Two-dimensional TLC Research Articles

Phytochemical and biological study of phenolic components from Geoffroea decorticans stem bark

The stem bark of Geoffroea decorticans(Gill.ex Hook. etArn.) Burk. was used medicinally to cure several skin affections; however, phytochemical and biological antecedents were not found. Analyses of purified methanolic extract from G. decorticansbark (PFGB), realized by silylation derivatization for GC/MS, C18-CC and HPLC followed by two-dimensional TLC and UV-Vis spectroscopy, allowed to characterize nine phenolic compounds, among these, two methoxy flavonoids. Antibacterial assays of PFGB showed the highest activity (MICs= 125 μg/mL) against Staphylococcus aureus(25923) and Enterococcus faecalis(29212) ATCC strains. Moreover, PFGB showed the highest intracellular antioxidant activity at low concentration (5 μg/mL), evaluated by using the fluorescent DA-H2DCF probe on lymphocyte culture; cytotoxic effects on lymphocytes activated or not by LPS were not observed, through Trypan Blue Exclusion and MTT colorimetric assays.The results obtained from the ethnomedicinal approach of this work contribute to the scientific validation of the vulnerary medicinal use of G. decorticans.

Phytochemical and biological study of phenolic fraction from Geoffroea decorticans bark

The stem bark of Geoffroea decorticans (Gill.ex Hook. et Arn.) Burk. (Fabaceae) was used medicinally to cure several skin affections; however, previous phytochemical and biological antecedents that supported its vulnerary property were not found yet. A purified methanolic fraction from G. decorticans bark (PFGB), was derivatized by silylation for GC/MS analysis, and complementary analyses by two-dimensional TLC and UV-Vis spectroscopy against detected standards compounds. Nine phenolic compounds were characterized as gallic acid, quercetin, kaempferol, apigenin, protocatechuic acid, taxifolin, 4-Hydroxybenzoic acid; among these two methoxy-flavonoids. PFGB showed significant antibacterial activity (MICs= 125 μg/mL) against Staphylococcus aureus (25923) and Enterococcus faecalis (29212) ATCC strains. Cytotoxic effects on human lymphocytes activated or not by LPS were not observed up to 100 μg/mL, by MTT colorimetric assay. The resultsobtained through the ethnopharmacological approach of this work contribute to the scientific validation of the vulnerary medicinal use of G. decorticans.

Evaluation of antioxidant and anticancer activity of crude extract and different fractions of Chlorella vulgaris axenic culture grown under various concentrations of copper ions

BackgroundChlorella vulgaris is a microalga potentially used for pharmaceutical, animal feed, food supplement, aquaculture and cosmetics. The current study aims to study the antioxidant and prooxidant effect of Chlorella vulgaris cultivated under various conc. of copper ions.MethodsThe axenic green microalgal culture of Chlorella vulgaris was subjected to copper stress conditions (0.00, 0.079, 0.158, 0.316 and 0.632 mg/L). The growth rate was measured at OD680 nm and by dry weight (DW). Moreover, the Antioxidant activity against DPPH and ABTS radical, pigments and phytochemical compounds of the crude extracts (methylene chloride: Methanol, 1:1) were evaluated. The promising Cu crude extract (0.316 mg/L) further fractionated into twenty-one fractions by silica gel column chromatography using hexane, chloroform and ethyl acetate as a mobile phase.ResultsThe obtained results reported that nine out of these fractions exhibited more than 50% antioxidant activity and anticancer activity against Hela cancer cell lines. Based on IC50, fraction No. 7 was found to be the most effective fraction possessing a significant increase in both antioxidant and anticancer potency. Separation of active compound (s) in fraction No 7 was performed using precoated silica gel plates (TLC F254) with ethyl acetate: hexane (9:1 v/v) as mobile phase. Confirmation of active compound separation was achieved by two-dimensional TLC and visualization of the separated compound by UV lamp. The complete identification of the separated active compound was performed by UV- Vis- spectrophotometric absorption, IR, MS, H1-NMRT C13-NMR. The isolated compound ((2E,7R,11R)-3,7,11,15-Tetramethyl-2-hexadecenol) have high antioxidant activity with IC50 (10.59 μg/ml) against DPPH radical assay and comparable to the capacities of the positive controls, Butylated hydroxy toluene [BHT] (IC50 11.2 μg/ml) and Vitamin C (IC50 12.9 μg/ml). Furthermore, pure isolated compound exhibited a potent anticancer activity against Hela cell line with IC50 (4.38 μg/ml) compared to Doxorubicin (DOX) as synthetic drug (13.3 μg/ml). In addition, the interaction of the pure compound with Hela cancer cell line and gene expression were evaluated.ConclusionsThe authors recommend cultivation of Chlorella vulgaris in large scale under various stress conditions for use the crude extracts and semi purified fractions for making a pharmaco-economic value in Egypt and other countries.

Pseudarthrobacter psychrotolerans sp. nov., a cold-adapted bacterium isolated from Antarctic soil.

A novel cold-tolerant bacterium, designated strain YJ56T, was isolated from Antarctic soil collected from the Cape Burk area. Phylogenetic analysis through 16S rRNA gene sequence similarity revealed that strain YJ56T was most closely related to the genus Pseudarthrobacter, including Pseudarthrobacter oxydans DSM 20119T (99.06 % similarity), Pseudarthrobacter polychromogenes DSM 20136T (98.98 %) and Pseudarthrobacter sulfonivorans ALLT (98.76 %). The genome size (5.2 Mbp) of strain YJ56T was the largest among all the published genomes of Pseudarthrobacter type strains (4.2-5.0 Mbp). The genomic G+C content of strain YJ56T (64.7 mol%) was found to be consistent with those of other Pseudarthrobacter strains (62.0-71.0 mol%). The average nucleotide identity and average amino acid identity values between strain YJ56T and P. sulfonivorans ALLT were estimated at 84.1 and 84.2 %, respectively. The digital DNA-DNA hybridization value between the two strains was calculated to be 28.0 %. This rod-shaped and obligate aerobic strain exhibited no swimming or swarming motility. It had catalase activity but no oxidase activity. Cells grew at 4-28 °C (optimum, 13 °C) and pH 5.0-11.0 (optimum, pH 7.0) and with 0-6.0 % (w/v) NaCl (optimum, 0%) in Reasoner's 2A medium. MK-9 (H2) was the sole menaquinone. Two-dimensional TLC results revealed that the primary polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two glycolipids and phosphatidylinositol. Fatty acid methyl ester analysis showed that anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0, C16 : 0 and iso-C16 : 0 were the major cellular fatty acids in strain YJ56T. Based on phenotypic and genotypic characteristics, strain YJ56T represents a novel species of the genus Pseudarthrobacter, and thus the name Pseudarthrobacter psychrotolerans sp. nov is proposed. The type strain is YJ56T (=JCM 33881T=KACC 21510T).

Quantitative analysis of acetaminophen, ethoxybenzamide, piroxicam, hydrochlorothiazide, caffeine, chlorzoxazone and nicotinamide illegally adulterated in Chinese medicinal pills

A traditional Chinese medicine, which was supplied as pills by a temple and used as Ch'i tonic, was obtained for testing the illegal adulteration with chemical drugs. In the qualitative analysis, two-dimensional TLC, with chloroform:ethanol(9:1,v/v) and ethyl acetate: methanol: ammonia water(8:1:1,v/v) as the solvent systems, established the presence of acetaminophen, piroxicam, hydrochlorothiazide, caffeine, ethoxybenzamide, chlorzoxazone and nicotinamide in the pills. In quantitative analysis, a reversed-phase HPLC method has been established for simultaneous determination of the seven chemical drugs. The analysis of the sample extract was performed on a Cosmosil 5C18-AR column (4.6 × 150mm) by a linear gradient elution with 1%triethylamine - 1% phosphoric acid buffer, acetonitrile and methanol(0 min, 90:5:5; 60min, 40:30:30; 70min,90:5:5) as the mobile phase at 1ml/min and with fenbufen as an internal standard. The monitor was set at 260nm. System suitability tests showed that the established method was quite suitable for the determination of the adulterants in pills. By using this method, 78mg of acetaminophen, 16mg of piroxicam, 30mg of caffeine, 30mg of ethoxybenzamide, 41mg of chlorzoxazone, 4mg of hydrochlorothiazide and 16mg of nicotinamide, were found in each pill of the sample.

Quantitative analysis of caffeine, ethoxybenzamide, chlorzoxazone, diazepam and indomethacine illegally adulterated in Chinese medical pills

A traditional Chinese medicine, which was brought by a consumer for treating heart disease and uricosuria, was obtained and tested the illegal adulteration with chemical drugs. In the qualitative analysis, two-dimensional TLC, with chloroform:ethanol (9:1,v/v) and ethyl acetate: methanol: ammonia water (8:1:1,v/v) as the solvent systems, established the presence of caffeine, indomethacin, ethoxybenzamide, chlorzoxazone and diazepam in the pills. For quantitative analysis, a reversed-phase HPLC method was established for simultaneous determination of the five chemical drugs. The analysis of the sample extract was performed on a Cosmosil 5C 18-AR column (4.6x150mm) by a linear gradient elution with the mobile phase of 0.05 M KH 2 PO 4-0.1% formic acid buffer and methanol, programmed from 60:40, v/v, to 40:60, v/v, for 30 min, with 30 mim hold and then 60:40, v/v, for the subsequent 15 min, at a flow rate of 0.7 ml/min and with propylparaben as an internal standard. The monitor wavelength was set at 230 nm. System suitability tests showed that the established method was quite suitable for the determination of the adulterants in the pills. By using this method, 30.8mg of caffeine, 71.4 mg of ethoxybenzamide, 35.7 mg of chlorzoxazone, 0.6 mg of diazepam and 9.3 mg of indomethacin, were found in each pill of the sample.

PREPARATION OF PHTHALONITRILE MONOMER WITH LOW MELTING POINT

This article presents the results of the synthesis of para – and meta-hydroxyphenoxyphalonitrile,phosphorus-containing chlorangidrides with various substituents in phosphorus and 4-(3-propargyloxyphenoxy)phthalonitrile, the introduction of which into the binder, allowed to achieve the necessary viscosity indicators withoutloss of heat resistance. At the first stage, it was proposed to obtain a monomer with a phenoxy group in phosphorus.For this purpose, we proposed the use of dimethylacetamide (DMAA) with K2CO3, but the product washydrolytically unstable in the main medium, so it was impossible to isolate the desired monomer. To solve thisproblem, we conducted a number of experiments on the selection of synthesis conditions using various solvents.Comparing the Tgt of the obtained monomers, we found higher values for para-oriented phthalonitriles, which isprobably due to higher structure symmetry, as well as an increase in glass transition temperature (Tgt) forphosphonate-based monomers. Due to the equally high thermal properties of the cured matrices, we decided to use4-(3-hydroxyphenoxy) phthalonitrile as the main precursor, as well as phosphate chlorangidrides. Based on thecalculated data, a pattern was identified that consists in a decrease in the Tgt of phosphorus-containing phthalonitrileswhen switching to aliphatic substituents in phosphorus. A decrease in the Tgt of monomers was found with anincrease in the size of the aliphatic substituent. As a result of the experiment, we obtained 9 new phosphoruscontaining phthalonitrile monomers with high yield that are resistant to hydrolysis, which is confirmed by the factthat the products were isolated by flash chromatography on silica gel, as well as two-dimensional TLC. Thesynthesized substances were glassy masses with Tgt = - 5-58 °C. The consistency of the approach involving theintroduction of phosphate bridges into the structure of phthalonitriles was shown.

Methylobacterium currus sp. nov., isolated from a car air conditioning system

A novel bacterial strain, designated PR1016AT, was isolated from a car air conditioning system. This rod-shaped strain showed catalase and oxidase activities, was aerobic and methylotrophic, and had a reddish pink colour. The genomic DNA G+C content of strain PR1016AT was 70.2 mol%, as determined by genome sequencing. Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain PR1016AT was most closely related to Methylobacterium aquaticum GR16T (98.86 %), M. variabile GR3T (98.43 %), M. platani PMB 02T (98.36 %) and M. tarhaniae N4211T (98.14 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain PR1016AT and M. aquaticum GR16T, M. platani PMB02T and M. variabile GR3T were 88.61, 88.14 and 87.88 %, and 36.4, 35.8 and 34.7 %, respectively. Numerous insertion sequences are present in the genome of strain PR1016AT, which has a larger genome than the four Methylobacterium species described above. Cells grew at 18-42 °C (optimum, 30 °C), at pH 4.0-9.0 (optimum, pH 7.0) and in the presence of 0-1.0 % (w/v) NaCl (optimum, 0 %). The major respiratory quinone was Q10. Fatty acid methyl ester analysis revealed that summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) was the predominant cellular fatty acid in strain PR1016AT. Two-dimensional TLC indicated that the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The genotypic and phenotypic characteristics indicate that strain PR1016AT represents a novel species of the genus Methylobacterium, for which the name Methylobacterium currus sp. nov. is proposed. The type strain is PR1016AT (=KACC 19662T=JCM 32670T).

Isolation of Alkaloid Compounds from Ethanol Extract of Rimpang Galang Merah (Alpinia purpurata (Vielli) K. Schum) and nanoparticle production from its Alkaloid Extract. Comparative Study of Antibacterial Properties on Staphylococcus aureus and Eschericia

Isolation of alkaloid compounds from ethanol extract of red galangal rhizomes to compare their antibacterial properties with extracts formed into nanoparticles has been performed. The isolation of the alkaloid compounds was performed successively by salting using (i) hydrochloric acid (HCl) to pH 3 (when in ethanol) and using (ii) ammonium hydroxide (NH4OH) to pH 10 (when in chloroform). The alkaloid concentrated extract was obtained after chloroform was evaporated using a rotary evaporator. This alkaloid extract was then carried out by TLC silica gel GF254 using chloroform eluent: ethyl acetate (9: 2) to obtain a specific alkaloid type as a single/pure compound. Test of alkaloid purity successfully isolated by one-dimensional or two-dimensional TLC using various types of eluents with varying degrees of polarity, ie acetone, chloroform, ethyl acetate, ethanol and chloroform: ethyl acetate mixture (9: 2). Isolates of pure alkaloids were analyzed using UV-Vis spectrophotometers), FTIR and (LC-MS). The obtained alkaloid isolates were isokuinolin group with UV-visible absorption characteristics at maximum wavelength (λmaks) of 212 nm, 227 nm and 261 nm. The alkaloid isolate also showed the presence of functional groups -OH, -C-H, C = O, C = C, C = N, C-N, and C-O with molecular weight of 262.90 g/mol. The production of ethanol extract nanoparticles containing alkaloid-containing red galangal (EEAlkNPs) was performed using a top-down approach using ultrason (40 kHz, 2x50 watt) for 10 minutes. The EEAlkNPs size was determined by the dynamic light scattering method (DLS) using the Particles size analyzer (PSA) tool and obtained an average particle size of 220.2 nm. The antibacterial properties of EEAlkNPs were tested by disc diffusion method for the culture of S.aureus and E.coli bacteria. It is known that the antibacterial properties of EEAlkNPs are relatively larger than the original ethanol extract at the same concentration, ie, 2000 parts per million (bpj) or g / L.

HPLC, Two-Dimensional TLC Determination of Phenolic Content, and anIn VitroPerspective to Antioxidant Potential ofEuonymus verrucosusScop. Extracts

Summary. The presence of phenolic content in overground extracts of Euonymus verrucosus Scop. – commonly growing in Europe – has been reported recently. The chromatographical and spectral data revealed the presence of several simple phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, vanillic, syringic, caffeic, p-coumaric, ferulic, and m-coumaric acids), both as free and conjugated with other secondary metabolites. The comparison of two-dimensional TLC systems on cellulose stationary phases with HPLC– DAD reversed-phase chromatography was performed to assess a cheap and rapid technique in the identification process of major phenolic constituents. 2,2-Diphenyl-1picrylhydrazyl (DPPH) radical-scavenging tests, expressed as IC50, revealed the most beneficial results for the fraction after alkaline hydrolysis and yielded 205 ± 8 μg mL−1.

Lipid Composition of the Birch Mushroom Inonotus obliquus

Inonotus obliquus (birch mushroom or chaga) is the sterile conk of the fungus. Preparations of chaga have been used in both folk and official medicine. Thus, the chaga preparation Befungin is described in the Russian pharmacopoeia. The basic composition of chaga preparations and characteristic water-soluble (pigment and polyphenol) complexes in addition to directions for the use of chaga preparations for oncological diseases were reported. Information on the chemical composition of I. obliquus, in particular its lipid composition, are exceedingly limited. A broad spectrum of activity was demonstrated for I. obliquus and its preparations [1–4]. Many researchers relate the effects of I. obliquus with compounds such as phenol complexes, polysaccharides, and melanin [4, 5]. We investigated the lipid composition of chaga because several of them also exhibit antitumor activities. They include glyceryl ethers (GE) [6, 7] and prostaglandins (PG) [8, 9]. There is also information that fungi, including I. obliquus, contain oxylipins [10]. The I. obliquus studied by us contained 1.25% total lipids. We found that the PG and lipid contents were subject to seasonal oscillations (depending on the collection time). Whereas the lipid content in autumn was 0.5%, it tripled in January and was 1.5%. Seasonal variations were also noted in the PG content. In October, PG was 0.9%; in January, 0.3%. The dependence was reverse of that of the lipids. Lipids from I. obliquus were analyzed by oneand two-dimensional TLC. The lipid extract contained neutral lipids such as diand triglycerides, phytosterols, fatty alcohols, free fatty acids (FA), and FA esters. Phosphoand glycolipids were not observed. According to the literature, I. obliquus exhibited antitumor activity [1, 5] that was related to a polysaccharide complex. The antitumor activity in the lipids was associated with GE [11]. The -GE were hydrocarbon derivatives of glycerin with an ether bond in the glycerin 1-position and were remarkable because one representative of this class, batyl alcohol, was described in the Russian pharmacopoeia as a radioprotective agent. Therefore, we determined the -GE content in the lipid extract of I. obliquus using the method proposed by us earlier [12]. The -GE made up 0.6% of the total lipids. The FA composition and content of I. obliquus are given below.

A Bifunctional Glycosyltransferase from Agrobacterium tumefaciens Synthesizes Monoglucosyl and Glucuronosyl Diacylglycerol under Phosphate Deprivation

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Studies on Medicago lupulina saponins. 6. Some chemical characteristics and biological activity of root saponins

The saponin fraction of black medic trefoil (<em>Medicago lupulina</em>) roots was isolated and purified. Its hydrolysis afforded several aglycones that have been identified by spectral and chemical properties as medicagenic acid, hederagenine and soyasapogenols: B, C, D, E and F. They made up, respectively. 46.9 and 45% of total sapogenins isolated. Two-dimensional TLC of <em>M. lupulina</em> root saponins revealed fourteen compounds, two of which were medicagenic acid glycosides. The haemolytic, antifungal and allelopathic activities of <em>M. lupulina</em> and <em>M. media</em> roots are compared and discussed.

Characterization of Ellagitannins, Gallotannins, and Bound Proanthocyanidins from California Almond (Prunus dulcis) Varieties

Extractable and bound proanthocyanidins and hydrolyzable tannins were characterized in Nonpareil, Carmel, and Butte almond varieties from California, with n = 3 samples/variety. Bound proanthocyanidins were recovered from extracted defatted almond residue by hydrolysis with 4 N sodium hydroxide and represented 3-21% of the total proanthocyanidin content among varieties. The bound proanthocyanidins were recovered primarily as monomers and dimers. In contrast, acid hydrolysis of extracted almond residue did not yield bound proanthocyanidins. Hydrolyzable tannins were characterized in aqueous acetone extracts of defatted almond using two-dimensional TLC and further quantitated by HPLC following acid hydrolysis. Almond hydrolyzable tannin content was 54.7 ± 2.3 mg ellagic acid and 27.4 ± 7.3 mg gallic acid per 100 g almond among varieties. The tannin contents of Nonpareil, Carmel, and Butte almond varieties were not significantly different. Thus, bound proanthocyanidins and hydrolyzable tannins significantly contribute to almond polyphenol content.

Exposure of Mycobacteria to Cell Wall-inhibitory Drugs Decreases Production of Arabinoglycerolipid Related to Mycolyl-arabinogalactan-peptidoglycan Metabolism

The "cell wall core" consisting of a mycolyl-arabinogalactan-peptidoglycan (mAGP) complex represents the hallmark of the mycobacterial cell envelope. It has been the focus of intense research at both structural and biosynthetic levels during the past few decades. Because it is essential, mAGP is also regarded as a target for several antitubercular drugs. Herein, we demonstrate that exposure of Mycobacterium bovis Bacille Calmette-Guérin or Mycobacterium marinum to thiacetazone, a second line antitubercular drug, is associated with a severe decrease in the level of a major apolar glycolipid. This inhibition requires MmaA4, a methyltransferase reported to participate in the activation process of thiacetazone. Following purification, this glycolipid was subjected to detailed structural analyses, combining gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance. This allowed to identify it as a 5-O-mycolyl-β-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1)-Gro, designated dimycolyl diarabinoglycerol (DMAG). The presence of DMAG was subsequently confirmed in other slow growing pathogenic species, including Mycobacterium tuberculosis. DMAG production was stimulated in the presence of exogenous glycerol. Interestingly, DMAG appears structurally identical to the terminal portion of the mycolylated arabinosyl motif of mAGP, and the metabolic relationship between these two components was provided using antitubercular drugs such as ethambutol or isoniazid known to inhibit the biosynthesis of arabinogalactan or mycolic acid, respectively. Finally, DMAG was identified in the cell wall of M. tuberculosis. This opens the possibility of a potent biological function for DMAG that may be important to mycobacterial pathogenesis.

Imaging Technology of Complex Lipid Molecular Species by a Combination of TLC-Blot and MALDI-TOF –Special Reference to Human Brain Ganglioside Molecular Species

Glycosphingolipids and phospholipids from white matter of human hippocampus, were analyzed by a sequential procedure of two-dimensional TLC (2d-TLC), transference of separated lipids to a PVDF membrane by a TLC-Blot equipment and Mass spectrometry (MS) analysis with an ion-trap type MALDI-TOF equipment. The method is simple and quick; very small amount of sample (0.1 mg of brain tissue) is enough to analyze all lipid components. The 2d-TLC provided excellent separation and the MS analyses allowed identifying the characteristic profile of molecular species for individual glycolipids and phospholipids. The results of MS analyses on gangliosides showed that di- and tri-sialogangliosides are richer in d20:1 sphingosine-containing ceramide than monosialogangliosides, suggesting the presence of sialylation selection after GM1 gangliosides. Then we analyzed ganglioside molecular species obtained from different brain regions by using MS imaging technology. The MS images of individual gangliosides provided clear visual profiles in terms of molecular species distribution. The imaging profiles were region dependent and also indicated that the sialyltransferase toward GM1 ganglioside prefers to select d20:1 sphingosine containing molecule. This technology provides visual characterization of individual phospholipid and glycosphingolipid molecular species and informs us about the metabolic characterization of target tissue, opening a new gate for colorful lipidomics research.

Phosphorylation of PhoP Protein Plays Direct Regulatory Role in Lipid Biosynthesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis PhoP is essential for virulence and intracellular growth of the tubercle bacilli. Genetic evidence suggests that PhoP regulates complex lipid biosynthesis, and absence of some of these lipid molecules in a phoP mutant partly accounts for its attenuated growth in macrophages and/or mice. To investigate the mechanism of regulation, here we demonstrate the essentiality of phosphorylation of PhoP in the regulation of complex lipid biosynthesis. We show that phosphorylated PhoP activates transcription of pks2 and msl3, gene(s) encoding polyketide β-ketoacyl synthases through direct DNA binding at the upstream regulatory region(s) of the target genes. Our results identify the genetic determinants recognized by PhoP and show that activation of target genes requires interaction(s) of the phosphorylated regulator at the cognate binding sites. The fact that these sites within the regulatory region of respective genes do not bind in vitro with either unphosphorylated or phosphorylation-deficient PhoP protein is consistent with phosphorylation-dependent assembly of the transcription initiation complex leading to in vivo transcriptional activation. Together, these results reveal so far unknown molecular mechanisms of how PhoP contributes to M. tuberculosis cell wall composition by regulating complex lipid biosynthesis.

Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure

Archaeal and eukaryotic tRNA (N(2),N(2)-guanine)-dimethyltransferase (Trm1) produces N(2),N(2)-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-l-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-l-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.

2D TLC separation of phenols by use of RP-18 silica plates with aqueous and non-aqueous mobile phases

Two-dimensional (2D) TLC of eleven phenols has been performed on octadecyl-silica. The most efficient systems were selected by analysis of retention data obtained by one-dimensional chromatography using aqueous and non-aqueous mobile phases. Correlation graphs were plotted to illustrate the separation in the chromatographic systems examined. Complete separation of the phenols was achieved by using an aqueous mobile phase in the first dimension and a non-aqueous mobile phase in the second dimension. Statistical methods (principal-components analysis and cluster analysis) were used for better characterization of the TLC systems.

Preparation of CIT-protein conjugates using a novel citrinin derivative as hapten

In this paper, a novel citrinin (CIT) derivative, 8-MeO-CIT, was synthesized by the method of methylation, with the protection of hydroxyl in CIT using methoxyl for the first time. The overall yield of 8-methoxyl-citrinin (8-MeO-CIT) was about 49.8%. 8-MeO-CIT was then purified by two-dimensional TLC, and its characteristics were analyzed by HPLC and ESI–MS. 8-MeO-CIT-BSA and CIT-BSA were prepared using the NHS-ester method and the Mannich-type method, respectively. Characteristics of both CIT-protein conjugates were determined by UV–Vis and FT-IR spectroscopy. Results showed that the conjugate molar ratio and utilized ratio of CIT in 8-MeO-CIT-BSA increased by 4.1 and 18.4% compared to CIT-BSA. It suggested that NHS-ester method using novel 8-MeO-CIT as hapten might prefer to traditional Mannich-type method for preparing CIT-protein conjugates.