Isolation and characterization of a sulfated glyceroglucolipid from alveolar lavage of rabbit.

A major acidic glycolipid of the rabbit alveolar lavage has been isolated and characterized. The isolation procedure involved extraction of lipids, column fractionation on DEAE-Sephadex and silicic acid, and thin-layer chromatography. Chemical analysis of the glycolipid revealed the presence of glucose, fatty acids, glycerol mono-ethers and sulfate. By partial acid and alkaline methanolysis, oxidation with periodate and chromium trioxide, and methylation analysis of the native and desulfated compound, the structure of this glycolipid is proposed to be: SO3H-6Glc alpha 1 leads to 6Glc alpha 1 leads to 6Glc alpha 1 leads to 6Glc alpha 1 leads to 3-1,(3)-O-alkyl-2-0-acylglycerol.     

Use of the photo-micronucleus assay in Chinese hamster V79 cells to study photochemical genotoxicity

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we investigated the metabolism of this carcinogen by prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, and lactoperoxidase as model mammalian peroxidases. Horseradish peroxidase (HRP)-mediated oxidation of o-anisidine was also determined and compared with the reactions catalyzed by mammalian peroxidases. All three peroxidases oxidized o-anisidine via a radical mechanism. Using HPLC combined with electrospray tandem mass spectrometry, we determined that peroxidases oxidized o-anisidine to a diimine metabolite, which subsequently hydrolyzed to form a quinone imine. Two additional metabolites were identified as a dimer linked by an azo bond and another metabolite consisting of three methoxybenzene rings, which exact structure has not been identified as yet. Using [¹⁴C]-labeled o-anisidine, we observed substantial peroxidase-dependent covalent binding of o-anisidine to DNA, tRNA and polydeoxynucleotides [poly(dX)]. The ³²P-postlabeling assay (a standard procedure and enrichment of adducts by digestion with nuclease P1 or by extraction into 1-butanol prior to ³²P-labeling) was employed as the second method to detect and quantitate binding of o-anisidine to DNA. Using these versions of the ³²P-postlabeling technique we did not observe any DNA adducts derived from o-anisidine. The o-anisidine-DNA adducts became detectable only when DNA modified by o-anisidine was digested using three times higher concentrations of micrococcal nuclease and spleen phosphodiesterase (MN/SPD). We found deoxyguanosine to be the target for o-anisidine binding in DNA using poly(dX) and deoxyguanosine 3′-monophosphate (dGp). A diimine metabolite of o-anisidine is the reactive species forming adducts in dGp. The results strongly indicate that peroxidases play an important role in o-anisidine metabolism to reactive species, which might be responsible for its genotoxicity, and its carcinogenicity to the urinary bladder in rodents. The limitation of the ³²P-postlabeling technique to analyze DNA adducts derived from o-anisidine as a means to estimate its genotoxicity is discussed.     

Consequences of quercetin methylation for its covalent glutathione and DNA adduct formation

This study investigates the pro-oxidant activity of 3′- and 4′-O-methylquercetin, two relevant phase II metabolites of quercetin without a functional catechol moiety, which is generally thought to be important for the pro-oxidant activity of quercetin. Oxidation of 3′- and 4′-O-methylquercetin with horseradish peroxidase in the presence of glutathione yielded two major metabolites for each compound, identified as the 6- and 8-glutathionyl conjugates of 3′- and 4′-O-methylquercetin. Thus, catechol-O-methylation of quercetin does not eliminate its pro-oxidant chemistry. Furthermore, the formation of these A-ring glutathione conjugates of 3′- and 4′-O-methylquercetin indicates that quercetin o-quinone may not be an intermediate in the formation of covalent quercetin adducts with glutathione, protein and/or DNA. In additional studies, it was demonstrated that covalent DNA adduct formation by a mixture of [4-¹⁴C]-3′- and 4′-O-methylquercetin in HepG2 cells amounted to only 42% of the level of covalent adducts formed by a similar amount of [4-¹⁴C]-quercetin. Altogether, these results reveal the effect of methylation of the catechol moiety of quercetin on its pro-oxidant behavior. Methylation of quercetin does not eliminate but considerably attenuates the cellular implications of the pro-oxidant activity of quercetin, which might add to the mechanisms underlying the apparent lack of in vivo carcinogenicity of this genotoxic compound. The paper also presents a new mechanism for the pro-oxidant chemistry of quercetin, eliminating the requirement for formation of an o-quinone, and explaining why methylation of the catechol moiety does not fully abolish formation of reactive DNA binding metabolites.     

Atromentic acid from Clitocybe illudens

Atromentic acid was isolated from culture liquids of Clitocybe illudens. Identification, based on analyses and spectra of the compound and a number of new derivatives, was confirmed by direct comparison of its tetramethylated derivative with a synthetic sample. Products, giving a blueing reaction have been detected in the same culture liquids, and an as yet unidentified compound has been isolated in crystalline form as its methylation product, C₄₂H₃₆O₁₂. The presence of pulvinic acid derivatives in Basidiomycetes other than the Boletaceae has been reported in only one other instance.     

Identification of cytochromes o and a3 as functional terminal oxidases in the thermophilic bacterium, PS3

Low-temperature photodissociation spectra of membranes from the thermophile PS3 reveal cytochromes o and a₃. The latter reacts with O₂ at −103°C to give a light-insensitive compound(s), but the initial stages of O₂ binding to cytochrome o could not be studied under these conditions. Photochemical action spectra identify cytochromes a₃ and o, but not a CO-binding c-type cytochrome, as functional terminal oxidases in this bacterium.     

Studies on the metabolism and toxicological detection of the designer drug 4-methylthioamphetamine (4-MTA) in human urine using gas chromatography-mass spectrometry

4-Methylthioamphetamine (4-MTA) is a scheduled designer drug that has appeared on the illicit drug market and led to several non-fatal or even fatal poisonings. Only few data are available on its metabolism. The first aim of this study was to identify the 4-MTA metabolites in human urine and then to study whether the authors' STA procedure is suitable for screening for and identification of 4-MTA and/or its metabolites in urine. After enzymatic cleavage of conjugates, solid-phase extraction (SPE) and acetylation the following metabolites could be identified by full-scan gas chromatography-mass spectrometry (GC-MS): deamino-oxo 4-MTA, deamino-hydroxy 4-MTA, ring hydroxy and beta-hydroxy 4-MTA. 4-MTA sulfoxide could be identified as possible artifact. In urine samples after enzymatic hydrolysis, acidic extraction, and methylation, 4-methylthiobenzoic acid could be identified. The authors' systematical toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction (LLE) and acetylation allowed detection of 4-MTA as target analyte plus all the above-mentioned metabolites with the exception of 4-methylthiobenzoic acid. The extraction efficiency of 4-MTA was approximately 70% and the limit of detection (LOD) was 30 ng/ml (S/N 3).     

Methyl esterification of glutamic acid residues of methyl-accepting chemotaxis proteins in Bacillus subtilis.

The amino acid residue modified in the reversible methylation of Bacillus subtilis methyl-accepting chemotaxis proteins was identified as glutamic acid; methylation results in the formation of glutamate 5-methyl ester. Identification was made by comparing the behaviour of a 3H-labelled compound isolated from proteolytically hydrolysed methyl-accepting chemotaxis proteins labelled in vivo with that of authentic methylated amino acids by chromatographic and electrophoretic techniques. Also, the isolated compound on mild alkaline hydrolysis shows behaviour identical with that of authentic glutamate 5-methyl ester. [3H]Methanol released by mild alkaline hydrolysis was made to react with 3,5-dinitrobenzyl chloride to form [3H]methyl 3,5-dinitrobenzoate, which was identified by reverse-phase high-pressure liquid chromatography.     

Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment.

Assessment of free fatty acid (FFA) concentration and isotopic enrichment is useful for studies of FFA kinetics in vivo. A new procedure to recover the major FFA from plasma for concentration and isotopic enrichment measurements is described and validated. The procedure involves extraction of plasma lipids with hexane, methylation with iodomethane (CH(3)I) to form fatty acid methyl esters (FAME), and subsequent purification of FAME by solid phase extraction (SPE) chromatography. The new method was compared with a traditional method using thin-layer chromatography (TLC) to recover plasma FFA, with subsequent methylation by BF(3)/methanol. The TLC method was found to be less reliable than the new CH(3)I method because of contamination with extraneous fatty acids, chemical fractionation of FFA species, and incomplete recovery of FFA associated with TLC. In contrast, the CH(3)I/SPE method was free of contamination, did not exhibit chemical fractionation, and had higher recovery. The iodomethane reaction was specific for free fatty acids; no FAME were formed when esterified fatty acids (triglycerides, cholesteryl esters, phospholipids) were subjected to the methylation reaction.We conclude that the CH(3)I/SPE method provides rapid and convenient recovery of plasma fatty acids for quantification or GC/MS analysis as methyl esters, and is not subject to the problems of contamination, reduced recovery, and chemical fractionation associated with recovery of FFA by TLC.     

Assay method for the carboxylic acid metabolite of clopidogrel in human plasma by gas chromatography–mass spectrometry

This paper describes a GC–MS method for the analysis of the carboxylic acid metabolite (SR26334, II) of methyl (+)-(S)--(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate hydrogensulfate (clopidogrel, SR 25990, I) in plasma and serum. The analytical procedure involves a robotic liquid–liquid extraction with diethyl ether followed by a solid–liquid extraction on C₁₈ cartridges. The derivatization process was performed using n-ethyl diisopropylethylamine and -bromo-2,3,4,5,6-pentafluoro toluene. A structural analogue (III) of II, was used as internal standard. The 1/X²; weighted calibration curve obtained in the range 5–250 ng/ml was well described by a quadratic equation. The extraction efficiency was better than 48% over the range studied; for the internal standard it averaged 51% at 50 ng/ml. Precision ranged from 3.6 to 15.8%, and accuracy was between 92 and 114%. Dilution has no influence on the performance of the method which could then be used to quantitate plasma samples containing up to 25 000 ng/ml. The limit of quantification was 5 ng/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay methods for use in pharmacokinetic studies.     

Design, synthesis, and inhibition of platelet aggregation for some 1-o-chlorophenyl-1,2,3,4-tetrahydroisoquinoline derivatives

Based on ticlopidine active as an ADP receptor antagonist for inhibiting platelet aggregation in clinical test, and upon finding (±)-1,2-substituted-7-sulfonylamide/amide-1,2,3,4-tetrahydroisoquinoline (11–31) inhibited of platelet aggregation, a series of (±)-1-o-chlorophenyl-2-substituted-tetrahydroisoquinoline derivatives was designed and synthesized. Four analogs proved to be potential antiplatelet aggregation agents, and compound 9 (TQP-3, applying for patent) which inhibits ADP-induced human platelet aggregation with IC₅₀ values of approximately 0.206 nM was the most active. Compound 2 is more active than compound 1, which (Type I) is similar to ticlopidine. This is because there is a spacial hindrance in compound 1, and the o-chloro group of compound 2 may play the same a role as o-chloro group of ticlopidine. On the other hand, with the different substitutions at different positions on the 2-substituted phenylacyl group, their inhibition of platelet aggregation differs. These compounds with m-substituted group (5, 7, 9) showed a higher IC₅₀ value for inhibiting ADP-induced human platelet aggregation than those with o-substituted group (4, 6) or p-substituted group (3, 8). It was observed that their inhibition is bromine-substituted derivative (9), chlorine-substituted derivative (7), and nitro-substituted derivative (5) in turn. Moreover, these compounds (Type II) may be more similar to clopidogrel than to ticlopidine due to the acyl group at 2 position of the nucleus playing a role as the ester group of clopidogrel. It was conjectured that these analogs function as a potential antiplatelet aggregation role by acting as ADP receptor antagonists.     

A disialoganglioside of the globo-series from chicken skeletal muscle

We have isolated a disialoganglioside of the globo-series from chicken pectoral muscle. The compound was obtained by extraction followed by ion-exchange and silicic acid column chromatography and judged to be pure by thin-layer chromatography in three solvent systems. The structure of the ganglioside was determined by carbohydrate and ceramide composition analysis, sequential exoglycosidase digestion, methylation analysis, and 500-MHz 1H-NMR spectroscopy to be: (formula; see text) Analysis of the ceramide moiety indicated d18:1 sphingosine as the long-chain base, and C16:0, C18:0, C18:1, and C20:0 as the prevalent fatty acids. This glycolipid is only the second ganglioside of the globo-series, and the first disialo member of the series, found in chicken muscle.     

Direct methylation from mouse plasma and from liver and brain homogenates

The analysis of fatty acid composition of plasma and tissue is important as a method for studying lipid nutrition. We investigated the possibility of direct methylation of fatty acids by BF(3)-methanol from plasma and from liver and brain homogenates without lipid extraction. There were no ghost peaks in the chromatogram produced by the direct methylation method. The 18:0 percentages were significantly higher in the direct methylation method than in the lipid extraction method. There were not remarkable differences in fatty acid composition in the direct methylation and methylation after lyophilization methods. Furthermore, the recovery ratio of the internal standard in the direct methylation method was higher than that in the lipid extraction method. The difference of fatty acid composition with lipid extraction may be caused by the change of lipid class extraction. Therefore, the direct methylation method without lipid extraction is the most suitable for determining fatty acid composition in plasma and tissue.     

Standardizing methylation method during phospholipid fatty acid analysis to profile soil microbial communities

Phospholipid fatty acid (PLFA) as biomarkers, is widely used to profile microbial communities in environmental samples. However, PLFA extraction and derivatization protocols are not standardized and have widely varied among published studies. Specifically investigators have used either HCl/MeOH or KOH/MeOH or both for the methylation step of PLFA analysis, without justification or research to support either one. It seems likely that each method could have very different outcomes and conclusions for PLFA based studies. Therefore, the objective of this study was to determine the effect of catalyst type for methylation on detecting PLFAs and implications for interpreting microbial profiling in soil. Fatty acid samples extracted from soils obtained from a wetland, an intermittently flooded site, and an adjacent upland site were subjected to HCl/MeOH or KOH/MeOH catalyzed methylation procedures during PLFA analyses. The methylation method using HCl/MeOH resulted in significantly higher concentrations of most PLFAs than the KOH/MeOH method. Another important outcome was that fatty acids with a methyl group (18:1ω,7c 11Me, TBSA 10Me 18:0, 10Me 18:0, 17:0 10Me and 16:0 10Me being an actinomycetes biomarker) could not be detected by HCl/MeOH catalyzed methylation but were found in appreciable concentrations with KOH/MeOH method. From our results, because the HCl/MeOH method did not detect the fatty acids containing methyl groups that could strongly influence the microbial community profile, we recommend that the KOH/MeOH catalyzed transesterification method should become the standard procedure for PLFA profiling of soil microbial communities.     

4-O-Methylglucuronoxylan Isolated from the Midrib of Nicotiana tabacum

An acidic xylan from the midrib of Nicotiana tabacum was isolated by alkaline extraction and fractionation on a DEAE-cellulose column. Based on the results of methylation analysis, partial acid hydrolysis and Smith degradation, the acidic xylan was concluded to be composed of a linear backbone of β-(1→4)-linked D-xylopyranosyl residues with approximately every ninth residue carrying a terminal 4-O-methyl-α-D-glucopyranosyluronic acid residue linked as a single side chain by (1→2) linkage.     

High-performance liquid chromatographic determination of acetyl-11-keto-alpha-boswellic acid, a novel pentacyclic triterpenoid, in plasma using a fluorinated stationary phase and photodiode array detection: application in pharmacokinetic studies

A rapid, sensitive and selective HPLC separation with photodiode array detection was developed for the analysis of the novel pentacyclic triterpenoid acetyl-11-keto-alpha-boswellic acid. Complete baseline separation of acetyl-11-keto-alpha-boswellic acid from the corresponding isomer acetyl-11-keto-beta-boswellic acid was achieved on a fluorinated stationary phase. The standard curve was linear from 0.98 nmol/l to 196 nmol/l acetyl-11-keto-alpha-boswellic acid. The compound was isolated from chick embryonic plasma using extraction on diatomaceous earth with an overall average extraction yield of 82%. This method was applied in a kinetic study on the chick chorioallantoic membrane model (CAM) and showed unequivocal separation between acetyl-11-keto-alpha-boswellic acid and acetyl-11-keto-beta-boswellic acid unachievable so far.     

Sequencing Batch Reactor Biodegradation of Hydrolyzed Sarin as Sole Carbon Source

The chemical nerve agent sarin (o-isopropyl methylphosphonofluoridate) was hydrolyzed at 7.18 weight percent in aqueous sodium hydroxide yielding primarily o-isopropyl methylphosphonic acid (IMPA). This hydrolysate was diluted and fed as sole carbon source to activated sludge in an aerobic sequencing batch reactor. Feed chemical oxygen demand (COD) concentrations ranged from approximately 2500 mg/L (initial) to 5000 mg/L (final). The reactor was operated essentially on a 15-day hydraulic residence time. Overall COD removal efficiency was 86.2% and the IMPA in the feed was converted to methylphosphonic acid. MICROTOX® and Daphnia magna aquatic toxicity testing showed the effluent to be of very low toxicity to aquatic test organisms. The final MPA product was effectively absorbed by Phoslock™, which is a lanthanide modified clay.     

Teichoic acids from streptomyces griseus

Summary During the Schmidt-Thannhauser fractionation procedure at nucleic acid determinations of Streptomyces griseus, a phosphorous-containing compound(s) was found which could not be taken as nucleic acid or polyphosphate. The hydrolisate of the isolated substance proved to have almost the same composition as teichoic acid from Staphylococcus aureus Duncan.Teichoic acid was found both intracellular and in the isolated cell wall of Streptomyces griseus mycelia.In the cell wall only the ribitol type of teichoic acids could be detected but not the glycerol type.     

Exploring the recognized bio-mimicry materials for gas sensing

This study was undertaken to synthesize peptides that are partially similar to the binding sites of human olfactory receptor protein. First, a putative 3-D model structure of human olfactory receptor protein (P30953) was modeled using a molecular simulation method. The computer docking simulation was then performed to determine the most plausible binding sites between the model structure and target gases, trimethylamine, ammonia, acetic acid, and o-xylene. According to the simulation result, a series of polypeptide sequences, horp61 for TMA, horp103 for o-xylene, horp109 for ammonia, and horp193 for acetic acid as recognized molecules were designed for gas sensing purposes. Preparing these peptides as corresponding gas sensing probes, the results showed a high relative sensitivity response of 6.7 for TMA (probe horp61), 5.1 for o-xylene (probe horp103), 11 for ammonia (probe horp109), and 28 for acetic acid (probe horp193), respectively. These results indicate that peptide mimicking of binding domain on olfactory receptor opens a new window and offers a novel strategy for the further development of recognized materials for gas sensing.     

Synthesis of carbon-11 labeled fluorinated 2-arylbenzothiazoles as novel potential PET cancer imaging agents

Fluorinated 2-arylbenzothiazoles are new potential antitumor drugs, which show potent and selective inhibitory activity against breast, lung, and colon cancer cell lines. Carbon-11 labeled fluorinated 2-arylbenzothiazoles may serve as novel probes for positron emission tomography (PET) to image tyrosine kinase in cancers. The preparation of 4-fluorinated 2-arylbenzothiazoles 4-fluoro-2-(3-benzloxy-4-methoxyphenyl)benzothiazole (6a) and 4-fluoro-2-(3,4-dimethoxyphenyl)benzothiazole (6b) was achieved by a modification of Jacobson thioanilide radical cyclization chemistry. Hydrogenolytic cleavage of the benzyl ether group of compound 6a using H₂/Pd–C provided the precursor 4-fluoro-2-(3-hydroxy-4-methoxyphenyl)benzothiazole (7) for radiolabeling. Synthesis of radiolabeling precursors and the reference standards 5- and 6-fluorinated arylbenzothiazoles (11c–n) was achieved via the reaction of o-aminothiophenol disulfides with substituted benzaldehydes under reducing conditions. The target radiotracers carbon-11 labeled 4-, 5-, and 6-fluorinated arylbenzothiazoles (3-[¹¹C]6b, 4-[¹¹C]11c, 3-[¹¹C]11c, 5-[¹¹C]11f, 4-[¹¹C]11f, 4-[¹¹C]11i, 3-[¹¹C]11i, 5-[¹¹C]11l, and 4-[¹¹C]11l) were prepared by O-[¹¹C]methylation of the phenolic hydroxyl precursors (7, 11d, 11e, 11g, 11h, 11j, 11k, 11m, and 11n) with [¹¹C]methyl triflate and isolated by solid-phase extraction (SPE) purification in 30–55% radiochemical yields.     

The cytochrome system of Bacillus megaterium KM. The presence and some properties of two CO-binding cytochromes

1. Difference spectra of Bacillus megaterium KM membrane preparations indicate the presence of two pigments which bind CO and which exhibit the spectral characteristics of cytochromes a₃ and o. Relative amounts of the pigments vary with growth stage of the organism, but both are present at all stages which have been investigated. The pigments are believed to be metabolically active because they are completely reducible by substrate (NADH) and are reoxidizable in the presence of air. CO difference spectra of whole cell suspensions are in agreement with spectra of the isolated membrane fragments. In particulate preparations and in whole cells, CO difference spectra suggest that the a₃ component binds CO much more readily than the o component; this behavior offers a possible explanation for the fact that cytochrome o has been detected in only a few other microorganisms, since CO binding is by definition the property used to identify this cytochrome.

2. A separation of the two CO-binding pigments is obtained by incubation of membrane preparations with pancreatic lipase. This treatment selectively removes the o pigment from the membrane, leaving the a₃ component associated with an enzymatically active particulate fraction.