Biosynthesis and isolation of selenoneine from genetically modified fission yeast.

Selenoneine, a naturally occurring form of selenium, is the selenium analogue of ergothioneine, a sulfur species with health relevance not only as a purported antioxidant but likely also beyond. Selenoneine has been speculated to exhibit similar effects. To study selenoneine's health properties as well as its metabolic transformation, the pure compound is required. Chemical synthesis of selenoneine, however, is challenging and biosynthetic approaches have been sought. We herein report the biosynthesis and isolation of selenoneine from genetically modified fission yeast Schizosaccharomyces pombe grown in a medium containing sodium selenate. After cell lysis and extraction with methanol, selenoneine was purified by three consecutive preparative reversed-phase HPLC steps. The product obtained at the mg level was characterised by high resolution mass spectrometry, NMR and HPLC/ICPMS. Biosynthesis was found to be a promising alternative to chemical synthesis, and should be suitable for upscaling to produce higher amounts of this important selenium species in the future.

Homoarsenocholine - A novel arsenic compound detected for the first time in nature.

The arsenic speciation was determined in macrofungi of the Ramaria genus with HPLC coupled to inductively coupled plasma mass spectrometry. Besides arsenic species that are already known for macrofungi, like arsenobetaine or arsenocholine, two compounds that were only known from marine samples so far (trimethylarsoniopropanate and dimethylarsinoylacetate) were found for the first time in a terrestrial sample. An unknown arsenical was isolated and identified as homoarsenocholine. This could be a key intermediate for further elucidation of the biotransformation mechanisms of arsenic.

Arsenolipid biosynthesis by the unicellular alga Dunaliella tertiolecta is influenced by As/P ratio in culture experiments.

The influence of arsenate and phosphate levels in water on the formation of arsenic-containing lipids (arsenolipids) and water-soluble arsenicals by a unicellular marine alga was investigated by exposing Dunaliella tertiolecta to five regimes of arsenic and phosphate, and determining the biosynthesized organoarsenicals with HPLC/mass spectrometry. Under all conditions, the major arsenolipid produced by D. tertiolecta was the novel phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside (AsSugPhytol546) representing ca. 35-65% of total arsenolipids. The new compound contains a phytol aglycone and a methoxy group replacing a sugar hydroxyl - two structural features not previously observed for arsenolipids. Minor arsenolipids were several previously reported arsenosugar phospholipids (AsSugPLs, in particular AsSugPL958 and the previously unknown AsSugPL978), the relative quantities of which increased with increasing phosphate exposure, and an arsenic-containing hydrocarbon (AsHC360), which remained unaffected by the different treatments. The relative amount of total arsenolipids produced by D. tertiolecta remained remarkably constant (ca. 45% of total As) and independent of the culture conditions. In contrast, with rising As-concentrations we observed an increase of hydrophilic arsenicals, which were dominated by arsenate and arsenosugars. The results highlight a possible major difference in arsenic biochemistry between macroalgae and unicellular algae with potential implications for how various algae handle their natural arsenic exposure in the world's oceans.

Mono-acyl arsenosugar phospholipids in the edible brown alga Kombu (Saccharina japonica).

Twenty one arsenolipids, including eight new compounds (AsSugPL 692, AsSugPL 706, AsSugPL 720, AsSugPL 734, AsSugPL 742, AsSugPL 746, AsSugPL 748, and AsSugPL 776) were identified in the edible brown alga Kombu, Saccharina japonica, by means of HPLC coupled with elemental and molecular mass spectrometry. The hitherto undescribed compounds are all mono-acyl arsenosugar phospholipids, differing from previously reported natural arsenic-containing phospholipids by containing only one fatty acid on the glycerol group. Collectively, this new group of mono-acyl compounds constituted about 30% of total lipid arsenic; other significant groups were the di-acyl arsenosugar phospholipids (50%) and arsenic hydrocarbons (20%). The origin and relevance of the mono-acyl arsenosugar phospholipids in Kombu, a commercial seafood product, is briefly discussed.

A 2-O-Methylriboside Unknown Outside the RNA World Contains Arsenic.

Lipid-soluble arsenic compounds, also called arsenolipids, are ubiquitous marine natural products of currently unknown origin and function. In our search for clues about the possible biological roles of these compounds, we investigated arsenic metabolism in the unicellular green alga Dunaliella tertiolecta, and discovered an arsenolipid fundamentally different from all those previously identified; namely, a phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside. The discovery is of particular interest because 2-O-methylribosides have, until now, only been found in RNA. We briefly discuss the significance of the new lipid in biosynthesis and arsenic biogeochemical cycling.

Arsenic-Containing Phosphatidylcholines: A New Group of Arsenolipids Discovered in Herring Caviar.

A new group of arsenolipids based on cell-membrane phosphatidylcholines has been discovered in herring caviar (fish roe). A combination of HPLC with elemental and molecular mass spectrometry was used to identify five arsenic-containing phosphatidylcholines; the same technique applied to salmon caviar identified an arsenic-containing phosphatidylethanolamine. The arsenic group in these membrane lipids might impart particular properties to the molecules not displayed by their non-arsenic analogues. Additionally, the new compounds have human health implications according to recent results showing high cytotoxicity for some arsenolipids.

Investigating the intra-individual variability in the human metabolic profile of urinary selenium.

Selenium is an essential micronutrient widely present in our diet. It plays its role through the selenoproteins. Previous reports have shown marked variation between individuals in the excretion of this trace element, but the intra-individual variability in selenium excretion has not been specifically investigated. The present study investigates the intra-individual variation in the urinary excretion of selenium in a group of healthy volunteers. We also discuss inter-individual variability trends. Urine samples were collected from healthy volunteers without selenium supplementation twice a day for 7 days and then once a week for an additional 7 weeks. A total of 168 urine samples were collected and analyzed for total selenium and individual selenium species using elemental mass spectrometry and HPLC/mass spectrometry, respectively. We found only modest day-to-day and week-to-week intra-individual variation of selenium excretion. Two commonly reported urine metabolites, selenosugar 1 and selenosugar 3, were detected in all urine samples, and our data suggest that selenosugar 3 is a deacetylated product of selenosugar 1 produced in a manner dependent on selenium intake. Trimethylselenonium displayed no intra-individual variability but considerable inter-individual variability in agreement with the involvement of genetic polymorphisms, as recently reported. Se-methylselenoneine was consistently detected in the urine of all volunteers and was a significant metabolite in one volunteer contributing up to 24% of total urinary selenium. Our data indicate that selenium urinary excretion is consistent within an individual, and that intra-individual variation in selenium excretion is unlikely to complicate future inter-individual variation studies.

A method for determining arsenolipids in seawater by HPLC-high resolution mass spectrometry.

Arsenic-containing lipids (arsenolipids), naturally occurring arsenicals in algae, have never been detected in seawater even though they might be introduced to the water column on senescence of marine algae or by active excretion. The complex nature of seawater presents an analytical challenge to detect these compounds and to monitor their environmental fate. We developed a simple sample preparation method using liquid-liquid extraction combined with HPLC-high resolution mass spectrometry (HRMS) capable of measuring six standard arsenolipids in seawater at the ng As/L level (<1% of the total arsenic in seawater). The method is suitable for studies on the biotransformation and pathways of arsenolipids in the marine environment. When we applied the method to four samples of natural seawater, however, we did not find any of the six standard arsenolipids.

Arsenic-Containing Phosphatidylcholines: A New Group of Arsenolipids Discovered in Herring Caviar.

A new group of arsenolipids based on cell-membrane phosphatidylcholines has been discovered in herring caviar (fish roe). A combination of HPLC with elemental and molecular mass spectrometry was used to identify five arsenic-containing phosphatidylcholines; the same technique applied to salmon caviar identified an arsenic-containing phosphatidylethanolamine. The arsenic group in these membrane lipids might impart particular properties to the molecules not displayed by their non-arsenic analogues. Additionally, the new compounds have human health implications according to recent results showing high cytotoxicity for some arsenolipids.

Human excretory products of selenium are natural constituents of marine fish muscle.

A selenosugar (selenosugar 1, methyl-2-acetamido-2-deoxy-1-seleno-ß-D-galactopyranoside) was identified in aqueous extracts of muscle tissue of three marine fish species, mackerel (Scomber scombrus), sardine (Sardina pilchardus), and tuna (Thunnus albacares), by high-performance liquid chromatography coupled to elemental and high-resolution molecular mass spectrometry. Selenoneine (2-selenyl-Nα, Nα, Nα-trimethyl-L-histidine), a known selenium compound in fish, was the major form of selenium in the aqueous extracts, and the methylated derivative of selenoneine, namely Se-methylselenoneine, was also identified as a minor natural constituent in the fish. Selenosugar 1, a major urinary excretion product of selenium often found in organs and body fluids related to selenium excretion, has so far not been reported in muscle tissue. Se-methylselenoneine has been proposed as the main urinary metabolite from selenoneine. This first report of selenosugar 1 and Se-methylselenoneine as natural constituents of fish muscle tissue opens up a new perspective on the role of these compounds in selenium metabolism and is relevant to selenium supplementation studies.

Arsenolipids in oil from blue whiting Micromesistius poutassou--evidence for arsenic-containing esters.

Arsenic-containing lipids in the oil from the blue whiting fish (Micromesistius poutassou) were separated into three broad polarity groups and investigated by HPLC and mass spectrometry. A total of 11 arsenolipids including 4 new compounds were identified. The polar lipid fraction constituting 24% of the total arsenolipid content (which totalled 2.16 µg As/g) contained four known dimethylarsinoyl fatty acids and three known dimethylarsinoyl hydrocarbons. The less polar fraction (ca 30% of the total arsenolipids) contained four new dimethylarsinoyl hydrocarbons with chain lengths 22-30 carbons, in addition to more complex arsenicals that hydrolysed to known dimethylarsinoyl fatty acids suggesting they were conjugated carboxylic acids, presumably esters. The rest of the lipid-soluble arsenic (ca 45% of the total) remained in the non-polar fraction together with the bulk of the fish oil lipids, a complex mixture of compounds that precluded identification of the small amounts of arsenolipids.

Quantification of arsenolipids in the certified reference material NMIJ 7405-a (Hijiki) using HPLC/mass spectrometry after chemical derivatization.

Arsenic-containing lipids (arsenolipids) are novel natural products recently shown to be widespread in marine animals and algae. Research interest in these arsenic compounds lies in their possible role in the membrane chemistry of organisms and, because they occur in many popular seafoods, their human metabolism and toxicology. Progress has been restricted, however, by the lack of standard arsenolipids and of a quantitative method for their analysis. We report that the certified reference material CRM 7405-a (Hijiki) is a rich source of arsenolipids, and we describe a method based on HPLC-ICPMS/ESMS to quantitatively measure seven of the major arsenolipids present. Sample preparation involved extraction with DCM/methanol, a cleanup step with silica, and conversion of the (oxo)arsenolipids originally present to thio analogues by brief treatment with H2S. Compared to their oxo analogues, the thioarsenolipids showed much sharper peaks on reversed-phase HPLC, which facilitated their resolution and quantification. The compounds were determined by HPLC-ICPMS and HPLC-ESMS, which provided both arsenic-selective detection and high resolution molecular mass detection of the arsenolipids. In this way, the concentrations of two arsenic-containing hydrocarbons and five arsenosugar phospholipids are reported in the CRM Hijiki. This material may serve as a convenient source of characterized arsenolipids to delineate the presence of these compounds in seafoods and to facilitate research in a new era of arsenic biochemistry.

Synthesis and Characterization of Arsenolipids: Naturally Occurring Arsenic Compounds in Fish and Algae.

Arsenic-containing lipids (arsenolipids) are natural products present in fish and algae. Because these compounds occur in foods, there is considerable interest in their human toxicology. We report the synthesis and characterization of seven arsenic-containing lipids, including six natural products. The compounds comprise dimethylarsinyl groups attached to saturated long-chain hydrocarbons (three compounds), saturated long-chain fatty acids (two compounds), and monounsaturated long chain fatty acids (two compounds). The arsenic group was introduced through sodium dimethylarsenide or bis(dimethylarsenic) oxide. The latter route provided higher and more reproducible yields, and consequently, this pathway was followed to synthesize six of the seven compounds. Mass spectral properties are described to assist in the identification of these compounds in natural samples. The pure synthesized arsenolipids will be used for in vitro experiments with human cells to test their uptake, biotransformation, and possible toxic effects.

Effects of bacterial inoculants on the indigenous microbiome and secondary metabolites of chamomile plants.

Plant-associated bacteria fulfill important functions for plant growth and health. However, our knowledge about the impact of bacterial treatments on the host's microbiome and physiology is limited. The present study was conducted to assess the impact of bacterial inoculants on the microbiome of chamomile plants Chamomilla recutita (L.) Rauschert grown in a field under organic management in Egypt. Chamomile seedlings were inoculated with three indigenous Gram-positive strains (Streptomyces subrutilus Wbn2-11, Bacillus subtilis Co1-6, Paenibacillus polymyxa Mc5Re-14) from Egypt and three European Gram-negative strains (Pseudomonas fluorescens L13-6-12, Stenotrophomonas rhizophila P69, Serratia plymuthica 3Re4-18) already known for their beneficial plant-microbe interaction. Molecular fingerprints of 16S rRNA gene as well as real-time PCR analyses did not show statistically significant differences for all applied bacterial antagonists compared to the control. In contrast, a pyrosequencing analysis of the 16S rRNA gene libraries revealed significant differences in the community structure of bacteria between the treatments. These differences could be clearly shown by a shift within the community structure and corresponding beta-diversity indices. Moreover, B. subtilis Co1-6 and P. polymyxa Mc5Re-14 showed an enhancement of the bioactive secondary metabolite apigenin-7-O-glucoside. This indicates a possible new function of bacterial inoculants: to interact with the plant microbiome as well as to influence the plant metabolome.

Arsenic-containing lipids are natural constituents of sashimi tuna.

Arsenic occurs naturally in many types of seafood as water- and fat-soluble organoarsenic compounds. Although water-soluble compounds have been well characterized, the fat-soluble compounds, so-called arsenolipids, have until recently remained unknown. We report that sashimi-grade tuna fish, with a total arsenic content of 5.9 microg of As/g dry mass, contains approximately equal quantities of water- and fat-soluble arsenic. The water-soluble arsenic comprised predominantly arsenobetaine (>95%) with a trace of dimethylarsinate. Two fat-soluble compounds, which together accounted for about 40% of the lipid-arsenic, were isolated and characterized. The first was identified as 1-dimethylarsinoylpentadecane [(CH(3))(2)As(O)(CH(2))(14)CH(3)] by comparison of HPLC/mass spectrometric data and accurate mass data with those of an authenticated synthesized standard. The second arsenolipid was postulated as 1-dimethylarsinoyl all-cis-4,7,10,13,16,19-docosahexane from mass spectrometric data and analogy with non-arsenic-containing lipids found in fish. The remaining fat-soluble arsenic consisted of less polar arsenolipids of currently unknown structure. This is the first identification of arsenolipids in commonly consumed seafood.

Arsenic-containing hydrocarbons: natural compounds in oil from the fish capelin, Mallotus villosus.

Arsenic-containing hydrocarbons have been identified for the first time as natural components of fish oil.

Design, synthesis and ribosome binding of chloramphenicol nucleotide and intercalator conjugates.

Molecular modelling based on X-ray structures of the antibiotic drug chloramphenicol bound in a bacterial ribosome has been used for design of chloramphenicol derivatives. Conjugates of the chloramphenicol amine through appropriate linkers to either a pyrene moiety or to a mono- or dinucleotide moiety were designed to improve binding to ribosomes by providing specific interactions in the peptidyl transferase site or to the P-loop in the ribosome. Specific binding of the conjugates were investigated by footprinting analysis using chemical modifications of accessible nucleotides in ribosomal RNA. The pyrene chloramphenicol conjugate shows enhanced binding to the chloramphenicol binding site compared to the native chloramphenicol, whereas the four nucleotide conjugates could not be shown to bind to the chloramphenicol binding site or to the P-loop.

Structural and Kinetic Studies of Spin Crossover in an Iron(II) Complex with a Novel Tripodal Ligand.

Configurational and ligand conformational influences on the kinetics of (1)A(1) right harpoon over left harpoon (5)T(2) spin crossover in the Fe(II) complex with the novel tripodal ligand, 1,1,1-tris((N-(2-pyridylmethyl)-N-methylamino)methyl)ethane (tptMetame), have been explored. Despite having six chelate rings and three chiral nitrogen atoms, only one enantiomeric pair of isomers, Delta, SSS, and Lambda, RRR, of the complex ion is observed. The conformation of the three rings forming the upper "cap" of the complex structure can be assigned delta or lambda with respect to the 3-fold molecular axis. X-ray data at 300 and 153 K, above and below the critical temperature for the spin transition, show that the conformation of the ligand "cap" is the same as the absolute configuration of the complex, with the same Lambdalambda(CAP)(or Deltadelta(CAP)) combination prevailing for both the LS ((1)A(1)) and HS ((5)T(2)) isomers. Molecular mechanics calculations further show that the ligand energy remains lowest for this Lambdalambda(CAP) (or Deltadelta(CAP)) combination at all Fe-N distances over the range spanning the LS and HS isomers. Measurements of the spin crossover relaxation time have been carried out in solution over the temperature range 293-170 K. The observed monophasic relaxation traces are also consistent with the absolute configuration of the complex remaining unaltered during the spin crossover.