New Pyridyl and Dihydroisoquinoline Alkaloids Isolated from the Chevron Nemertean Amphiporus angulatus.

Nemertean worms contain toxins that are used to paralyze their prey and to deter potential predators. Hoplonemerteans often contain pyridyl alkaloids like anabaseine that act through nicotinic acetylcholine receptors and crustacean chemoreceptors. The chemical reactivity of anabaseine, the first nemertean alkaloid to be identified, has been exploited to make drug candidates selective for alpha7 subtype nAChRs. GTS-21, a drug candidate based on the anabaseine scaffold, has pro-cognitive and anti-inflammatory actions in animal models. The circumpolar chevron hoplonemertean Amphiporus angulatus contains a multitude of pyridyl compounds with neurotoxic, anti-feeding, and anti-fouling activities. Here, we report the isolation and structural identification of five new compounds, doubling the number of pyridyl alkaloids known to occur in this species. One compound is an isomer of the tobacco alkaloid anatabine, another is a unique dihydroisoquinoline, and three are analogs of the tetrapyridyl nemertelline. The structural characteristics of these ten compounds suggest several possible pathways for their biosynthesis.

Discovery of the Nicotinic Receptor Toxin Anabaseine in a Polystiliferan Nemertean.

Nemerteans (also called Nemertines) are a phylum of predominantly marine worms that use toxins to capture prey and to defend themselves against predators. Hoplonemerteans have a proboscis armed with one or more stylets used in prey capture and are taxonomically divided into Order Monostilifera, whose members possess a single large proboscis stylet, and Order Polystilifera, whose members have multiple small stylets. Many monostiliferans contain alkaloidal toxins, including anabaseine, that stimulate and then desensitize nicotinic acetylcholine receptors that are present in all animals. These compounds also interact with pyridyl chemoreceptors in crustaceans, reducing predation and larval settlement. Anabaseine has been a lead compound in the design of alpha7 nicotinic acetylcholine receptor agonists like GTS-21 (also called DMXBA) to treat disorders of cognition such as Alzheimer's disease and schizophrenia. These drug candidates also display anti-inflammatory activities of potential medical importance. Most polystiliferans live deep in open oceans and are relatively inaccessible. We fortunately obtained two live specimens of a large benthic polystiliferan, Paradrepanophorus crassus (Pc), from the coast of Spain. MS and NMR analyses of the Ehrlich's reagent derivative allowed identification of anabaseine. A spectrophotometric assay for anabaseine, also based on its reaction with Ehrlich's reagent, revealed high concentrations of anabaseine in the body and proboscis. Apparently, the biosynthetic mechanism for producing anabaseine was acquired early in the evolution of the Hoplonemertea, before the monostiliferan-polystiliferan divergence.

Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic.

Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.

Nitrogen starvation-induced accumulation of triacylglycerol in the green algae: evidence for a role for ROC40, a transcription factor involved in circadian rhythm.

Microalgal triacylglycerol (TAG), a promising source of biofuel, is induced upon nitrogen starvation (-N), but the proteins and genes involved in this process are poorly known. We performed isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics to identify Chlorella proteins with modulated expression under short-term -N. Out of 1736 soluble proteins and 2187 membrane-associated proteins identified, 288 and 56, respectively, were differentially expressed under -N. Gene expression analysis on select genes confirmed the same direction of mRNA modulation for most proteins. The MYB-related transcription factor ROC40 was the most induced protein, with a 9.6-fold increase upon -N. In a previously generated Chlamydomonas mutant, gravimetric measurements of crude total lipids revealed that roc40 was impaired in its ability to increase the accumulation of TAG upon -N, and this phenotype was complemented when wild-type Roc40 was expressed. Results from radiotracer experiments were consistent with the roc40 mutant being comparable to the wild type in recycling membrane lipids to TAG but being impaired in additional de novo synthesis of TAG during -N stress. In this study we provide evidence to support the hypothesis that transcription factor ROC40 has a role in -N-induced lipid accumulation, and uncover multiple previously unknown proteins modulated by short-term -N in green algae.

Conversion of membrane lipid acyl groups to triacylglycerol and formation of lipid bodies upon nitrogen starvation in biofuel green algae Chlorella UTEX29.

Algal lipids are ideal biofuel sources. Our objective was to determine the contributors to triacylglycerol (TAG) accumulation and lipid body formation in Chlorella UTEX29 under nitrogen (N) deprivation. A fivefold increase in intracellular lipids following N starvation for 24 h confirmed the oleaginous characteristics of UTEX29. Ultrastructural studies revealed increased number of lipid bodies and decreased starch granules in N-starved cells compared to N-replete cells. Lipid bodies were observed as early as 3 h after N removal and plastids collapsed after 48 h of stress. Moreover, the identification of intracellular pyrenoids and differences in the expected nutritional requirements for Chlorella protothecoides (as UTEX29 is currently classified) led us to conduct a phylogenetic study using 18S and actin cDNA sequences. This indicated UTEX29 to be more phylogenetically related to Chlorella vulgaris. To investigate the fate of different lipids after N starvation, radiolabeling using ¹4C-acetate was used. A significant decrease in ¹4C-galactolipids and phospholipids matched the increase in ¹4C-TAG starting at 3 h of N starvation, consistent with acyl groups from structural lipids as sources for TAG under N starvation. These results have important implications for the identification of key steps controlling oil accumulation in N-starved biofuel algae and demonstrate membrane recycling during lipid body formation.

Polyphenol oxidase inhibitor from blue mussel (Mytilus edulis) extract.

UNLABELLED: Enzymatic browning remains a problem for the fruit and vegetable industry, especially new emerging markets like pre-cuts. A crude inhibitor from blue mussel (Mytilus edulis) showed broad inhibition for apple (58%), mushroom (32%), and potato (44%) polyphenol oxidase (PPO) and was further characterized. Inhibition increased as the concentration of inhibitor increased in the reaction mixture eventually leveling off at a maximum inhibition of 92% for apple PPO. The inhibitor was capable of bleaching the brown color formed in the reaction mixture with apple PPO. Identification of the inhibitor by mass spectrometry and high-performance liquid chromatography revealed it to be hypotaurine (C2 H7 NO2 S). Hypotaurine and other sulfinic acid analogs (methane and benzene sulfinic acids) showed very good inhibition for apple PPO at various concentrations with the highest inhibition occurring at 500 µM for hypotaurine (89%), methane sulfinic acid (100%), and benzene sulfinic acid (100%). PRACTICAL APPLICATION: An inhibitor found in the expressed liquid from blue mussel shows very good inhibition on enzymatic browning. Since this enzyme is responsible for losses to the fruit and vegetable industry, natural inhibitors that prevent browning would be valuable. Finding alternative chemistries that inhibit browning and understanding their mode of action would be beneficial to the fruit and vegetable industries and their segments such as pre-cuts, juices, and so on. Inhibitors from products ingested by consumers are more acceptable as natural ingredients.

Bilirubin present in diverse angiosperms.

BACKGROUND AND AIMS: Bilirubin is an orange-yellow tetrapyrrole produced from the breakdown of heme by mammals and some other vertebrates. Plants, algae and cyanobacteria synthesize molecules similar to bilirubin, including the protein-bound bilins and phytochromobilin which harvest or sense light. Recently, we discovered bilirubin in the arils of Strelitzia nicolai, the White Bird of Paradise Tree, which was the first example of this molecule in a higher plant. Subsequently, we identified bilirubin in both the arils and the flowers of Strelitzia reginae, the Bird of Paradise Flower. In the arils of both species, bilirubin is present as the primary pigment, and thus functions to produce colour. Previously, no tetrapyrroles were known to generate display colour in plants. We were therefore interested in determining whether bilirubin is broadly distributed in the plant kingdom and whether it contributes to colour in other species. METHODOLOGY: In this paper, we use HPLC/UV and HPLC/UV/electrospray ionization-tandem mass spectrometry (HPLC/UV/ESI-MS/MS) to search for bilirubin in 10 species across diverse angiosperm lineages. PRINCIPAL RESULTS: Bilirubin was present in eight species from the orders Zingiberales, Arecales and Myrtales, but only contributed to colour in species within the Strelitziaceae. CONCLUSIONS: The wide distribution of bilirubin in angiosperms indicates the need to re-assess some metabolic details of an important and universal biosynthetic pathway in plants, and further explore its evolutionary history and function. Although colour production was limited to the Strelitziaceae in this study, further sampling may indicate otherwise.

Benzotriazole-assisted solid-phase assembly of Leu-enkephalin, amyloid beta segment 34-42, and other "difficult" peptide sequences.

Microwave-assisted solid-phase syntheses of six "difficult" peptides, H-VVSVV-NH(2) (3), H-VVVSVV-NH(2) (4), H-VIVIG-OH (5), H-TVTVTV-NH(2) (6), H-VKDGYI-NH(2) (7), and H-VKDVYI-NH(2) (8), were achieved utilizing N-(Fmoc-alpha-aminoacyl)benzotriazoles. Extension to the syntheses of Leu-enkephalin (9) and amyloid-beta (34-42) (10) demonstrates that this strategy comprises an efficient route to new and known "difficult" peptides.

Fluorescent labeling of peptides on solid phase.

N(alpha)-Fmoc-N(epsilon)-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine (N(alpha)-Fmoc-L-Lys(Mca)-OH) 3 is conveniently prepared by benzotriazole methodology (52% over two steps). N-Acylbenzotriazoles Mca-Bt 2, N(alpha)-Fmoc-L-Lys(Mca)-Bt 4, coumarin-3-ylcarbonyl (Cc)-Bt 5, N(alpha)-Fmoc-L-Lys(Cc)-Bt 7 and N(alpha)-(Cc)-L-Lys(Fmoc)-Bt 9 enable the efficient microwave enhanced solid-phase fluorescent labeling of peptides.

Microwave-assisted solid-phase peptide synthesis utilizing N-Fmoc-protected (alpha-aminoacyl)benzotriazoles.

A novel microwave-assisted solid-phase peptide synthesis utilizing N-Fmoc-protected(alpha-aminoacyl)benzotriazoles was applied in the preparation of tri-, tetra-, penta-, hexa-, and heptapeptides in 71% average crude yield.

Degradation and effects of the potential mosquito larvicides methazolamide and acetazolamide in sheepshead minnow (Cyprinodon variegatus).

To test for environmental persistence in order to determine the potential of carbonic anhydrase inhibitors as larvicides, the decomposition and degradation of samples containing methazolamide (MTZ) and acetazolamide (ACZ) in aqueous solution were monitored under different conditions. Additionally, nontarget species impact was assessed in an acute toxicity test using sheepshead minnow (Cyprinodon variegatus). The fish were exposed for 120 h to 10(-3) and 10(-4) M each compound in replicate seawater tanks. In the high-MTZ treatment, all fish died within 48 h, while mortality in the low-MTZ treatment was 27% at 120 h. In the high-ACZ treatment mortality reached 83% at 120 h. We observed no mortality for the lowest dose of ACZ. Tissue samples were collected from the fish to investigate absorption of the compounds. In the gills, MTZ concentrations were around 40 microg g(-1) and ACZ reached concentrations up to 80 microg g(-1). Liver concentrations were low for MTZ probably due to metabolism.

Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC-ESI-MS.

Ellagic acid, ellagic acid glycosides, and ellagitannins found in various fruits and nuts, including muscadine grape, are reported to have potential health-promoting benefits and antioxidant properties. This study isolated and identified several ellagic acid derivatives present in muscadine grapes and determined their relative antioxidant properties (AOX). Compounds were extracted from grape skins and pulp using methanol, and the solvent was evaporated. Isolates were dissolved in citric acid buffer (pH 3.5) and absorbed onto C18 cartridges. Nonretained polyphenolics were collected separately and again partitioned from Sephadex LH-20, whereas retained polyphenolics were first eluted with ethyl acetate followed by methanol. Ellagic acid derivatives were identified on the basis of UV and mass spectra, and the presence of ellagitannins was confirmed by a significant increase in free ellagic acid with HPLC followed by acid hydrolysis. Muscadine grapes contained phenolic acids, flavonols, anthocyanins, ellagic acid, and numerous ellagic acid derivatives. AOX varied in the order ethyl acetate > methanol > C18 nonretained fractions; each correlated to both total phenolics (r = 0.90) and total ellagic acid (r = 0.99) contents. Results of this study revealed previously unidentified ellagic acid derivatives in muscadine grapes.

Mass spectral fragmentation of the intravenous anesthetic propofol and structurally related phenols.

Propofol (2,6-diisopropyl phenol) is a widely used intravenous anesthetic. To define its pharmacokinetics and pharmacodynamics, methods for its quantitation in biological matrixes have been developed, but its pattern of mass spectral fragmentation is unknown. We found that fragmentation of the [M - H](-) ion (m/z 177) of propofol in both APCI MS/MS and ESI MS/MS involves the stepwise loss of a methyl radical and a hydrogen radical from one isopropyl side chain to give the most intense product ion, [M -H - CH(4)](-), at m/z 161. This two-step process is also the preferred mode of fragmentation for similar branched alkyl substituted phenols. This mode of fragmentation of the [M - H](-) ion is supported by three independent lines of evidence: (1) the presence of the intermediary [M - H - CH(3)](-) radical ion under conditions of reduced collision energy, (2) the determination of the mass of the predominant [M - H - CH(4)](-) product ion by high resolution mass spectrometry, and (3) the pattern of product ions resulting from further fragmentation of the [M - H - CH(4)](-) product ion. Phenols with a single straight chain alkyl substituent, in contrast, undergo beta elimination of the alkyl radical irrespective of the length of the alkyl chain, yielding the most intense product ion at m/z 106. This product ion represents a special case of a stable intermediary radical for the two-step process described for branched side chains, because further elimination of a hydrogen radical from the beta carbon is not possible.

Simultaneous quantification of poly-dispersed anionic, amphoteric and nonionic surfactants in simulated wastewater samples using C18 high-performance liquid chromatography-quadrupole ion-trap mass spectrometry.

This paper describes the development of a guantitative method for direct and simultaneous determination of three frequently encountered surfactants, amphoteric (cocoamphoacetate, CAA), anionic (sodium laureth sulfate, SLES), and nonionic (alcohol ethoxylate, AE) using a reversed-phase C18 HPLC coupled with an ESI ion-trap mass spectrometer (MS). Chemical composition, ionization characteristics and fragmentation pathways of the surfactants are presented. Positive ESI was effective for all three surfactants in agueous methanol buffered with ammonium acetate. The method enables rapid determinations in small sample volumes containing inorganic salts (up to 3.5 g L(-1)) and multiple classes of surfactants with high specificity by applying surfactant specific tandem mass spectrometric strategies. It has dynamic linear ranges of 2-60, 1.5-40, 0.8-56 mg L(-1) with R2 egual or greater than 0.999, 0.98 and 0.999 (10 microL injection) for CAA, SLES, and AE, respectively.

Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries.

Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009; Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1' library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1'-Gln-(Xaa). In both cases, P1 and P1' were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1' residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatography-mass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1'-Phe pools revealed the favored amino acids at the P3, P2, P2', and P3' positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.

Methionine oxidation by peroxymonocarbonate, a reactive oxygen species formed from CO2/bicarbonate and hydrogen peroxide.

Kinetic and thermodynamic evidence is reported for the role of the peroxymonocarbonate ion, HCO4-, as a reactive oxygen species in biology. Peroxymonocarbonate results from the equilibrium reaction of hydrogen peroxide with bicarbonate via the perhydration of CO2. The kinetic parameters for HCO4- oxidation of free methionine have been obtained (k1 = 0.48 +/- 0.08 M(-1)s(-1) by a spectrophotometric initial rate method). At the physiological concentration of bicarbonate in blood ( approximately 25 mM), it is estimated that peroxymonocarbonate formed in equilibrium with hydrogen peroxide will oxidize methionine approximately 2-fold more rapidly than plasma H2O2 itself. As an example of methionine oxidation in proteins, the bicarbonate-catalyzed hydrogen peroxide oxidation of alpha1-proteinase inhibitor (alpha1-PI) has been investigated via its inhibitory effect on porcine pancreatic elastase activity. The second-order rate constant for HCO4- oxidation of alpha1-PI (0.36 +/- 0.06 M(-1)s(-1)) is comparable to that of free methionine, suggesting that methionine oxidation is occurring. Further evidence for methionine oxidation, specifically involving Met358 and Met351 of the alpha1-PI reactive center loop, has been obtained through amino acid analyses and mass spectroscopic analyses of proteolytic digests of the oxidized alpha1-PI. These results strongly suggest that HCO4- should be considered a reactive oxygen species in aerobic metabolism.

HPLC/ESI-quadrupole ion trap mass spectrometry for characterization and direct quantification of amphoteric and nonionic surfactants in aqueous samples.

An amphoteric (cocamidopropylbetaine, CAPB) and a nonionic (alcohol polyethoxylate, AE) surfactant were characterized by electrospray ionization quadrupole ion trap mass spectrometry (ESI-MS) as to their homologue distribution and ionization/fragmentation chemistry. Quantitative methods involving reversed-phase gradient HPLC and (+)ESI-MSn were developed to directly determine these surfactants in hydroponic plant growth medium that received simulated graywater. The predominant homologues, 12 C alkyl CAPB and 9 EO AE, were monitored to represent the total amount of the respective surfactants. The methods demonstrated dynamic linear ranges of 0.5-250 ng (r2 > 0.996) for CAPB and 8-560 ng (r2 > 0.998) for AE homologue mixture, corresponding to minimum quantification limits of 25 ppb CAPB and 0.4 ppm AE with 20-microL injections. This translated into an even lower limit for individual components due to the polydispersive nature of the surfactants. The procedure was successfully employed for the assessment of CAPB and AE biodegradation in a hydroponic plant growth system used as a graywater bioreactor.

Intestinal and hepatic microsomal metabolism of testosterone and progesterone by a 3 alpha-hydroxysteroid dehydrogenase to the 3 alpha-hydroxy derivatives in the channel catfish, Ictalurus punctatus.

Intestinal or hepatic microsomes from channel catfish converted [4-14C]-testosterone to three major metabolites: 6 beta-hydroxytestosterone, androstenedione and a third metabolite. Formation of the unknown metabolite required NADPH as cofactor. When incubated with 200 microM testosterone, the rate of formation of the unknown metabolite was 265+/-158 pmol/(min mg) protein (mean+/-S.D.) in microsomes from the proximal intestine, 515+/-93 pmol/(min mg) protein in distal intestine and 226+/-42 pmol/(min mg) protein in hepatic microsomes. Comparison of the chromatographic and spectral properties of the unknown metabolite with those of authentic testosterone derivatives showed that this metabolite corresponded to 4-androstene-3 alpha,17 beta-diol. No 3 alpha-reduced metabolite was formed in incubations of testosterone with catfish intestinal cytosol. Testosterone was reduced to 5 alpha-dihydrotestosterone primarily in the cytosolic fraction and not in microsomes. Incubation of progesterone with intestinal microsomes resulted in the formation of a metabolite with properties similar to that of the 3 alpha-reduced testosterone, and this metabolite was identified by co-chromatography with authentic standard as 3 alpha-reduced progesterone. Thus, 3 alpha-hydroxysteroid dehydrogenase is an important pathway in intestinal microsomes of the channel catfish.