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Thin-layer Chromatography (thin-layer + chromatography)
Kinds of Thin-layer Chromatography Selected AbstractsThe Rubino test for leprosy is a ,2 -glycoprotein 1-dependent antiphospholipid reactionIMMUNOLOGY, Issue 1 2000A. Panunto-Castelo Summary We describe the isolation and identification of three components required for the Rubino reaction (RR), which is the rapid sedimentation of formalinized sheep red-blood cells (SRBC) initiated by serum from leprosy patients with defective Mycobacterium leprae -specific cell immunity. The Rubino reaction factor (RRF) required for this phenomenon, previously identified as an immunoglobulin M (IgM), was purified from leprosy patient serum by adsorption to formalinized SRBC. Purified RRF IgM, when added to formalinized SRBC, did not produce a positive RR. However, when the contact was carried out in the presence of normal human serum (NHS), cells rapidly sedimented. The purified cofactor from NHS contained two components of 70 000 and 50 000 molecular weight (MW), as determined by sodium dodecyl sulphate,polyacrylamide gel electrophoresis (SDS,PAGE). The latter was recognized by the RRF IgM on immunoblot and its N-terminal sequence indicated that it was ,2 -glycoprotein 1 (,2 -GP1), an anionic phospholipid-binding protein. Methanol-treated formalinized SRBC did not support the RR. Thin-layer chromatography of an extract of membranes indicated that the SRBC ligand was a cell-surface phospholipid. Cardiolipin inhibited the RR. These data demonstrate that the RR involves a trimolecular interaction in which IgM, ,2 -GP1 and an SRBC phospholipid participate. By analogy with the antiphospholipid antibodies (anti-PL) that occur in autoimmune processes, serum samples from 29 systemic lupus erythematosus patients with high levels of anticardiolipin antibodies were submitted to the RR. A positive RR was obtained for 45% (13 of 29 patients). These results modify the paradigm of the absolute specificity of the RR for leprosy and demonstrate that RRF IgM is a ,2 -GP1-dependent anti-PL. [source] Possible mechanisms for the relative efficacies of ortho -phthalaldehyde and glutaraldehyde against glutaraldehyde-resistant Mycobacterium chelonaeJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2001S.E. Walsh Aims: This investigation compared glutaraldehyde (GTA)-sensitive and -resistant strains of Mycobacterium chelonae and examined the effects of pretreatment of GTA-sensitive and -resistant strains of Myco. chelonae with chemical agents that interfere with cell wall synthesis. Methods and Results: When exposed to 2% (v/v) GTA at 25°C, GTA-resistant strains of Myco. chelonae dried on to glass carriers were not inactivated to any significant extent. By contrast, GTA-sensitive strains of Myco. chelonae and a strain of Myco. terrae suffered a > 6 log reduction in viability in 5 min. However, ortho -phthalaldehyde (OPA; 0·5% w/v) achieved a corresponding inactivation against two GTA-resistant strains within 5,10 and 10,20 min, respectively. Electron microscopy, using a non-aldehyde fixation process and also negative staining, failed to detect any extensive changes in GTA-sensitive and -resistant cultures exposed to GTA or OPA. Thin-layer chromatography was unsuccessful in detecting differences between GTA-resistant and -sensitive strains of Myco. chelonae. However, pretreatment of GTA-resistant cells with mycobacterial cell wall synthesis inhibitors increased their subsequent susceptibility further to OPA but not to GTA. Conclusions:Ortho -phthalaldehyde is an effective new biocidal agent that, at its in-use concentration, is rapidly bactericidal to non-sporulating bacteria, including GTA-sensitive and -resistant mycobacteria. Significance and Impact of the Study: Pretreatment of GTA-resistant cells with mycobacterial cell wall synthesis inhibitors increased their subsequent susceptibility to OPA but not to GTA. [source] Thin-layer chromatography combined with diode laser desorption/atmospheric pressure chemical ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2004Song Peng The desorption of an analyte by a continuous wave diode laser from a porous surface of a thin-layer plate covered with a graphite suspension is presented. The thermally desorbed analyte molecules are ionized in the gas phase by a corona discharge at atmospheric pressure. Therefore, both essential processes,the desorption and the ionization of analyte molecules, which are often performed in one step,are separated. The target preparation is easy and fast since no additional extraction process is required. The mass spectrometric background signal was mostly limited to the low mass range showing no interference with typical compounds of interest. In this study, the calmative and antihypertensive drug reserpine was chosen as model analyte, which is often used for specification of mass spectrometers. No fragmentation was observed because of efficient collisional cooling under atmospheric pressure. The influence of diode laser power and the composition of the graphite suspension were investigated, and a primary optimization was performed. Copyright © 2004 John Wiley & Sons, Ltd. [source] FS01.3 Disperse (yes), orange (yes), 3 (no): what do we test in textile dye dermatitis?CONTACT DERMATITIS, Issue 3 2004Christophe J Le Coz Introduction:, Patients sensitized to para-phenylenediamine (PPD) have a high degree of patch test reactivity to Disperse Orange 3 (DO3), and a lesser one to Disperse Red 1 and Red 17. Two successive patients positive to PPD, Disperse Red 1 and 17, negative to DO3 were real eye-openers for our considerations about purity of our current allergen DO3. Materials and methods:, We realized comparative thin-layer chromatography (TLC), with DO3 from Chemotechnique®(DO3-Chem) and Trolab®(both extracted from petrolatum), and "pure" DO3 from two chemical providers. TLC clearly indicated that DO3-Chem was not DO3. HPLC analysis with pure DO3 from Chemotechnique® and comparison of structures by NMR with samples of DO3, revealed that DO3-Chem was Disperse Orange 31 (DO31). In addition, signals through the GERDA network allowed the collection of test materials and observations. Among other members, only 2 used DO3-Chem (from 2 different batches) that was DO31 too, according to TLC Results: According to their data, they observed no or a lower reactivity to DO3 than expected (4 patients DO3-Chem + among 23 PPD+ e.g.). Finally, the error was proved to be due to the provider of the dye to Chemotechnique®, who likely deleted the 1 of Disperse Orange 31 on his packaging. Discussion:, Chemical structure of DO31 indicates a possible in vivo hydrolysis into nitroaniline and a second compound, a substituted PPD derivative that clearly does not frequently react in PPD positive patients. Like drugs, patch tests are submitted to post-commercialization controls. In addition to allergens providers who should enhance their quality controls, dermato-allergologists have to be vigilant, and must active networks when they observe a rare bird. [source] Cover Picture: Electrophoresis 21'2009ELECTROPHORESIS, Issue 21 2009Article first published online: 27 OCT 200 Issue no. 21 is a regular issue with Emphasis on "Nucleic Acids". The first part has 7 articles on nucleic acids covering various topics, e.g., sequencing, genotyping, PCR, insertion, mutation, etc. The remaining 11 articles are concerned with monoliths, pseudo-phases, coating, and sample pretreatment such as derivatization and concentration. Selected articles are: Applications of MALDI-TOF MS to large-scale human mtDNA population-based studies ((10.1002/elps.200900294)) Visual DNA as a diagnostic tool ((10.1002/elps.200900273)) Preliminary results for two-dimensional separation with high performance thin-layer chromatography and pressurized planar electrochromatography ((10.1002/elps.200900471)) [source] Comparative in vitro and in vivo genotoxicities of 7H -benzo[c]fluorene, manufactured gas plant residue (MGP), and MGP fractionsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2004Leslie Cizmas Abstract Manufactured gas plant residue (MGP) is a complex mixture of polycyclic aromatic hydrocarbons (PAHs) that is tumorigenic in the lungs of mice. This study compared the relative genotoxicity of 7H -benzo[c]fluorene (BC), a PAH component of MGP, with MGP and MGP fractions in order to assess the contribution of BC to the genotoxicity of MGP. An MGP sample was separated into seven fractions (F1,F7) using silica gel column chromatography with petroleum ether (PE) followed by PE:acetone (99:1 v/v, then 98:2). PAHs were quantified using gas chromatography/mass spectrometry. An aliquot of F2, the fraction with the highest BC concentration and highest weighted mutagenic activity in Salmonella typhimurium strain TA98, was further separated using silica gel thin-layer chromatography with hexane. The first F2 subfraction, sF2-a, was enriched in BC and coeluting compounds and contained 35,000 ppm BC and 216,109 ppm carcinogenic PAHs (cPAHs, the sum of seven PAHs categorized by the U.S. EPA as class B2 carcinogens). The second F2 subfraction, sF2-b, contained a ninefold lower concentration of BC, with 3,900 ppm BC and 45,216 ppm cPAHs. Female ICR mice received topical application of crude MGP, crude MGP spiked with analytical-grade BC, F2, sF2-a, sF2-b, or analytical-grade BC. DNA adduct levels were analyzed by nuclease P1-enhanced 32P-postlabeling. In lung DNA of mice receiving 0.48 or 3.0 mg/mouse, net total RAL × 109 values were F2, 30.8 and 87.2; sF2-a, 24.8 and 106.7; and sF2-b, 19.6 and 151.0, respectively. Mice dosed with 0.10 mg analytical-grade BC (the mass of BC in 3.0 mg sF2-a) exhibited a net total RAL × 109 value of 7.03 in lung DNA. This was equal to approximately 7% of the total RAL × 109 value produced by 3.0 mg sF2-a. Thus, although BC appears to make an appreciable contribution to pulmonary adduct formation, the results suggest that MGP components other than BC play an important role in lung DNA adduct formation following topical MGP administration. Environ. Mol. Mutagen. 43:159,168, 2004. © 2004 Wiley-Liss, Inc. [source] Fatty acid metabolism assessed by 125I-iodophenyl 9-methylpentadecanoic acid (9MPA) and expression of fatty acid utilization enzymes in volume-overloaded heartsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2004T. Miyamoto Abstract Background, The peroxisome proliferator-activated receptor (PPAR) , is a member of the nuclear receptor superfamily and regulates gene expression of fatty acid utilization enzymes. In cardiac hypertrophy and heart failure by pressure-overload, myocardial energy utilization reverts to the fetal pattern, and metabolic substrate switches from fatty acid to glucose. However, myocardial metabolism in volume-overloaded hearts has not been rigorously studied. The aim of the present study was to examine fatty acid metabolism and protein expressions of PPAR, and fatty acid oxidation enzymes in volume-overloaded rabbit hearts. Methods, Volume-overload was induced by carotid-jugular shunt formation. Sham-operated rabbits were used as control. Chronic volume-overload increased left ventricular weight and ventricular cavity size, and relative wall thickness was decreased, indicating eccentric cardiac hypertrophy. 125I-iodophenyl 9-methylpentadecanoic acid (9MPA) was intravenously administered, and animals were sacrificed at 5 min after injection. The 9MPA was rapidly metabolized to iodophenyl-3-methylnonanoic acid (3MNA) by ,-oxidation. Lipid extraction from the myocardium was performed by the Folch method, and radioactivity distribution of metabolites was assayed by thin-layer chromatography. The protein was extracted from the left ventricular myocardium, and levels of PPAR, and fatty acid oxidation enzymes were examined by Western blotting. Results, Myocardial distribution of 9MPA tended to be more heterogeneous in shunt than in sham rabbits (P = 0·06). In volume-overloaded hearts by shunt, the conversion from 9MPA to 3MNA by ,-oxidation was faster than the sham-control hearts (P < 0·05). However, protein levels of PPAR, and fatty acid utilization enzymes were unchanged in shunt rabbits compared with sham rabbits. Conclusions, These data suggest that myocardial fatty acid metabolism is enhanced in eccentric cardiac hypertrophy by volume-overload without changes in protein expressions of PPAR, and fatty acid utilization enzymes. Our data may provide a novel insight into the subcellular mechanisms for the pathological process of cardiac remodelling in response to mechanical stimuli. [source] A simplified method for HPLC-MS analysis of sterols in vegetable oilEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 12 2008Antonio Segura Carretero Abstract We have developed a liquid-chromatographic method using atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) detection in positive mode. This method was used to separate and identify 15,sterols and 2,dihydroxy triterpenes in saponified oils, enabling the analysis of these compounds directly from saponified samples without recourse to thin-layer chromatography; this fact thus significantly simplifies the process. The analyses were made using a Waters Atlantis 5,µm dC18 150×2.1,mm column with a gradient of acetonitrile/water (0.01% acetic acid) at a flow rate of 0.5,mL/min and a column temperature of 30,°C. The quantification of several of these compounds in soybean oil, palm oil, seed oil, sunflower oil, olive-pomace oil and virgin olive oil was carried out using their commercial standards, and the results were compared satisfactorily with the official method. [source] Positional distribution of fatty acids in triacylglycerols and phospholipids from adzuki beans (Vigna angularis)EUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 2 2008Hiromi Yoshida Abstract The fatty acid distributions of triacylglycerols (TAG) and major phospholipids (PL) obtained from adzuki beans (Vigna angularis) were investigated. The total lipids extracted from the beans were separated by thin-layer chromatography (TLC) into eight fractions. The major lipid components were PL (63.5,wt-%), TAG (21.2,wt-%), steryl esters (7.5,wt-%) and hydrocarbons (5.1,wt-%), while free fatty acids, diacylglycerols (1,3-DAG and 1,2-DAG) and monoacylglycerols were also present in minor proportions (0.2,1.1,wt-%). The major PL components isolated from the beans were phosphatidylcholine (45.3,wt-%), phosphatidylethanolamine (25.8,wt-%) and phosphatidylinositol (21.5,wt-%). Phosphatidylinositol was unique in that it had the highest saturated fatty acid content among the three PL. With a few exceptions, however, the principal characteristics of the fatty acid distribution in the TAG and three PL were evident in the beans: Unsaturated fatty acids were predominantly concentrated in the sn -2 position while saturated fatty acids primary occupied the sn -1 or sn -3 position in the oils of the adzuki beans. In general, these results could be useful to both consumers and producers for the manufacture of traditional adzuki foods in Japan. [source] Photoinduced Formation of Reactive Oxygen Species from the Acid Form of 6-(Hydroxymethyl)pterin in Aqueous SolutionHELVETICA CHIMICA ACTA, Issue 6 2006Andrés Abstract The photochemistry of 6-(hydroxymethyl)pterin (HPT; 1) in aqueous solution (pH 5,6) was investigated by irradiation at 350,nm at room temperature. The photochemical reactions of the acidic form 1a were followed by UV/VIS spectrophotometry, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and enzymatic methods for the determination of the superoxide anion radical (O) and hydrogen peroxide (H2O2). When 1a is exposed to UV-A radiation, the intermediates 4 and 4, are formed reacting with O2 to yield 6-formylpterin (FPT; 5) and 6-carboxypterin (CPT; 6) under formation of O and H2O2 (Scheme,3). The quantum yields of the disappearance of HPT (1a) and of the formation of the photoproducts 5 and 6 were determined. HPT was investigated for its efficiency in singlet-oxygen (1O2) production in acidic aqueous solution. The corresponding quantum yield of 1O2 production (,,) was 0.15,±,0.02, as measured by the 1O2 luminescence in the near-IR (1270,nm) upon continuous excitation of the sensitizer. However, 1O2 does not participate in the actual photooxidation of HPT (1a) to FPT (5) and CPT (6). [source] Efficient method of cyclic imides synthesis under ozone influence by the example of ,-caprolactam oxidation reactionHETEROATOM CHEMISTRY, Issue 7 2008Olga Alekseeva The process of ,-caprolactam oxidation by ozone in a CCl4 solution was investigated. Stoichiometric coefficient (1:1) and the mechanism of oxidation were identified. The composition and structure of the ozonolysis reaction products and the ozone attack on the caprolactam molecule were determined by IR and 1H NMR spectroscopy methods, as well as using thin-layer chromatography. It was found that the ozonolysis reaction is rather selective. The main reaction product,azepane-2,7-dione (Scharf et al., Angew Makromol Chem 1979, 79, 193),was formed with a yield of more than 90% and can be used as an intermediate during the production of commercially important antibiotics. Kinetics of the reaction obeyed the bimolecular law, the effective rate constant of the reaction in the CCl4 solution is equal to kr = 22.7 l/(mol s). The method of fast conversion of lactame to cycloimide under the ozone influence is reported. © 2008 Wiley Periodicals, Inc. Heteroatom Chem 19:661,666, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.20492 [source] Analysis of hair lipids and tensile properties as a function of distance from scalpINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 4 2005L. Duvel Synopsis Cuticle cells form the outer covering surrounding and protecting the cortex. The cuticle cells are thin, flat and overlap, and intercellular lipid lamellae are found in the gaps between the cell boundaries. The lipid lamellae are also found within the cortex in the cell boundaries between the long fribrous corticle cells. In addition, the outer surfaces of the cuticle cells are covered by a monolayer of covalently bound fatty acids, a major component of which is 18-methyleicosanoic acid. The fatty acids are thought to be attached through thio-ester linkages. Together these lipids are thought to be major determinants of the physical properties of the hair. The present study tested the hypothesis that both free and covalently bound lipids are progressively lost during normal environmental exposures. This progressive loss within the cuticle layers may, in part, lead to an increased susceptibility of the protein and lipid lamellae in the cortex to degradation. This degradation, in turn, would contribute to a progressive decrease in the tensile properties of the hair. Research grade hair was cut into five segments from the root to the distal end. Lipids from each segment were extracted and analyzed by thin-layer chromatography in conjunction with photodensitometry. The major free polar lipid classes in the hair included ceramides, glucosylceramides and cholesterol sulfate. The concentrations of all of the free polar lipids as well as the covalently bound fatty acids decreased in going from the root to the distal end of the hair. In addition, there was a significant reduction in tensile properties of the hair from the root to distal end. In conclusion, the progressive loss of endogenous free and covalently bound lipids from hair, which are probably related to normal weathering of the hair and grooming practices, may help contribute to a marked decrease in tensile properties to the hair. Résumé Les cellules de la cuticule forment le revêtement externe qui protège le cortex des cheveux. Les cellules de la cuticule sont minces, plates et se chevauchent. De fines couches de lipides sont présentes dans le matériau assurant la jonction entre les cellules cuticulaires. D'autres fines couches de lipides sont également présentes dans les espaces intercellulaires du cortex, entre les longues cellules corticales fibreuses. De plus, les surfaces externes des cellules de la cuticule sont recouvertes d'une couche monomoléculaire d'acides gras liés par covalence, un des composants majoritaires étant l'acide 18-méthyleicosanoique. On pense que ces acides gras sont fixés par liaisons thioesters. On pense également que l'ensemble de ces lipides joue un rôle important sur les propriétés physiques du cheveu. L'hypothèse testée dans cette étude est que les lipides libres et ceux liés par covalence sont progressivement éliminés lors de l'exposition normale des cheveux à l'environnement extérieur. Cette délipidation progressive de la cuticule pourrait, en partie, entraîner une plus grande sensibilité des constituants lipidiques et protéiniques du cortex aux agressions externes et accroître leur dégradation. Cette dégradation, à son tour, contribuerait à une diminution progressive des propriétés mécaniques en extension des cheveux. Des cheveux de provenance commerciale ont été coupés en cinq segments de leur racine à leur extrémité distale. Les lipides de chaque segment ont été extraits, séparés par chromatographie couche mince et dosés par densitométrie photographique. Les classes majoritaires de lipides polaires libres sont constituées de céramides, de glucosylcéramides et de sulfate de cholestérol. Les teneurs de tous les lipides polaires libres ainsi que des acides gras liés par covalence diminuent de la racine à l'extrémité distale du cheveu. De plus, on constate une réduction considérable des propriétés mécaniques en extension des cheveux de la racine à l'extrémité distale.-.En conclusion, la perte progressive des lipides endogènes libres et liés par covalence, probablement attribuables aux expositions à l'environnement et au stress des traitements capillaires peut aider à contribuer à une baisse marquée des propriétés mécaniques en extension des cheveux. [source] Anti-Fenton reaction activity of three taxa of water yam (Dioscorea alata L.)INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 9 2007Tsu-Shing Wang Summary In the present study, we compared the anti-Fenton reaction activity of three taxa of water yam (Dioscorea alata L.): DS2, TN2 and PSY [D. alata L. var. purpurea (Roxb.) M. Pouch]. Anti-Fenton reaction activity was evaluated by measuring the damage inflicted on calf thymus DNA by copper ions combined with hydrogen peroxide with the use of an ethidium bromide binding assay and agarose gel electrophoresis. We found that extracts of tuber pulp from all three taxa of yam had significant anti-Fenton reaction activity. The protection pattern of the three tuber pulp extracts was similar to that of EDTA, a typical divalent metal ion chelator, which displayed a significant protection lag-phase. With the use of thin-layer chromatography, we found that a common, major ansialdehyde-sulphuric acid stained spot (possibly a polysaccharide mucilage) with an Rf of 0.09 may be the most likely contributor to the anti-Fenton reaction activities of the yam tuber extracts investigated. The present study identifies the mechanism of the health benefit of the Dioscorea family. The copper-chelating and absorbing capability of yam tuber pulp extracts may be useful in functional screening. [source] Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of ochratoxin AINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 8 2004Won-Bo Shim Summary A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the determination of ochratoxin A (OTA) was developed. Fluorescein-labelled OTA derivative (tracer) was synthesized and purified by thin-layer chromatography. The optimized OTA FPIA had a dynamic range from 5 to 200 ng mL,1 with IC50 value of 30 ng mL,1 and a detection limit of 3 ng mL,1. The method developed was characterized by high specificity and reproducibility. Cross-reactivity with other mycotoxins (zearalenone, aflatoxins, patulin and T-2 toxin) was negligible (<0.1%). Methanol extracts of barley samples were used for the analysis. The results of OTA determination in barley were compared with those determined by indirect competitive enzyme-linked immunosorbent assay (ELISA). Recoveries for the samples spiked at 50, 100 and 500 ng g,1 levels were 91, 90 and 97%, respectively, for FPIA, and 98, 98 and 102%, for ELISA. Naturally contaminated barley samples were analysed by these methods but some disagreement was observed between the results. The FPIA method can be applied for screening of food samples for OTA residues without a complicated clean-up. [source] Stable maintenance of 5, -reductase activity in long-term subcultures of fibroblasts derived from the foreskinINTERNATIONAL JOURNAL OF UROLOGY, Issue 6 2002Kazumi Nakae Abstract Background: There is up to a 50-fold variation in control subjects in current assays of 5,-reductase activity which makes interpretation difficult. It was therefore attempted in this study to establish an assay method which produced stable 5,-reductase activity in long-term subcultured foreskin fibroblasts. Methods: Foreskin fibroblasts were obtained from three boys with phimosis (control subjects), three patients with Reifenstein syndrome and one patient with 5,-reductase deficiency (due to mutation L113P in exon 2 of the SRD5A2 gene). To maximize the number of cells in the DNA synthesis phase, cells were subcultured consistently to approximately 70% confluency. Thawed cells, frozen after the third subculture, were incubated for 24 h with [1,,2,- 3H] testosterone. 5,-Reductase activity was expressed as the sum of formed [3H] 5,-reduced metabolites (separated by thin-layer chromatography). Results: The full range of 5,-reductase activity in controls and patients with Reifenstein syndrome was 3.44,15.59 pmol/h per mg protein: a 4.53-fold variation. The activity in the patient with 5,- reductase deficiency was 0.52 pmol/h per mg protein. Conclusion: By the cell culture methods used in this study, which aimed to increase the number of cells in the DNA synthesis phase, foreskin fibroblasts maintained a considerably stable level of 5,-reductase activity during long-term subculture. Therefore, this assay method can be used for differential diagnosis of 5,-reductase deficiency from other relevant entities. [source] Antibacterial activity of plant extracts from northwestern ArgentinaJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007J.R. Soberón Abstract Aims:, To determine the antibacterial and cytotoxic activities of aqueous and ethanolic extracts of northwestern Argentinian plants used in folk medicine. To compare the mentioned activities with those of five commercial antibiotics. To identify the compounds responsible for the antibacterial activity. Methods and Results:, Plant extracts were prepared according to traditional uses in northwestern Argentina. Antibacterial activity was assayed by agar dilution in Petri dishes and broth dilution in 96-well plates. Lethal dose 50 (LD50) was determined by the Artemia salina assay. Phytochemical analysis was performed by sample adsorption on silica gel, thin-layer chromatography (TLC), bioautography and UV-visible spectra. The results showed that Tripodanthus acutifolius aqueous extracts have lower minimal inhibitory concentrations (MIC) (502 and 506 ,g of extracted material (EM) per ml for infusion and decoction, respectively) than cefotaxim MIC (640 ,g ml,1) against Acinetobacterfreundii (303). These data were lower than their LD50. Tripodanthus acutifolius tincture showed lower MIC (110 ,g of EM per ml) and minimal bactericidal concentration (MBC) (220 ,g of EM per ml) than cefotaxim (MIC and MBC of 320 ,g ml,1) for Pseudomonasaeruginosa. This extract also showed a MIC/MBC of 110/220 ,g of EM per ml, lower than oxacillin (MIC/MBC of 160/220 ,g ml,1) for Staphylococcus aureus (ATCC 25923). The cytotoxicity of all extracts were compared with that of commercial antibiotics. Rutin (3,3,,4,,5,7-pentahydroxyflavone 3- , -rhamnosilglucoside), iso -quercitrin (3,3,,4,,5,7-pentahydroxyflavone 3- , -glucoside) and a terpene would be partially responsible for the antibacterial activity of T. acutifolius infusion. Conclusions:,Tripodanthus acutifolius extracts had the ability to inhibit bacterial growth. The antibacterial activity differs with the applied extractive method, and it could be partially attributed to glycoflavonoids. This paper contributes to the knowledge of antibacterial capacity of plants from northwestern Argentina. Significance and Impact of the Study:, These antibacterial activities support further studies to discover new chemical structures that can contribute to alleviate or cure some illnesses. [source] Xylanolytic complex from Aspergillus giganteus: production and characterizationJOURNAL OF BASIC MICROBIOLOGY, Issue 4 2003Glauciane Danusa Coelho An Aspergillus giganteus strain was isolated as an excellent producer of xylanase associated with low levels of cellulase. Optimal xylanase production was obtained in liquid Vogel medium containing xylan as carbon source, pH 6.5 to 7.0, at 25 °C and under shaking at 120 rpm during 84h. Among the several carbon sources tested, higher xylanase production was verified in xylan, xylose, sugar-cane bagasse, wheat bran and corn cob cultures, respectively. Optimal conditions for activity determination were 50 °C and pH 6.0. The xylanolytic complex of A. giganteus showed low thermal stability with T50 of 2 h, 13 min and 1 min when it was incubated at 40, 50 and 60 °C, respectively, and high stability from pH 4.5 to 10.5, with the best interval between 7.0 to 7.5. This broad range of stability in alkali pH indicates a potential applicability in some industrial processes, which require such condition. Xylanolytic activity of A. giganteus was totally inhibited by Hg+2, Cu+2 and SDS at 10 mM. The analysis of the products from the oat spelts xylan hydrolysis through thin-layer chromatography indicated endoxylanase activity, lack of debranching enzymes and ,-xylosidase activity in assay conditions. [source] EVALUATION OF GLOBAL YIELD, COMPOSITION, ANTIOXIDANT ACTIVITY AND COST OF MANUFACTURING OF EXTRACTS FROM LEMON VERBENA (ALOYSIA TRIPHYLLA[L'HÉRIT.] BRITTON) AND MANGO (MANGIFERA INDICA L.) LEAVESJOURNAL OF FOOD PROCESS ENGINEERING, Issue 2 2007CAMILA G. PEREIRA ABSTRACT In this work, the global yields, composition and antioxidant activity (AA) of extracts from lemon verbena (Aloysia triphylla) and mango (Mangifera indica) leaves obtained by different separation processes were determined. Lemon verbena extracts were obtained by supercritical fluid extraction (SFE), while mango leaf extracts were obtained by SFE, low-pressure solvent extraction (LPSE) and hydrodistillation. The extract's constituents were analyzed by thin-layer chromatography, gas chromatography/mass spectrometry and gas chromatography/flame ionization detector. The AA of the extracts was evaluated by the coupled reaction of , -carotene/linolenic acid. The cost of manufacturing (COM) was estimated for the SFE extracts. Higher global yields were obtained using SFE at 350 bar/45C (1.49%) for lemon verbena and LPSE (3.04%) for mango. The AAs of the extracts were larger than that of the , -carotene for both plants. The minimum values of COM were U.S.$26.96 and 52.45/kg of extract for lemon verbena and mango, respectively. [source] Lipid Changes of Freeze-Dried Spinach by Various Kinds of OxidationJOURNAL OF FOOD SCIENCE, Issue 8 2000J. Lee ABSTRACTM : Lipid changes in freeze-dried spinach by various oxidations were studied by using thin-layer chromatography and gas chromatography. There were no characteristic changes in lipids by the oxidation except decrease in esterified sterols in NL and phosphatidic acid and phosphatidylcholine in PL. Stability of the freeze-dried spinach lipid to the oxidation was higher when it was in the form of total lipid than when separated into NL, GL, or PL. Autoxidation and enzyme-catalyzed oxidation resulted in the highest fatty-acid composition change in NL. However, photooxidation showed the biggest change in PL and GL. C16 fatty acids tended to be more stable than C18 fatty acids to the oxidation. [source] Synthesis and biological evaluation of a novel asymmetrical 99mTc-nitrido complex of metronidazole derivativeJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 13 2007Dejing Kong Abstract The novel dithiocarbamate derivative of metronidazole, potassium 2-(2-methyl-5-nitro-1H -imidazolyl)-ethyl-dithiocarbamate (MNIE-DTC), was synthesized as the pharmacophore-containing bifunctional ligand. The corresponding asymmetrical 99mTc-nitrido complex, expected as a tumor hypoxia marker, had been successfully obtained by the addition of the biphosphine ligand PNP5 (PNP5 = N -ethoxethyl- N,N -bis[2-(bis(3-methoxypropyl)phosphino)ethyl]-amine) and the dithiocarbamate ligand (MNIE-DTC) to the 99mTc-nitrido precursor solution at 100°C for 15 min. The radiochemical purity of the product was above 95% as measured by thin-layer chromatography and high-performance liquid chromatography. In vitro studies showed that the complex possessed good stability under physiological conditions. Its partition coefficient studies indicated that it was a lipophilic complex. The electrophoresis results showed that the complex was cationic. Biological evaluation of the complex [99mTcN(PNP5)(MNIE-DTC)]+ performed in Kunming mice bearing H22 tumor showed that the complex had a moderate tumor uptake (0.57±0.06%ID/g at 1h), and the ratios of tumor/blood and tumor/muscle were 2.46 and 1.31 at 1h p.i., and reached 4.52 and 2.86 at 4h p.i., respectively. Copyright © 2007 John Wiley & Sons, Ltd. [source] Hypoglycaemic and hypolipidaemic effects of fractions from kolaviron, a biflavonoid complex from Garcinia Kola in streptozotocin-induced diabetes mellitus ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2006O. A. Adaramoye In the search for natural hypoglycaemic agents as alternatives to synthetic ones that are expensive and not easily accessible, and to justify the use of Garcinia kola seeds in traditional African medicine to treat diabetes, the hypoglycaemic and hypolipidaemic effects of fractions from kolaviron (KV) (a Garcinia kola seed extract) were investigated in normal and streptozotocin (STZ)-diabetic rats. KV, a biflavonoid complex from Garcinia kola seed, was separated by thin-layer chromatography into three fractions; Fraction I (FI), Fraction II (FII) and Fraction III (FIII) with RF values of 0.48, 0.71 and 0.76, respectively. In normoglycaemic rats, KV, FI and FII administered at a dose of 100 mg kg,1 body weight elicited significant (P < 0.05) hypoglycaemic activity within 4 h of oral administration. Precisely, KV, FI and FII decreased blood glucose levels of normoglycaemic rats by 66%, 50% and 61%, respectively, when compared with controls 30 min after oral administration of the extracts. In hyperglycaemic rats, KV, FI and FII significantly (P < 0.05) reduced blood sugar levels in STZ-diabetic rats within 4 h of oral administration. Furthermore, KV alone produced a significant (P < 0.05) anti-diabetic effect from day 3 to day 7 of oral intubation of STZ-diabetic rats. In addition, the extracts showed favourable effect on the plasma lipid profile of STZ-diabetic rats, and also decreased significantly (P < 0.05) the STZ-induced increase in the activity of microsomal glucose-6-phosphatase and lipid peroxidation (LPO) products. This study confirms the anti-diabetic and hypo-lipidaemic effects of KV in STZ-diabetic rats. These observed effects of KV are attributed to two of its fractions, FI and FII, with RF values of 0.48 and 0.71, respectively. [source] Synthesis of a model cyclic triblock terpolymer of styrene, isoprene, and methyl methacrylateJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 10 2002Dimitris Pantazis Abstract The synthesis of a model cyclic triblock terpolymer [cyclic(S- b -I- b -MMA] of styrene (S), isoprene (I), and methyl methacrylate (MMA) was achieved by the end-to-end intramolecular amidation reaction of the corresponding linear ,,,-amino acid precursor [S- b -I- b -MMA] under high-dilution conditions. The linear precursor was synthesized by the sequential anionic polymerization of S, I, and MMA with 2,2,5,5-tetramethyl-1-(3-lithiopropyl)-1-aza-2,5-disilacyclopentane as an initiator and amine generator and 4-bromo-1,1,1-trimethoxybutane as a terminator and carboxylic acid generator. The separation of the unreacted linear polymer from the cyclic terpolymer was facilitated by the transformation of the unreacted species into high molecular weight polymers by the evaporation of the reaction solvent and the continuation of the reaction under high-concentration conditions. The intermediate materials and the final cyclic terpolymer, characterized by size exclusion chromatography, vapor pressure osmometry, thin-layer chromatography, IR and NMR spectroscopy, exhibited high molecular weight and compositional homogeneity. Dilute-solution viscosity measurements were used as an additional proof of the cyclic structure. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1476,1483, 2002 [source] Quantitative determination of haloperidol in tablets by high performance thin-layer chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 5 2007Sigrid Mennickent Abstract A densitometric high performance thin-layer chromatography (HPTLC) method was developed and validated for the quantitative analysis of haloperidol in tablets. Chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of acetone/chloroform/n -butanol/acetic acid glacial/water (5:10:10:2.5:2.5 v/v/v/v/v) as the mobile phase. Quantitative analysis was carried out at a wavelength of 254 nm. The method was linear in the 10,100 ng/,L range, with a determination coefficient of 0.999. The coefficients of variation for precision were not higher than 2.35%. The detection limit was 0.89 ng/,L, and the quantification limit was 2.71 ng/,L. The accuracy ranged from 97.76 to 100.33%, with a CV not higher than 4.50%. This method was successfully applied to quantify haloperidol in real pharmaceutical samples, including the comparison with HPLC measurements. The method was fast, specific, with a good precision and accuracy for the quantitative determination of haloperidol in tablets. [source] High-resolution magic-angle spinning NMR for the identification of reaction products directly from thin-layer chromatography spotsMAGNETIC RESONANCE IN CHEMISTRY, Issue 10 2007Scott A. Bradley Abstract We have investigated the prospect of identifying organic reaction products directly from separated thin-layer chromatography (TLC) spots with high-resolution magic-angle spinning (HRMAS) NMR. The concept is to use the TLC spots for NMR analysis so that spectra can be obtained before the reaction is worked up, but without having to elute the product from the TLC stationary phase. Thus, the separated spot is scraped from the plate, transferred to an HRMAS sample rotor, and suspended with a deuterated solvent. Herein, we describe the effects of having the stationary phase present during NMR acquisition. Using a Varian 4 mm gHX Nanoprobe and rotenone as a test compound, we found that the presence of the stationary phase during NMR acquisition resulted in (i) a large, broad ,background' signal near 4.6 ppm and (ii) a decrease in the signal-to-noise ratio due to the adsorption of the product molecules to the adsorbent. However, both effects could be adequately and conveniently eliminated. The background signal was removed by using either a CPMG pulse sequence or chemical exchange. The adsorption was avoided by using a more polar solvent system. Finally, we found that spectra with good signal-to-noise ratio and resolution could be acquired in a matter of minutes even for cases of limited product concentration. Therefore, we believe the technique has value and provides the organic chemist with another option to obtain NMR data critical for structural elucidation or verification. Copyright © 2007 John Wiley & Sons, Ltd. [source] Advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometryMASS SPECTROMETRY REVIEWS, Issue 3 2010Johannes Müthing Abstract Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including infectious diseases and the development of cancer. Knowledge of the number and sequence of monosaccharides and their anomeric configuration and linkage type, which make up the principal items of the glyco code of biologically active carbohydrate chains, is essential for exploring the function of GSLs. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of novel biochemical and biophysical technologies. Mass spectrometry (MS) takes part in the network of collaborations to further unravel structural and functional aspects within the fascinating world of GSLs with the ultimate aim to better define their role in human health and disease. However, a single-method analytical MS technique without supporting tools is limited yielding only partial structural information. Because of its superior resolving power, robustness, and easy handling, high-performance thin-layer chromatography (TLC) is widely used as an invaluable tool in GSL analysis. The intention of this review is to give an insight into current advances obtained by coupling supplementary techniques such as TLC and mass spectrometry. A retrospective view of the development of this concept and the recent improvements by merging (1) TLC separation of GSLs, (2) their detection with oligosaccharide-specific proteins, and (3) in situ MS analysis of protein-detected GSLs directly on the TLC plate, are provided. The procedure works on a nanogram scale and was successfully applied to the identification of cancer-associated GSLs in several types of human tumors. The combination of these two supplementary techniques opens new doors by delivering specific structural information of trace quantities of GSLs with only limited investment in sample preparation. © 2009 Wiley Periodicals, Inc. Mass Spec Rev 29:425-479, 2010 [source] Changes in phosphatidylinositol and phosphatidylinositol monophosphate kinase activities during the induction of somatic embryogenesis in Coffea arabicaPHYSIOLOGIA PLANTARUM, Issue 2 2003María Julissa Ek-Ramos Evidence was obtained for the presence of phosphatidylinositol (PIK) and phosphatidylinositol monophosphate kinase (PIPK) at different developmental stages during somatic embryogenesis in Coffea arabica L. by in vitro phosphorylation of endogenous lipids in the presence of [,- 32P]ATP followed by thin-layer chromatography. The results indicate the existence of a relationship between the development stages that were analysed and the kinases found. In cells without differentiated structures (EC, embryogenic calli) phosphatidylinositol kinase and phosphatidylinositol monophosphate 5-kinase (EC 2.7.1.68) activities were present. These activities increased significantly in the first differentiated stage (PREG, preglobular structures) and decreased as the development stages advanced. Phosphatidylinositol monophosphate (PIP) formation decreased from the globular (GLO) to the cotyledonary (COT) stage. The PIP fraction contained both isomers, PI 3-P and PI 4-P. This demonstrates PI3K (EC 2.7.1.137) and PI4K (EC 2.7.1.67) activity during somatic embryogenesis in Coffea arabica L. When wortmannin, an inhibitor of PI3K and PI4K activities, was included in an in vitro assay, a dose-dependent inhibition of the formation of both isomers was observed. The addition of wortmannin to the induction medium during the PREG stage reduced the number of normal embryos. Our results suggest that PI and PIP kinases and the formation of certain phosphoinositides may play roles in the regulation of somatic embryo development in Coffea arabica L. [source] On the structural diversity of Shiga toxin glycosphingolipid receptors in lymphoid and myeloid cells determined by nanoelectrospray ionization tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 15 2010Petra Hoffmann Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Gal,4Gal,4Glc,1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAc,3Gal,4Gal,4Glc,1Cer), respectively, which represent the targets of Stxs on many different cell types. B-cell-derived Raji cells and THP-1 cells of monocytic origin are widely used for the investigation of Stx-mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP-1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) with collision-induced dissociation (CID) in conjunction with Stx1 as well as anti-Gb3Cer and anti-Gb4Cer antibodies. Using the combination of a thin-layer chromatography (TLC) overlay assay and MS1 and MS2 analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1-receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP-1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP-1 cells we observed the expression of Gb4Cer in THP-1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short- and long-chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx-susceptible cells. Copyright © 2010 John Wiley & Sons, Ltd. [source] Improved imaging resolution in desorption electrospray ionization mass spectrometry,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 17 2008Vilmos Kertesz The imaging resolution of desorption electrospray ionization mass spectrometry (DESI-MS) was investigated using printed patterns on paper and thin-layer chromatography (TLC) plate surfaces. Resolution approaching 40,µm was achieved with a typical DESI-MS setup, which is approximately 5 times better than the best resolution reported previously. This improvement was accomplished with careful control of operational parameters (particularly spray tip-to-surface distance, solvent flow rate, and spacing of lane scans). In addition, an appropriately strong analyte/surface interaction and uniform surface texture on the size scale no larger than the desired imaging resolution were required to achieve this resolution. Overall, conditions providing the smallest possible effective desorption/ionization area in the DESI impact plume region and minimizing the analyte redistribution on the surface during analysis led to improved DESI-MS imaging resolution. Published in 2008 by John Wiley & Sons, Ltd. [source] [2H/H] Isotope ratio analyses of [2H5]cholesterol using high-temperature conversion elemental analyser isotope-ratio mass spectrometry: determination of cholesterol absorption in normocholesterolemic volunteersRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 3 2004Jean-Philippe Godin This paper validates the use of high-temperature conversion elemental analyser isotope-ratio mass spectrometry (TC-EA/IRMS) for measuring the [2H/H] enrichment of plasma [2H5]cholesterol. From a molecular point of view, the free cholesterol is initially separated from plasma by thin-layer chromatography (TLC) and then injected onto the TC-EA reactor which converts cholesterol molecules into CO and H2 gases. The slope of the curve of the experimental mole percent excess (MPE(exp.)) versus MPE(theor.) was very close to 1, demonstrating that no significant isotopic fractionation was observed during all processing of the samples (i.e., isolation of plasma free cholesterol by TLC and pyrolysis in the TC-EA reactor). Excellent linearity (r2,=,0.9994, n,=,4) of , (,) of [2H/H] isotopic measurements versus mole percent (MP) was assessed over the range 0 to 0.1 MP. The precision of the [2H/H] measurement, evaluated with two calibration points processed with TLC, was ,2HV-SMOW,=,,192.5,±,3.4, and ,2HV-SMOW,=,,136.9,±,2.9,. The standard deviations of the within-assay and between-assay repeatabilities of the analytical process, evaluated using the quality control (QC) of plasma samples, were 4.6 and 6.1,, respectively. Plant sterols are known to reduce cholesterol absorption and therefore were used as a positive control in a clinical study performed with normocholesterolemic volunteers. This present method produces biological results consistent with those already reported in the literature. Copyright © 2004 John Wiley & Sons, Ltd. [source] Extracellular cross-linking of maize arabinoxylans by oxidation of feruloyl esters to form oligoferuloyl esters and ether-like bondsTHE PLANT JOURNAL, Issue 4 2009Sally J. Burr Summary Primary cell walls of grasses and cereals contain arabinoxylans with esterified ferulate side chains, which are proposed to cross-link the polysaccharides during maturation by undergoing oxidative coupling. However, the mechanisms and control of arabinoxylan cross-linking in vivo are unclear. Non-lignifying maize (Zea mays L.) cell cultures were incubated with l- [1- 3H]arabinose or (E)-[U- 14C]cinnamate (radiolabelling the pentosyl and feruloyl groups of endogenous arabinoxylans, respectively), or with exogenous feruloyl-[3H]arabinoxylans. The cross-linking rate of soluble extracellular arabinoxylans, monitored on Sepharose CL-2B, peaked suddenly and transiently, typically at ,9 days after subculture. This peak was not associated with appreciable changes in peroxidase activity, and was probably governed by fluctuations in H2O2 and/or inhibitors. De-esterified arabinoxylans failed to cross-link, supporting a role for the feruloyl ester groups. The cross-links were stable in vivo. Some of them also withstood mild alkaline conditions, indicating that they were not (only) based on ester bonds; however, most were cleaved by 6 m NaOH, which is a property of p- hydroxybenzyl,sugar ether bonds. Cross-linking of [14C]feruloyl-arabinoxylans also occurred in vitro, in the presence of endogenous peroxidases plus exogenous H2O2. During cross-linking, the feruloyl groups were oxidized, as shown by ultraviolet spectra and thin-layer chromatography. Esterified diferulates were minor oxidation products; major products were: (i) esterified oligoferulates, released by treatment with mild alkali; and (ii) phenolic components attached to polysaccharides via relatively alkali-stable (ether-like) bonds. Thus, feruloyl esters participate in polysaccharide cross-linking, but mainly by oligomerization rather than by dimerization. We propose that, after the oxidative coupling, strong p- hydroxybenzyl,polysaccharide ether bonds are formed via quinone-methide intermediates. [source] | |||||||||||||||||||||||||||||||