Tetrahydropyran ring-containing fatty acid-combined taurine (tetrathermoyltaurine) in the taurolipid fraction of Tetrahymena thermophila.

A tetrahydropyran ring-containing fatty acid-combined taurine (tetrathermoyltaurine) was found in the taurolipid fraction of Tetrahymena thermophila. Tetrathermoyltaurine accounted for about 0.6% of the total taurolipid of the cells. The compound was subjected to methanolic hydrochloric acid hydrolysis, and the structures of the hydrolysis products were identified by nuclear magnetic resonance and mass spectrometries, as taurine and 2,3-dihydroxy-9,13-oxy-7-trans-octadecenoic acid (tetrathermic acid). The chemical structure of tetrathermoyltaurine was identified as 2-(2,3-dihydroxy-9,13-oxy-7-trans-octadecenoylamino) ethanesulfonic acid. This structure suggests that tetrathermoyltaurine may be derived from taurolipid B as the major taurolipid of the cells. When cells of HL 60, as a human lymphoma, were cultured with tetrathermoyltaurine, 88% of the cell growth was inhibited at the concentration of 100 micrograms/ml.     

Identification of pentahydroxystearic acid-containing taurolipid (taurolipid C) isolated from Tetrahymena thermophila

A pentahydroxystearic acid-containing taurolipid (taurolipid C) was found in cells of Tetrahymena thermophila. The lipid accounted for about 1.2% of the total taurolipids of the cells. The lipid was subjected to mild alkaline and methanolic hydrochloric acid hydrolyses, and the structures of the hydrolyses products were identified by mass and nuclear magnetic resonance spectrometries, as taurine, non-hydroxy fatty acid and 2,3,7,12,13-pentahydroxystearic acd, a novel fatty acid. The NMR spectra of the intact and acetylated lipid showed that the carboxyl group of the pentahydrxyostearic acid was combined with the amino group of taurine, and the hydroxy group at C-3 was esterified with non-hydroxy fatty acids. From these results, the pentahydroxystearic acid-containing taurolipid (taurolipid C) isolated from T. thermophila was identified as 2-(3-acyloxy-2,7,12,13-tetrahydroxyoctadecanoylamino)ethanesulf oni c acid.     

2-(7,13-Dihydroxy-2-trans-octadecenoylamino) ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis

A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila. Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid. By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid. When cells of T. thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from [2(n)-³H]taurine and [1-¹⁴C]stearic acid, both the radioactivities were detected in taurolipid A, B and C. Furthermore, the ratio of the radioactivities of ³H and ¹⁴C in the lysotaurolipids were the same as that of the lipotaurine. From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.     

2-(7,13-Dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis

A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila. Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid. By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid. When cells of T. thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from [2(n)-3H]taurine and [1-14C]stearic acid, both the radioactivities were detected in taurolipid A, B and C. Furthermore, the ratio of the radioactivities of 3H and 14C in the lysotaurolipids were the same as that of the lipotaurine. From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.     

Isolation and Characterization of a Dibenzofuran-Degrading Yeast: Identification of Oxidation and Ring Cleavage Products

We characterized the ability of a yeast to cleave the aromatic structure of the dioxin-like compound dibenzofuran. The yeast strain was isolated from a dioxin-contaminated soil sample and identified as Trichosporon mucoides. During incubation of glucose-pregrown cells with dibenzofuran, six major metabolites were detected by high-performance liquid chromatography. The formation of four different monohydroxylated dibenzofurans was proven by comparison of analytical data (gas chromatography-mass spectrometry) with that for authentic standards. Further oxidation produced 2,3-dihydroxydibenzofuran and its ring cleavage product 2-(1-carboxy methylidene)-2,3-dihydrobenzo[b]furanylidene glycolic acid, which were characterized by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. These two metabolites are derived from 2-hydroxydibenzofuran and 3-hydroxydibenzofuran, as shown by incubation experiments using these monohydroxylated dibenzofurans as substrates.     

Products Formed from Analogues of 1,1,1-Trichloro-2,2-Bis(p-Chlorophenyl) Ethane (DDT) Metabolites by Pseudomonas putida

Cultures of Pseudomonas putida growing in solutions with diphenylmethane as sole carbon source formed 1,1,1′,1′-tetraphenyldimethyl ether. The product was identified by gas chromatography, mass spectrometry, and infrared and nuclear magnetic resonance spectrometry. The formation of benzophenone, benzhydrol, and phenylglycolic acid was established by gas chromatography and mass spectrometry. Similar techniques also revealed that phenylacetic acid was a major metabolite. Resting cell suspensions converted benzhydrol to phenyl-glycolic acid and products tentatively identified as hydroxybenzhydrols and a hydroxybenzophenone. Cell suspensions of the bacterium also converted the tetraphenyldimethyl ether to benzhydrol and benzophenone. Possible pathways for the degradation of these analogues of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolites are discussed.     

Determination of phosphorus impurity that directly affects quantification of microbial genomic DNA using inductively coupled plasma optical emission spectrometry

We prepared genomic DNA from human placenta, Escherichia coli, and Bacillus subtilis using various DNA extraction methods and quantified the genomic DNA using ultraviolet (UV) spectrophotometry, capillary electrophoresis (CE), and inductively coupled plasma optical emission spectrometry (ICP–OES). Application of ICP–OES unexpectedly led to a serious overestimation of phosphorus in B. subtilis genomic DNA prepared using cetyltrimethyl ammonium bromide (CTAB). Further investigations using reversed-phase high-performance liquid chromatography (RP–HPLC), ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC–ESI–MS/MS), and ³¹P nuclear magnetic resonance (NMR) identified the phosphorus impurity as lipoteichoic acid (LTA).     

Pathway of taurolipid B formation from exogenous taurolipid A by Tetrahymena thermophila

When Tetrahymena thermophila was incubated with taurolipid A isolated from T. pyriformis NT-1, the exogenously added taurolipid A was deacylated rapidly, and taurolipid B content in the cells was increased. The deacylated taurolipid A (lysotaurolipid A) content reached a maximum early on during incubation, and then declined. Taurolipid B and lysotaurolipid B contents in the cells were increased continuously during the incubation. These observations suggest that lysotaurolipid A was an intermediate for taurolipid B formation. When cells were incubated with lysotaurolipid A, newly formed lysotaurolipid B and taurolipid B were observed. Furthermore, when cells were incubated with lysotaurolipid B, only taurolipid B was newly formed. In contrast, newly formed lysotaurolipid B was observed when cells were incubated with exogenous taurolipid B. From the results, we have postulated the biosynthetic pathway of taurolipid B from exogenous taurolipid A in cells of Thermophila.     

Effects of taurolipids on lysosomal enzymes in Tetrahymena

Effects of novel taurolipid A and B localized in Tetrahymena lysosomes on the activities of lysosomal enzymes purified from Tetrahymena were investigated. Both taurolipids activated acid phosphatase, while they did not affect α-glucosidase and β-hexosaminidase. The acid phosphatase activity was activated approximately 3-fold by both taurolipids A and B, with the half-maximum activations for taurolipid A and B being at approximately 1.03·10⁻⁴ and 0.72·10⁻⁴ M, respectively. When the purified acid phosphatase was incubated at 37°C in citrate-phosphate buffer (pH 5.0) its activity was rapidly inactivated, but the inactivation was prevented to a remarkable extent by the addition of taurolipids to the incubation medium. These results thus suggest that the taurolipids may be involved in activating and stabilizing acid phosphatase in Tetrahymena lysosomes.     

Degradation of Quercetin and Luteolin by Eubacterium ramulus

The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by ¹H and ¹³C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation of E. ramulus. The degradation of luteolin by resting cells of E. ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E. ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.     

Isolation and Quantitation of beta-d-Glucopyranosyl Abscisate from Leaves of Xanthium and Spinach

From previous work (Zeevaart 1980 Plant Physiol 66: 672-678) Xanthium leaves are known to contain a high level of alkali-hydrolyzable conjugated abscisic acid. This abscisic acid conjugate has been isolated and identified by mass spectrometry, nuclear magnetic resonance, and chemical and enzymic degradation techniques, as the glucosyl ester of abscisic acid, β-d-glucopyranosyl abscisate. The glucosyl ester of abscisic acid was the only abscisic acid conjugate found in Xanthium leaves. It was also isolated from spinach leaves.     

The chemical nature of the defensive larval secretion of the moth, Catochria catocaloides

The defensive larval secretion of Catochria catocaloides was determined by nuclear magnetic resonance and mass spectrometry as a solution of up to 38% formic acid in water.     

2-Acetamidoglucal,a new metabolite isolated from the urine of a patient with sialuria

A new metabolite, namely 2-acetamidoglucal, has been found in the urine of a patient with sialuria in addition to the metabolites N-acetylneuraminic acid, N-acetylmannosamine, N-acetylglucosamine and N-deoxy-2,3-dehydro-Nacetylneuraminic acid reported earlier. The structure has been identified by mass spectrometry and 360 MHz proton nuclear magnetic resonance spectroscopy and verified by synthesis. All accumulated compounds fit into the metabolic pathway for the biosynthesis of CMP-N-acetylneuraminic acid. Sialuria is discussed in terms of a failure of regulation of UDP-N-acetyl-glucosamine 2-epimerase.     

Production of 10(S)-hydroxy-8(E)-octadecenoic and 7,10(S,S)-hydroxy-8(E)-octadecenoic ethyl esters by Novozym 435 in solvent-free media

Novozym 435, lipase B from Candida antarctica, was used in this study for the production of ethyl esters. For the first time, trans-hydroxy-fatty acid ethyl esters were synthesized in vitro in solvent-free media. We studied the effects of the substrate–ethanol molar ratio and enzyme synthetic stability of the biocatalyst. To determine the structure of the formed compounds, Fourier transformed infrared spectroscopy, nuclear magnetic resonance, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry were used, three less time-consuming structural techniques. trans-Hydroxy-fatty acid ethyl esters were synthesized with a reaction yield of 90 % or higher with optimal reaction conditions.     

Degradation of Phenanthrene and Anthracene by Cell Suspensions of Mycobacterium sp. Strain PYR-1

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2′-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.     

Discovery of Infection Associated Metabolic Markers in Human African Trypanosomiasis

Human African trypanosomiasis (HAT) remains a major neglected tropical disease in Sub-Saharan Africa. As clinical symptoms are usually non-specific, new diagnostic and prognostic markers are urgently needed to enhance the number of identified cases and optimise treatment. This is particularly important for disease caused by Trypanosoma brucei rhodesiense, where indirect immunodiagnostic approaches have to date been unsuccessful. We have conducted global metabolic profiling of plasma from T.b.rhodesiense HAT patients and endemic controls, using ¹H nuclear magnetic resonance (NMR) spectroscopy and ultra-performance liquid chromatography, coupled with mass spectrometry (UPLC-MS) and identified differences in the lipid, amino acid and metabolite profiles. Altogether 16 significantly disease discriminatory metabolite markers were found using NMR, and a further 37 lipid markers via UPLC-MS. These included significantly higher levels of phenylalanine, formate, creatinine, N-acetylated glycoprotein and triglycerides in patients relative to controls. HAT patients also displayed lower concentrations of histidine, sphingomyelins, lysophosphatidylcholines, and several polyunsaturated phosphatidylcholines. While the disease metabolite profile was partially consistent with previous data published in experimental rodent infection, we also found unique lipid and amino acid profile markers highlighting subtle but important differences between the host response to trypanosome infections between animal models and natural human infections. Our results demonstrate the potential of metabolic profiling in the identification of novel diagnostic biomarkers and the elucidation of pathogenetic mechanisms in this disease.     

Bacteriohopanetetrol: abundant lipid in frankia cells and in nitrogen-fixing nodule tissue

An unusual class of lipid with amphiphilic properties has been detected in nodule tissue of Alnus and Ceanothus. High levels of the same lipid (20-50% of total cell lipids) were detected in solvent extracts of Frankia spp. cells. However, the lipid was absent in host roots. The lipid was purified and quantified by high-performance liquid chromatography/flame ionization detector. Phenol-sulfuric acid determinations and proton nuclear magnetic resonance indicated that the purified lipid is not a glycolipid. Mass spectra of the predominant species are consistent with published spectra for bacteriohopanetetrol (C₃₅H₆₂O₄), a pentacyclic triterpenoid, or hopanoid.     

Molecular Rearrangement of Longifolene by Arthrobacter ilicis T2

Arthrobacter ilicis T₂ brings about a unique type of cometabolic structural rearrangement of longifolene, a sesquiterpene, resulting in the formation of an acid. Infrared, nuclear magnetic resonance, mass spectrometry, and decoupling studies indicate that the acid product has a sativenelike structure, which is confirmed by conversion of the acid to its methyl ester and hydrocarbon.     

Identification and characterization of antifungal active substances of Streptomyces hygroscopicus BS-112

An antifungal Actinomyces BS-112 strain, with Aspergillus flavus as the target pathogen, was isolated from soil in the forest land of Mountain Tai. This strain showed a strong antagonistic activity against various mold fungi in food and feed. Strain BS-112 was identified as Streptomyces hygroscopicus based on its morphologic, cultural, physiological, biochemical characteristics, cell wall components and 16S rDNA sequence. Four active components were separated and purified from strain BS-112. These four antifungal components were identified as tetrins A and B and tetramycins A and B using spectroscopic analysis including mass spectrometry and nuclear magnetic resonance spectroscopy. Tetrins A and B and tetramycins A and B strongly inhibited the growth of A. flavus, A. alutaceus, A. niger, and A. fumigatus in vitro.     

Novel Sulfonolipid in the Extremely Halophilic Bacterium Salinibacter ruber

Salinibacter ruber is an extremely halophilic bacterium, phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria. Electrospray mass analyses (negative ion) of the total lipid extract of a pure culture of S. ruber shows a characteristic peak at m/z 660 as the most prominent peak in the high-mass range of the spectrum. A novel sulfonolipid, giving rise to the molecular ion [M-H]⁻ of m/z 660, has been identified. The sulfonolipid isolated and purified by thin-layer chromatography was shown by chemical degradation, mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance analysis to have the structure 2-carboxy-2-amino-3-O-(13′-methyltetradecanoyl)-4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid. This lipid represents about 10% of total cellular lipids, and it appears to be a structural variant of the sulfonolipids found as main components of the cell envelope of gliding bacteria of the genus Cytophaga and closely related genera (W. Godchaux and E. R. Leadbetter, J. Bacteriol. 153:1238-1246, 1983) and of diatoms (R. Anderson, M. Kates, and B. E. Volcani, Biochim. Biophys. Acta 528:89-106, 1978). Since this sulfonolipid has never been observed in any other extreme halophilic microorganism, we consider the peak at m/z 660 the lipid signature of Salinibacter. This study suggests that this novel sulfonolipid may be used as a chemotaxonomic marker for the detection of Salinibacter within the halophilic microbial community in saltern crystallizer ponds and other hypersaline environments.