Profiling of lipids in Thermus thermophilus HB8 grown under various conditions.

The membrane lipids of Thermus species have unique structures. Only four polar lipid species have so far been identified in Thermus thermophilus HB8; namely, are two phosphoglycolipids and two glycolipids, both of which have three branched fatty acid chains. Other lipid molecules may be present; however, they have not been identified so far. To clarify the whole lipid profile of T. thermophilus HB8, we cultured this organism under four different growth (temperature and/or nutrition) conditions and analyzed the compositions of polar lipids and fatty acids by high-performance thin-layer chromatography (HPTLC) and gas chromatograph-mass spectrometry (GCï½°MS), respectively. Thirty-one lipid spots were detected on HPTLC plates and profiled in terms of the presence or absence of phosphate, amino, and sugar groups. Then, we allocated ID numbers to all the spots. Comparative analyses of these polar lipids showed that the diversity of lipid molecules increased under high temperature and minimal medium conditions. In particular, aminolipid species increased under high temperature conditions. As for the fatty acid comparison by GC-MS, iso-branched even-numbered carbon atoms, which are unusual in this organism, significantly increased under the minimal medium condition, suggesting that kinds of branched amino acids at the fatty acid terminus varies under different nutrition conditions. In this study, several unidentified lipids were detected, and elucidation of the lipid structures will provide important information on the environmental adaptation of bacteria.

Analysis of human skin sebum and animal meats by heat pulse desorption/mass spectrometry using proximity corona discharge ionization.

Recently, we have developed heat pulse desorption/mass spectrometry (HPD/MS). In HPD/MS, a heated N2 gas pulse was directed to the sample surface and desorbed analytes were mass analyzed by corona discharge ionization/mass spectrometry using an Orbitrap mass spectrometer. In this work, HPD/MS was applied to the analysis of skin surface components sampled from the forehead, nose, and jaw of three volunteers. It was found that various kinds of biological compounds such as squalene, free fatty acids, wax esters, triacylglycerols, and amino acids were detected. The simultaneous detection of compounds with a wide range of proton affinities suggests that the occurrence of consecutive proton transfer reactions is less likely to occur in the present experimental system. This is mainly due to the short distance of 1.5 mm between the tip of the corona needle and the inlet of the mass spectrometer (i.e., proximity corona discharge ion source). Under this condition, the transition time of the primary reactant ions (e.g., H3O+) from the tip of the corona discharge needle to the ion sampling orifice is roughly estimated to be ∼20 µs. This value nearly corresponds to the reaction lifetime of exoergic proton transfer reactions with a rate constant: ∼10-9 cm3 s-1 for the analytes of 1 ppm. Accordingly, analytes with concentrations less than 1 ppm would be ionized semi-quantitatively by the present method, making this method highly suitable for the rapid analysis of samples composed of complex mixture of compounds, e.g., non-target lipidomics.

PGLN: A newly identified amino phosphoglycolipid species in Thermus thermophilus HB8.

Thermus thermophilus has several minor lipid molecules with structures that have not been described yet. In this study, we identified a new lipid molecule in T. thermophilus HB8 with an amino group at the polar head, by detecting lipid spots with HPTLC and mass spectrometry. The structure of the lipid resembles an amino sugar phospholipid, except for the glucosamine that lacks an acetyl group. We named this amino phosphoglycolipid PGLN, and proposed its synthetic pathway from a precursor, phosphatidyl-glyceric alkylamine. The primary amine structure of PGLN may contribute to high temperature adaptation through electrostatic interactions between the head groups.

Heat Pulse Desorption of Low-Volatility Compounds by a Heated N2 Gas Pulse with Mass Spectrometry.

For the thermal desorption of low-volatility compounds, rapid heating followed by instant cooling is desirable to suppress thermal decomposition. In this work, a new thermal desorption method, heat pulse desorption (HPD), was developed. A heated N2 gas pulse (350 °C, 50 ms) was directed to the solid sample surface, and desorbed analytes were ionized by DC corona discharge and mass analyzed by an Orbitrap mass spectrometer. Because heat transfer from the heated N2 gas to the solid surface is not very efficient, desorption of the solid sample occurs at a certain temperature before reaching 350 °C. In short, there is a self-controlling desorption depending on the volatility of each analyte. Because the exit of the copper tube for gas blowing is separated from the sample surface, no carryover occurs, enabling the repetitive analysis of samples. HPD was applied to various compounds such as narcotics, pharmaceutical tablets, and explosives. Because analysis is completed within a few seconds per sample, this method is highly useful for quick and consecutive analysis of real samples, having potential utility in food quality control, counterfeit drugs analysis, and the detection of explosives for safety and security.

Corona Discharge and Field Electron Emission in Ambient Air Using a Sharp Metal Needle: Formation and Reactivity of CO3 -• and O2 -•.

CO3 -• and O2 -• are known to be strong oxidizing reagents in biological systems. CO3 -• in particular can cause serious damage to DNA and proteins by H• abstraction reactions. However, H• abstraction of CO3 -• in the gas phase has not yet been reported. In this work we report on gas-phase ion/molecule reactions of CO3 -• and O2 -• with various molecules. CO3 -• was generated by the corona discharge of an O2 reagent gas using a cylindrical tube ion source. O2 -• was generated by the application of a 15 kHz high frequency voltage to a sharp needle in ambient air at the threshold voltage for the appearance of an ion signal. In the reactions of CO3 -•, a decrease in signal intensities of CO3 -• accompanied by the simultaneous increase of that of HCO3 - was observed when organic compounds with H-C bond energies lower than ∼100 kcal mol-1 such as n-hexane, cyclohexane, methanol, ethanol, 1-propanol, 2-propanol, and toluene were introduced into the ion source. This clearly indicates the occurrence of H• abstraction. O2 -• abstracts H+ from acid molecules such as formic, acetic, trifluoroacetic, nitric and amino acids. Gas-phase CO3 -• may play a role as a strong oxidizing reagent as it does in the condensed phase. The major discharge product CO3 -• in addition to O2 -•, O3, and NO x • that are formed in ambient air may cause damage to biological systems.

High-resolution Kendrick Mass Defect Analysis of Poly(ethylene oxide)-based Non-ionic Surfactants and Their Degradation Products.

Matrix assisted laser desorption ionization (MALDI) high-resolution mass spectrometry (HRMS) and the recently introduced high-resolution Kendrick mass defect (HRKMD) analysis are combined to thoroughly characterize non-ionic surfactants made of a poly(ethylene oxide) (PEO) core capped by esters of fatty acids. A PEO monostearate surfactant is first analyzed as a proof of principle of the HRKMD analysis conducted with a fraction of EO as the base unit (EO/X with X being an integer) in lieu of EO for a regular KMD analysis. Data visualization is greatly enhanced and the distributions detected in the MALDI mass spectrum are assigned to a pristine (H, OH)-PEO as well as mono- and di-esterified PEO chains with palmitate and stearate end-groups in HRKMD plots computed with EO/45. The MALDI-HRMS/HRKMD analysis is then successfully applied to the more complex case of ethoxylated hydrogenated castor oil (EHCO) found to contain a large number of hydrogenated ricinoleate moieties (up to 14) in its HRKMD plot computed with EO/43, departing from the expected triglyceride structure. The exhaustiveness of the MALDI-HRMS/HRKMD strategy is validated by comparing the so-obtained fingerprints with results from alternative techniques (electrospray ionization MS, size exclusion and liquid adsorption chromatography, ion mobility spectrometry). Finally, aged non-ionic surfactants formed upon hydrolytic degradation are analyzed by MALDI-HRMS/HRKMD to easily assign the degradation products and infer the associated degradation routes. In addition to the hydrolysis of the ester groups observed for EHCO, chain scissions and new polar end-groups are observed in the HRKMD plot of PEO monostearate arising from a competitive oxidative ageing.

Rapid Analysis of Ingredients in Cream Using Ultrasonic Mist-Direct Analysis in Real-Time Time-of-Flight Mass Spectrometry.

A novel method for the simultaneous detection of ingredients in pharmaceutical applications such as creams and lotions was developed. An ultrasonic atomizer has been used to produce a mist containing ingredients. The analyte molecules in the mist can be ionized by using direct analysis in real time (DART) at lower temperature than traditionally used, and we thus solved the problem of normal DART-MS measurement using a high-temperature gas. Thereby, molecular-related ions of heat-unstable components and nonvolatile components became detectable. The deprotonated molecular ion of glycyrrhizic acid (m/z 821), which is unstable at high temperatures, was detected without pyrolysis by ultrasonic mist-DART-MS using unheated helium gas, although it was not detected by normal DART-MS using heated helium gas. The cationized molecular ions of derivatives of polyethylene glycol fatty acid monoesters, which are nonvolatile compounds, were also detected as m/z peaks observed from 800 to 2300. Although the protonated molecular ion of tocopherol acetate was not detected in ionization by ultrasonic mist, it was detected by ultrasonic mist-DART-MS even in the emulsion. It was not necessary to dissolve a sample completely to detect its ions. This method enabled us to obtain the composition of pharmaceutical applications simply and rapidly. Graphical Abstract ᅟ.

Stability of heterochiral hybrid membrane made of bacterial sn-G3P lipids and archaeal sn-G1P lipids.

The structure of membrane lipids in Archaea is different from those of Bacteria and Eucarya in many ways including the chirality of the glycerol backbone. Until now, heterochiral membranes were believed to be unstable; thus, no cellular organism could have existed before the separation of the groups of life. In this study, we tested the formation of heterochiral hybrid membrane made of Bacterial sn-glycerol-3-phosphate-type polar lipid and Archaeal sn-glycerol-1-phosphate-type polar lipid using the fluorescence probe. The stability of the hybrid liposomes made of phosphatidylethanolamines or phosphatidylcholines or polar lipids of thermophilic Bacteria and polar lipids of Archaea were investigated. The hybrid liposomes are all stable compared with homochiral liposome made of dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. However, the stability was drastically changed with increasing carbon chain length. Accordingly, "chirality" may not be but chain length is important. From these results, we suggest that the heterochiral hybrid membrane could be used as the membrane lipid for the last universal common ancestor (Commonote) before the emergence of Archaea and Bacteria.

Dietary glucosylceramide enhances cornified envelope formation via transglutaminase expression and involucrin production.

In this study, we investigated whether dietary glucosylceramide (GlcCer) and its metabolite sphingoid bases, sphingosine (SS), phytosphingosine (PS), sphingadienine (SD) and 4-hydroxysphingenine (4HS), influence cornified envelope (CE) formation. CE is formed during terminal differentiation of the epidermis through crosslinking of specific precursor proteins by transglutaminases (TGases), and is essential for the skin's barrier function. Oral administration of GlcCer (0.25 mg/day) for 14 consecutive days dramatically reduced transepidermal water loss, an indicator of the skin barrier condition, in hairless mice with barrier perturbation induced by single-dose ultraviolet B (UVB) irradiation. The GlcCer treatment also increased the level of TGase-1 mRNA in UVB-irradiated murine epidermis approximately 1.6-fold compared with the control. Further, all four sphingoid bases at 1 µM concentration enhanced CE formation of cultured normal human keratinocyte cells. Among them, SS, PS and SD, but not 4HS, stimulated production of involucrin, one of the CE major precursor proteins. SD increased the expression of TGase-1 mRNA, while SS increased the expression of TGase-3 mRNA. These results indicate that the skin barrier improvement induced by oral GlcCer treatment might be at least partly due to a reinforcement of CE formation in the epidermis mediated by sphingoid bases metabolically derived from GlcCer.

Deinococcus aetherius sp. nov., isolated from the stratosphere.

A pink-red pigmented, non-motile, coccoid bacterial strain, ST0316(T), was isolated from dust samples collected from the stratosphere in Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that it belonged to the genus Deinococcus. DNA G+C content (69.8 mol%), desiccation tolerance, and resistance to gamma-rays [D(10) (dose required to reduce the bacterial population by 10-fold) >8 kGy] and UV radiation (D(10) 1000 J m(-2)) supported the affiliation of strain ST0316(T) to the genus Deinococcus. The major peptidoglycan amino acids were d-glutamic acid, glycine, d-alanine, l-alanine and ornithine. Predominant fatty acids were C(16 : 1)omega7c, C(16 : 0), C(17 : 0) and iso-C(17 : 0). Strain ST0316(T) diverged from recognized species of the genus Deinococcus, showing less than 93.0 % similarity values to its closest relatives Deinococcus apachensis, D. aerius, D. geothermalis and D. murrayi. Strain ST0316(T) also differed from the type strains of closely related species in its polar lipid profile, nitrate reduction and carbon-source assimilation tests. Therefore, we propose a new species of the genus Deinococcus, Deinococcus aetherius sp. nov. (type strain, ST0316(T) =JCM 11751(T) =DSM 21230(T)).

Deinococcus aerius sp. nov., isolated from the high atmosphere.

An orange-pigmented, non-motile, coccoid bacterial strain, designated TR0125T, was isolated from dust samples collected in the high atmosphere above Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was within the radiation of Deinococcus species. The major peptidoglycan amino acids were D-glutamic acid, glycine, D-alanine, L-alanine and ornithine. The predominant fatty acids were iso-C17:0, iso-C17:1omega9c and iso-C15:0. Strong resistance to desiccation, UV-C and gamma radiation and high DNA G+C content also supported the affiliation of strain TR0125T to the genus Deinococcus. Strain TR0125T showed the highest 16S rRNA gene sequence similarity value (95.7%) to the type strain of Deinococcus apachensis, and phylogenetic analysis showed that it was further separated from D. apachensis than from Deinococcus geothermalis, indicating that strain TR0125T was not a member of these two Deinococcus species. In addition, phenotypic differences were found between strain TR0125T and the type strains of these two Deinococcus species. Therefore, a novel species of the genus Deinococcus, Deinococcus aerius sp. nov. (type strain, TR0125T=JCM 11750T=DSM 21212T), is proposed to accommodate this isolate.

Effects of pH and temperature on the composition of polar lipids in Thermoplasma acidophilum HO-62.

Thermoplasma acidophilum HO-62 was grown at different pHs and temperatures, and its polar lipid compositions were determined. Although the number of cyclopentane rings in the caldarchaeol moiety increased when T. acidophilum was cultured at high temperature, the number decreased at low pHs. Glycolipids, phosphoglycolipids, and phospholipids were analyzed by high-performance liquid chromatography with an evaporative light-scattering detector. The amount of caldarchaeol with more than two sugar units on one side increased under low-pH and high-temperature conditions. The amounts of glycolipids increased and those of phosphoglycolipids decreased under these conditions. The proton permeability of the liposomes obtained from the phosphoglycolipids that contained two or more sugar units was lower than that of the liposomes obtained from the phosphoglycolipids that contained one sugar unit. From these results, we propose the hypothesis that T. acidophilum adapts to low pHs and high temperatures by extending sugar chains on their cell surfaces, as well as by varying the number of cyclopentane rings.

Specific and rapid analysis of ubiquinones using Craven's reaction and HPLC with postcolumn derivatization.

A new method for the analysis of ubiquinones in various samples was developed using an HPLC system with postcolumn derivatization. Craven's reaction, a specific color reaction for the analysis of ubiquinones, was used in the system. Because the reaction progressed in organic solvents that contained ubiquinones and ethylcyanoacetate under an alkaline condition, the selectivity for ubiquinone detection was higher than that for ubiquinone detection using the nonderivatized ultraviolet detection system at 275 nm, a system widely used for the analysis of ubiquinones. The new detection system can avoid the adverse effects of impurities. Furthermore, it can confirm specificity by stopping the color reaction under a neutral condition. The detection limit for ubiquinone-10 was 1 ng (1.2 pmol). A good linearity for the calibration curve was observed in the range of 11.7 pmol to 11.7 nmol. To investigate the possible application of this method, various samples, such as soybean capsules used as a dietary supplement and biological materials (rice as well as bovine plasma and liver samples), were applied to the system and their ubiquinone contents were quantified. This method is thought to be widely and conveniently applicable for determining the level of ubiquinones because of its high selectivity for ubiquinone detection.

Characterization of the precursor of tetraether lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum.

Polar lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum was analyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [(14)C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chromatography. Lipids labeled with [(14)C]mevalonic acid, [(14)C]glycerol, and [(32)P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophosphatidylcaldarchaetidylglycerol, caldarchaetidylglycerol, and beta- l-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetraether lipid molecule is synthesized by head-to-head condensation of two molecules of archaetidylglycerol and that a sugar group of tetraether phosphoglycolipid is expected to attach to the tetraether lipid core after head-to-head condensation in T. acidophilum. A precursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The relative height of the peak m/z 806 decreased after removal of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form.

Complete polar lipid composition of Thermoplasma acidophilum HO-62 determined by high-performance liquid chromatography with evaporative light-scattering detection.

Polar ether lipids of Thermoplasma acidophilum HO-62 were purified by high-performance liquid chromatography with an evaporative light-scattering detector. Structures of purified lipids were investigated by capillary gas chromatography, mass spectrometry, and nuclear magnetic resonance. Three types of ether lipids were found: phospholipids, glycolipids, and phosphoglycolipids. The two phospholipids had glycerophosphate as the phosphoester moiety. The seven glycolipids had different combinations of gulose, mannose, and glucose, which formed mono- or oligosaccharides. The eight phosphoglycolipids with two polar head groups contained glycerophosphate as the phosphoester moiety and gulose alone or gulose and mannose, which formed mono- or oligosaccharides, as the sugar moiety. Although gulose is an unusual sugar in nature, several glyco- and phosphoglycolipids contained gulose as one of the sugar moieties in Thermoplasma acidophilum. All the ether lipids had isopranoid chains of C(40) or C(20) with zero to three cyclopentane rings. The structures of these lipids including four new glycolipids and three new phosphoglycolipids were determined, and a glycosylation process for biosynthesis of these glycolipids was suggested.