A glutamic acid residue is found in the identical position in the anti-nicotine antibody and may play a role in binding nicotine. ^

Effect of a Traditional Processing Method on the Chemical Composition of Local White Lupin (Lupinus albus L.) Seed in North-Western Ethiopia

The effect of a traditional Ethiopian lupin processing method on the chemical composition of lupin seed samples was studied. Two sampling districts, namely Mecha and Sekela, representing the mid- and high-altitude areas of north-western Ethiopia, respectively, were randomly selected. Different types of traditionally processed and marketed lupin seed samples (raw, roasted, and fi nished) were collected in six replications from each district. Raw samples are unprocessed, and roasted samples are roasted using fi rewood. Finished samples are those ready for human consumption as snack. Thousand seed weight for raw and roasted samples within a study district was similar (P > 0.05), but it was lower (P < 0.01) for fi nished samples compared to raw and roasted samples. The crude fi bre content of fi nished lupin seed sample from Mecha was lower (P < 0.01) than that of raw and roasted samples. However, the different lupin samples from Sekela had similar crude fi bre content (P > 0.05). The crude protein and crude fat contents of fi nished samples within a study district were higher (P < 0.01) than those of raw and roasted samples, respectively. Roasting had no effect on the crude protein content of lupin seed samples. The crude ash content of raw and roasted lupin samples within a study district was higher (P < 0.01) than that of fi nished lupin samples of the respective study districts. The content of quinolizidine alkaloids of fi nished lupin samples was lower than that of raw and roasted samples. There was also an interaction effect between location and lupin sample type. The traditional processing method of lupin seeds in Ethiopia has a positive contribution improving the crude protein and crude fat content, and lowering the alkaloid content of the fi nished product. The study showed the possibility of adopting the traditional processing method to process bitter white lupin for the use as protein supplement in livestock feed in Ethiopia, but further work has to be done on the processing method and animal evaluation.

A New Family of High-Affinity Transporters for Adenine, Cytosine, and Purine Derivatives in Arabidopsis

In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant Ki = 20 to 35 μM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.

Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness

The G protein-coupled μ-opioid receptor (μOR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of μOR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate μOR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the μOR. Although both etorphine and morphine effectively activate the μOR, only etorphine elicits robust μOR phosphorylation followed by plasma membrane translocation of β-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause μOR internalization, morphine is unable to either elicit μOR phosphorylation or stimulate β-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce μOR phosphorylation, accompanied by the rescue of β-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote μOR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.

Female choice increases offspring fitness in an arctiid moth (Utetheisa ornatrix)

In Utetheisa ornatrix (Lepidoptera, Arctiidae), the female mates preferentially with larger males. Having a larger father results in the eggs being more richly endowed with defensive pyrrolizidine alkaloid (which the female receives from the male with the sperm package, in quantity proportional to the male's body mass, and passes on to the eggs); having a larger father also results in the sons and daughters themselves being larger (body mass is heritable in Utetheisa). We provide evidence herein that these consequences enhance the fitness of the offspring. Eggs sired by larger males are less vulnerable to predation (presumably because of their higher alkaloid content), whereas sons and daughters, by virtue of being larger, are, respectively, more successful in courtship and more fecund. The female Utetheisa, therefore, by being choosy, reaps both direct phenotypic and indirect genetic benefits.

Nature knows best: An amazing reaction cascade is uncovered by design and discovery

The Daphniphyllum alkaloids are a group of highly complex polycyclic alkaloids. Examination of the structures if several members of this family of natural products led to a hypothesis about their mode of biosynthesis (depicted in Scheme SI). Based on this hypothetical biosynthetic pathway, a laboratory synthesis was designed that incorporated as a key transformation the novel one-pot transformation of dialdehyde 24 to pentacyclic unsaturated amine 25. This process turned out to be an exceptionally efficient way to construct the pentacyclic nucleus of the Daphniphyllum alkaloids. However, a purely fortuitous discovery, resulting from accidental use of methylamine rather than ammonia, led to a great improvement in the synthesis and suggests an even more attractive possible biosynthesis.

Synergy in a medicinal plant: Antimicrobial action of berberine potentiated by 5′-methoxyhydnocarpin, a multidrug pump inhibitor

Multidrug resistance pumps (MDRs) protect microbial cells from both synthetic and natural antimicrobials. Amphipathic cations are preferred substrates of MDRs. Berberine alkaloids, which are cationic antimicrobials produced by a variety of plants, are readily extruded by MDRs. Several Berberis medicinal plants producing berberine were found also to synthesize an inhibitor of the NorA MDR pump of a human pathogen Staphylococcus aureus. The inhibitor was identified as 5′-methoxyhydnocarpin (5′-MHC), previously reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy. 5′-MHC is an amphipathic weak acid and is distinctly different from the cationic substrates of NorA. 5′-MHC had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of ethidium bromide and berberine from S. aureus cells was completely inhibited by 5′-MHC. The level of accumulation of berberine in the cells was increased strongly in the presence of 5′-MHC, indicating that this plant compound effectively disabled the bacterial resistance mechanism against the berberine antimicrobial.

Shifts of Intracellular pH Distribution as a Part of the Signal Mechanism Leading to the Elicitation of Benzophenanthridine Alkaloids1 : Phytoalexin Biosynthesis in Cultured Cells of Eschscholtzia californica

Cultured cells of Eschscholtzia californica (Californian poppy) respond to a yeast elicitor preparation or Penicillium cyclopium spores with the production of benzophenanthridine alkaloids, which are potent phytoalexins. Confocal pH mapping with the probe carboxy-seminaphthorhodafluor-1-acetoxymethylester revealed characteristic shifts of the pH distribution in challenged cells: within a few minutes after elicitor contact a transient acidification of cytoplasmic and nuclear areas occurred in parallel with an increase of the vacuolar pH. The change of proton concentration in the vacuole and in the extravacuolar area showed a nearly constant relation, indicating an efflux of vacuolar protons into the cytosol. A 10-min treatment with 2 mm butyric or pivalic acid caused a transient acidification of the cytoplasm comparable to that observed after elicitor contact and also induced alkaloid biosynthesis. Experimental depletion of the vacuolar proton pool reversibly prevented both the elicitor-triggered pH shifts and the induction of alkaloid biosynthesis. pH shifts and induction of alkaloid biosynthesis showed a similar dependence on the elicitor concentration. Net efflux of K+, alkalinization of the outer medium, and browning of the cells were evoked only at higher elicitor concentrations. We suggest that transient acidification of the cytoplasm via efflux of vacuolar protons is both a necessary and sufficient step in the signal path toward biosynthesis of benzophenanthridine alkaloids in Californian poppy cells.

Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine Decarboxylase Promoters from Opium Poppy Are Conserved in Transgenic Tobacco1

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus1

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.

Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.

Análise química e atividades biológicas de Guatteria elliptica R. E. Fries (Annonaceae)

A espécie endêmica G. elliptica R. E. Fries não apresentava estudos fitoquímicos e biológicos detalhados na literatura. Assim, o objetivo desse trabalho foi avaliar a composição química e as propriedades biológicas dos óleos essenciais, extratos brutos, alcaloides totais, tortas, frações das tortas, amostras isoladas dessa espécie. O material vegetal foi coletado em Paranapiacaba (Santo André, SP, Brasil). O óleo essencial extraído das folhas por destilação à vapor apresentou um rendimento de 0,2%. A análise histológica das folhas encontrou óleo em células oleíferas localizadas no parênquima esponjoso. A composição do óleo (CG-EM) indicou espatulenol e óxido de cariofileno como compostos majoritários. Os alcaloides totais foram obtidos dos extratos brutos das folhas e dos galhos e analisados por CG-EM, identificando quatro aporfinas (nornuciferina, estefarina, corituberina e asimilobina) e duas protoberberinas (discretamina e caseadina). Os alcaloides totais foram fracionados em coluna cromatográfica ou por Extração em Fase Sólida e purificados por cromatografia em camada preparativa, originando duas amostras (Amostra 9 e 10). Na Amostra 9, foram identificados dois alcaloides aporfinicos nornuciferina e asimilobina (CG-EM e RMN-1H). Na Amostra 10, foram identificados (LC-EM/EM) cinco alcaloides aporfínicos (desidronantenina, glaunidina, liriodenina, N-óxido de oliverina e telikovina) e um alcaloide protoberberínico (caseadina). Caseadina, glaunidina, N-óxido de oliverina e telikovina não foram previamente identificados em Guatteria. Os resíduos dos extratos brutos livres de alcaloides foram fracionados pelo método de partição com solventes de polaridade crescente. Os extratos brutos e as frações acetato de etila e butanólicas de folhas e galhos apresentaram flavonoides (NP-PEG). Nos ensaios biológicos, a melhor atividade antioxidante (sequestro do radical DPPH) foi encontrada para a fração clorofórmica dos galhos (EC50=24,25±1,14 µg/mL) e a torta dos galhos (EC50=26,23±4,20 µg/mL). No ensaio antimicrobiano pelo método turbidimétrico a atividade mais importante foi obtida contra Staphylococcus aureus (ATCC 6538) para os alcaloides totais dos galhos (CIM/CBM=0,12±0,01/0,26 mg/mL) e das folhas (CIM/CBM=0,21±0,01/0,28 mg/mL), e fração hexânica das folhas (CIM/CBM=0,24±0,02/>1 mg/mL). Uma alta atividade antitumoral foi observada frente a células humanas de mama (MCF-7) para Amostra 10 (IC50=2,28±0,18 µg/mL), fração de acetato de etila das folhas (IC50=4,47±0,40 µg/mL), óleo essencial (IC50=7,01±0,23 µg/mL) e os alcaloides totais das folhas (IC50=9,32±0,36 µg/mL). Para as células de próstata (PC-3), foi encontrada atividade para a Amostra 10 (IC50=1,37±0,36 µg/mL) e o óleo essencial (IC50=5,32±0,35 µg/mL). A futura aplicação dos extratos e frações de G. elliptica como um agente antitumoral parece ser segura, pois mantiveram uma viabilidade celular maior do que 90% no ensaio de citotoxicidade com culturas de fibroblasto murino (BALB/c 3T3, ATCC CCL-163) nas concentrações onde a atividade antitumoral foi promissora (<30 µg/mL) contra MCF-7 e/ou PC-3.