An acidic morpholine derivative containing glyceride from thraustochytrid, Aurantiochytrium.

A novel acidic morpholine-derivative containing glyceride (M-glyceride) was isolated from the cells of two strains of the thraustochytrid, Aurantiochytrium. The glyceride accounted for approximately 0.1 -0.4% of the lyophilized cells. The glyceride consisted of peaks I (85%) and II (15%). The structures of the intact and acetylated glycerides were elucidated by liquid chromatography-quadrupole time-of-flight chromatograph mass spectrometer (LC-Q/TOF) and NMR spectroscopy. The hydrate type of M-glyceride was detected as a minor component by LC-MS/MS. By 2D-NMR experiments, peaks I of the intact M-glyceride were elucidated as 1,2-didocosapentaenoyl-glyceryl-2'-oxy-3'-oxomorpholino propionic acid, and peak II was estimated 1,2-palmitoyldocosapentaenoyl- and/or 1,2-docosapentaenoylpalmitoyl-glyceryl-2'-oxy-3'-oxomorpholino propionic acid. The double bond position of docosapentaenoic acid was of the ω - 6 type (C22 = 5.ω - 6). The M-glyceride content varied by the cell cycle. The content was 0.4% of lyophilized cells at the mid logarithmic phase, and decreased to 0.1% at the mid stationary phase. When cells were grown in 1.0 µM M-glyceride-containing growth media, cell growth was stimulated to 110% of the control. With 0.1 µM acetyl M-glyceride, stimulation of 113% of the control was observed. Finding morpholine derivatives in biological components is rare, and 2-hydroxy-3-oxomorpholino propionic acid (auranic acid) is a novel morpholine derivative.

Effects of soybean curd wastewater on the growth and hydrocarbon production of Botryococcus braunii strain BOT-22.

A laboratory study was conducted to evaluate the possibility of using wastewater from a soybean curd manufacturing plant as a growth promoter of Botryococcus braunii strain BOT-22. Soybean curd wastewater (SCW) were added to AF-6 medium to set the final concentration to 0% (control), 1%, 2%, 5%, and 10% (v/v). The growth and hydrocarbon production observed in the cultures with 1% and 2% SCW were significantly higher than that observed in the control. It was postulated that proteins and/or reducing sugars in SCW could enhance the growth. A major finding was a shift in the chemical composition of hydrocarbons from C(34)H(58) to C(32)H(54) in association with increased concentrations of SCW. Considering the inorganic ions in SCW, it was presumed that a mixture of nitrate, 1-2% SCW, and secondarily treated SCW can be applied for mass cultivation of Botryococcus.

Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production.

Optimum conditions of temperature, salinity and glucose concentration were investigated for squalene production of the strain of Aurantiochytrium sp. 18 W-13a, with a high content of squalene. Squalene production by this strain was optimum at 25 °C, 25-50% seawater concentration and 2-6% glucose concentration. When this strain was grown in the optimum condition, the squalene content and production of approximately 171 mg/g dry weight and 0.9 g/L were much higher than that previously reported in thraustochytrids, plants and yeasts, respectively. Therefore, 18 W-13a could be used as an alternative source of commercial squalene.

Comprehensive study of proteins that interact with microcystin-LR.

We carried out a comprehensive study of proteins that exhibit specific interactions with a naturally occurring toxin, microcystin (MC)-LR, in order to gain insight into the unknown underlying mechanism of MC virulence. This audacious study employed a simple affinity test that used MC-LR immobilized on an original ethylene oxide based monolithic solid phase (Moli-gel), and swine liver lysate. Some of the proteins that interacted with MC-LR on this original affinity resin were separated by SDS-PAGE, measured by nano-LC/MS/MS after trypsin digestion, and identified using a Mascot database search. Protein sequence analyses revealed that glutathione S-transferase (GST) was one of the candidate target proteins for MC-LR. This protein was confirmed as a target protein for MC-LR based on the results of for the inhibition of an enzymatic reaction by Dhb-MC-LR. Moreover, L-3-hydroxyacyl coenzyme A dehydrogenase (HDHA) was shown to be one of the proteins that specifically interacts with MC-LR. Our results demonstrated that our analytical systems based on an original affinity resin and nano-LC/MS/MS were effective for target protein research.

Thraustochytrid Aurantiochytrium sp. 18W-13a accummulates high amounts of squalene.

Here we report on the 18W-13a strain of Aurantiochytrium sp., which accumulates very high amounts of squalene. The squalene contents and production at 4 d of culture were 198 mg/g and 1.29 ± 0.13 g/L, respectively, exceptionally high values compared to previous reports.

Accurate LC-MS analyses for microcystins using per-15N-labeled microcystins.

Per-(15)N-labeled microcystins were prepared for use as surrogates for accurate liquid chromatography-mass spectrometry analysis. Two strains of Microcystis aeruginosa were cultured in (15)NO(3)-containing TS-15 medium. To change from the incorporation of (14)N to (15)N into all cell components, cells of Microcystis aeruginosa were precultured in Na(15)NO(3)-containing medium for more than 6 months. After mass cultivation of the strains, cells of each strain were harvested and lyophilized. Microcystin variants were extracted from the lyophilized cells and per-(15)N-labeled microcystin variants were purified using high-performance liquid chromatography and high-performance thin-layer chromatography. The structures of per-(15)N-labeled microcystin variants were confirmed by their mass spectrometry spectra and NMR spectra. When per-(15)N-labeled microcystins were used as surrogates for quantitative analysis of these toxins in cyanobacterial cells, excellent accuracy (98-106%) was obtained, with the m/z of M(+), [M+1](+), and [M+2](+) of both microcystins and the per-(15)N-labeled microcystins as surrogates being completely separated. In conclusion, per-(15)N-labeled microcystins are excellent surrogates for microcystin analysis using liquid chromatography-mass spectrometry.

A novel retinoic acid analogue, 7-hydroxy retinoic acid, isolated from cyanobacteria.

BACKGROUND: All-trans retinoic acid (RA) is a low-molecular compound derived from vitamin A. It induces events in various ways by binding with the retinoic acid receptor (RAR), a nuclear receptor, in animal cells. RA and its metabolites have been found in animal tissues. In this paper, we report a novel RA analogue found in cyanobacterial cells, describe the method for its isolation, and compare its photo-stability with that of all-trans RA. METHODS: The new A analogue was extracted from cells of Microcystis aeruginosa and Spirulina sp. and fractionated by high-performance liquid chromatography. The analogue was analysed using a yeast two-hybrid assay method to measure in vitro RAR-agonistic activity. Liquid chromatography-mass spectrometry/mass spectrometry analyses was performed to elucidate the chemical structure of this RA analogue. RESULTS: The results of the analysis of the fragments revealed that the novel RA analogue was 7-hydroxy RA. The yields from 3.5 µg (4.5% of the total RAR-agonistic activity of Spirulina sp. cells) of 7-hydroxy RA was a mixture of 4 isomers due to cis-trans isomerisation coupled with keto-enol tautomerism; its relative RAR agonistic activity was 0.49 ± 0.01 (n=3) when the activity of all trans RA was set up to 1.00. Under fluorescent light, the mixture of 7-hydroxy RA isomers was more stable than all- trans RA. CONCLUSIONS: We isolated a novel RAR-activating compound, 7-hydroxy RA, from cyanobacteria. GENERAL SIGNIFICANCE: 7-hydroxy RA is more stable than all-trans RA under UV-A.

Novel separation medium spongy monolith for high throughput analyses.

Sponge-like material was utilized as novel chromatographic media for high throughput analyses. The pore size of the sponge-like material was several dozen micrometer, and was named spongy monolith because it consists of continuous structured copolymers, which was made of poly(ethylene-co-vinyl acetate), such as monolithic materials including silica monoliths and organic polymer monoliths. The spongy monolith was packed into a stainless steel column (100 mm x 4.6 mm I.D.) and evaluated in liquid chromatography (LC) with an on-line column-switching LC concentration system. The results indicate that the packed column could be used with high flow rates and low back pressure (9.0 mL/min at 0.5 MPa). Furthermore, bisphenol A was quantitatively recovered by on-line column-switching LC concentration with the spongy monolithic column. Additionally, the adsorption capacity and physical strength of the media was enhanced via chemical modification of spongy monoliths using glycerol dimethacrylate. The results compared with original spongy monolith demonstrated that a higher adsorption capacity was achieved on a shorter column, and a stable low back pressure was obtained at high throughput elution even with a longer column.

Importance of surface properties of affinity resin for capturing a target protein, cyclooxygenase-1.

We have prepared affinity resins based on two kinds of solid phases, including a commercially available solid phase, to re-realize the importance of surface properties of affinity resins such as controlled ligand density as well as existential surroundings of the ligand. Affinity resins were prepared using non-steroidal anti-inflammatory drugs, such as Ketoprofen, Ibuprofen, and Aspirin, having different activities as ligands. The ligand density was controlled through two different strategies: one strategy was that the solid phases having different amino group densities (20, 60, 100, 125 micromol/ml) were utilized then, Ketoprofen was fully immobilized through condensation reaction to amino groups; another strategy was that a solid phase having amino group density (125 micromol/ml) was utilized then, each ligand was immobilized with controlled immobilization rate. In addition, a typical hydrophobic group, stearoyl group (C(18) group), was immobilized on the affinity resin with controlled ligand immobilization rate to change the existential surroundings of the ligand. Affinity tests were performed for Cyclooxgenase-1 (COX-1) as it was the target protein in this work. The amount of captured COX-1 was evaluated utilizing each affinity resin. It was suggested that the density of surface ligand tends to relate to the amount of captured COX-1 on our solid phase-based affinity resins; however, several exceptions occurred according to the surface properties of affinity resins in the case of commercial one.

Selective adsorption of water-soluble ionic compounds by an interval immobilization technique based on molecular imprinting.

Uniformity recognition sites for water-soluble ionic compounds were constructed onto the pores of porous polymer particles. This concept is based on a modified interval immobilization technique, which was used for the selective recognition of paralytic shellfish poison, saxitoxins (STXs). The results of batch adsorption and solid-phase extraction for one of the STX analogues, a prepared polymer that had special sites for the recognition of STXs, showed that the analogue could selectively recognize and concentrate STXs. Selective recognition was facilitated by interval-immobilized functional groups.

NIES certified reference material for microcystins, hepatotoxic cyclic peptide toxins from cyanobacterial blooms in eutrophic water bodies.

A certified reference material (CRM) for microcystins has been prepared by the National Institute for Environmental Studies (NIES). Microcystins are hepatotoxic cyclic peptides produced by cyanobacteria in eutrophic water bodies. At least seven microcystin variants were found by HPLC analysis of the NIES CRM, of which [Dha(7)]microcystin-RR and -LR were the major microcystins present. Because of the lack of available standards we determined the total microcystin concentration in the CRM by the MMPB method, and elucidated the structures of the main individual microcystin variants following their isolation. Analyses of NMR and MS spectra indicated that the remaining minor variants in the CRM were [D-Asp(3), Dha(7)]microcystin-RR and -LR, and [Dha(7)]microcystin-YR, -ThTyrR, and -HilR. The CRM is valuable not only as a standard material for the quantitation of total microcystins but also for the identification of individual [Dha(7)]microcystin variants.

Effective determination method for a cyanobacterial neurotoxin, beta-N-methylamino-L-alanine.

We developed a simple and effective analysis procedure that includes pretreatment and determination methods for beta-N-methylamino-l-alanine (BMAA), a cyanobacterial neurotoxin. BMAA may be produced by all known groups of cyanobacteria living in freshwater as well as marine environments. In this paper, we report a novel determination method for BMAA. A cation-exchange resin was effective for the selective concentration of BMAA from cyanobacterial extracts and yielded a high recovery rate. Moreover, liquid chromatography (LC)-electrospray ionization mass spectrometry with a hydrophilic LC column was effective for determining BMAA levels. The quantitation limit for BMAA based on selected ion monitoring (SIM) was determined as 0.5 ng at a signal/noise ratio of 5.

New values of molecular extinction coefficient and specific rotation for cyanobacterial toxin cylindrospermopsin.

The molecular extinction coefficient epsilon of cylindrospermopsin (CYN) purified by the anion exchange and the normal-phase HPLC procedures was determined to be 9800 at 262 nm. This epsilon is significantly higher than those (epsilon, 5800-6250) reported previously. In order to determine CYN concentrations in solutions using UV absorption, the epsilon-value of CYN should be corrected from 5800 to 9800. Further, the [alpha](D) value of CYN should be corrected from +12.5 degrees to +17.0 degrees .

A novel chip device based on wired capillary packed with high performance polymer-based monolith for HPLC: reproducibility in preparation processes to obtain long columns.

This report describes the development of novel wired chip devices for mu-HPLC analyses. The monolithic capillary column to be wired was prepared using a tri-functional epoxy monomer, tris(2,3-epoxypropyl)isocyanurate with a diamine, 4-[(4-aminocyclohexyl)methyl]cyclohexylamine. The prepared column was evaluated by SEM observation of the sectional structure of column and micro-HPLC. In addition, the reproducibility in the preparation of long capillary columns having nearly 1 m length was extensively examined for applications of novel wired chip devices. The authors demonstrated that the monolithic structure of the prepared long capillary could be finely controlled under the strictly maintained operational conditions and thus the relative standard deviation (RSD) of the column properties such as the number of theoretical plates, retention factor, and permeability could be well controlled to become less than 10%. Furthermore, the wired chip device column showed that its high performance was kept even after chip preparation.

Properties of flaky affinity resin with co-continuous structure.

A new type of flaky affinity resin for capture of the target proteins was prepared to discuss its properties compared with those of a particulate affinity resin. The resin prepared had totally co-continuous structure (monolith) and was utilized in the shape of flake. The concentration of surface amino groups for immobilization of ligand was determined to be 22.3 micromol/ml. Immobilizations of ligand such as Sulfonamide, Ketoprofen, Captopril, or Methotrexate (MTX) on the affinity resin were quantitatively proceeded to afford fully covered (100%) affinity resins. Control of the immobilization rate of affinity resin using Sulfonamide or Ketoprofen was successfully achieved with the calculated immobilization rate. The flaked shape of affinity resin (100-400 microm) presumably simplified affinity experimental procedures and the affinity resin immobilizing Sulfonamide effectively captured one of the target proteins, CAII, without non-specifically bound proteins. The observed properties of the flaky affinity resin as well as ease in handling are really useful for capture of the target proteins of possible rare ligands.

Simple and effective 3D recognition of domoic acid using a molecularly imprinted polymer.

We have developed a simple and effective molecular imprinting technique to target compounds with flexible structure. Domoic acid (DA), an amnesic shellfish poison, was used as the target compound while many acidic compounds (mono-, di-, and tricarboxylic acids) were used as template molecules for molecularly imprinted polymers (MIPs). Evaluation of selective recognition abilities using liquid chromatography revealed that the highest selective recognition ability for DA was found when pentane-1,3,5-tricarboxylic acid (1,3,5-PeTA) was used as the template. Computer modeling studies of the DA structure suggested that the observed selective recognition depended on the structural changes in DA at the recognition site of the MIPs as well as spatial distance between the COOH groups in DA and 1,3,5-PeTA. Using the 1,3,5-PeTA-MIP, we could easily purify DA from blue mussel extracts by solid-phase extraction.

Selective separation of hydroxy polychlorinated biphenyls (HO-PCBs) by the structural recognition on the molecularly imprinted polymers: direct separation of the thyroid hormone active analogues from mixtures.

We developed novel separation media for hydroxy polychlorinated biphenyls (HO-PCBs) using the molecular imprinting techniques. The results of evaluation for the molecularly imprinted polymers (MIPs) by the liquid chromatography (LC) suggested that MIPs had selective separation ability for certain HO-PCB analogues. The results of the LC evaluations and molecular modeling indicated that the molecular volumes and pK(a) values of template molecules were related with the retention factor of HO-PCBs. Additionally, according to the detail evaluation toward the selective separation behaviors of MIPs, these HO-PCB analogues have low pK(a) values dependent on their chemical structures. In other words, the prepared MIPs had selective recognition ability against the analogues, which have an OH group on a phenyl carbon and two chlorine atoms on the both neighboring carbons of the carbon attached with the OH group. Moreover, these analogues may have a potential for thyroid hormone activities so that we attempted to separate these analogues directly from mixtures of HO-PCBs using a prepared MIP.

Cylindrospermopsin determination using 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) as the internal standard.

Cylindrospermopsin (CYN) was determined by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) using 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) as the internal standard. In the selected ion monitoring of LC/ESI-MS, m/z 414 for CYN and 237 for HEPES were monitored using the negative mode; the retention times of CYN and HEPES were 12.41 and 14.21 min, respectively. CYN was determined from peak area ratios of m/z 414/237. By the treatment of an anion exchange cartridge using a buffer at pH 10.5, CYN was isolated and condensed. No interfering peak was observed. Linearity of this method was observed at the range of 0.10-31.12 ng. Total coefficients of variation were 5.1 and 2.9% at 104 and 1038 microg CYN L(-1). The quantitative limit at a signal-to-noise (S/N) ratio of 10 was 0.16 ng. CYN concentration in natural waters is low. CYN in waters should be condensed for determination. This method including the treatment for isolation and condensation of CYN is useful for determination of CYN in environmental and/or drinking waters.