Detection of toxigenic Fusarium isolates by thin layer chromatography

A simple screening method was used to investigate mycotoxin production among 114 Fusarium isolates. Zearalenone, T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, deoxynivalenol, nivalenol, fusarenon-X, butenolide, moniliformin and equisetin were detected. The importance of the use of a range of different substrates for mycotoxin production was proved.     

Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography

We describe methods for the complete analysis of cellular nucleotides from as few as 10(6) 32Pi-labeled cells in a simple 2-day experiment. Nucleotides are extracted with acid, neutralized, and resolved by two-dimensional thin layer chromatography on polyethyleneimine cellulose. In the first dimension the nucleotides are separated based on the negative charge of their phosphate groups (i.e. cyclic, mono-, di, and triphosphates) and in the second dimension on their content of nucleobases (i.e. Ura, Cyt, Thy, Gua, and Ade). Because the separation is logical, one can predict the chromatographic migration of most nucleotides. By running standards we have determined the chromatographic location of over 90 biologically important nucleotides, nucleotide derivatives, and modified nucleotides from tRNA. We also developed a set of enzymatic and chemical methods to be used in conjunction with the chromatographic separations for verifying the identity of nucleotides and characterizing novel nucleotides. In this paper we use these methods to analyze and inventory the nucleotide content of Salmonella typhimurium in balanced log phase growth. Other potential uses of the method are also described.     

The efficiency of interaction of deoxyribonucleoside-5'-mono-, di- and triphosphates with the active centre of E. coli DNA polymerase I Klenow fragment

The interaction of deoxyribonucleoside-5'-mono-, di- and triphosphates with E. coli DNA polymerase I Klenow fragments was examined. Dissociation constants of the enzyme complex with nucleotides were determined from the data on the enzyme inactivation by adenosine 2',3'-riboepoxide 5'-triphosphate. The role of nucleotide bases, phosphate groups and sugar moieties in the complex formation of nucleotides with the enzyme was elucidated. The necessity of complementary interaction of nucleotides with templates for template-controlled 'adjusting' of complementary dNTP to its reactive state was found. The crucial role of the interaction of dNTP gamma-phosphate with the enzyme in this process is discussed.     

Determination of plasma progesterone using thin layer chromatography during pregnancy and normal menstrual cycle]

Plasma progesterone levels were measured during pregnancy and the normal human menstrual cycle by thin-layer densitometry of a specific fluorescence reaction for delta4-3-ketosteroids. 94 determinations were carried out from the 7th week of pregnancy until term. 84 plasma progesterone concentrations were estimated between day 16 and day 26 and other 10 during the preovulatory phase of the normal menstrual cycle.     

Separation of fuchsin analogs using thin layer chromatography

The four analogs comprising basic fuchsin have been separated using thin layer chromatography (TLC). Mixtures spotted on reverse phase TLC plates were developed with a solution of 25% methanol, 10% ammonium hydroxide, and 65% distilled water. The Rf values of the analogs were for pararosaniline, 0.54; rosaniline, 0.41; magenta II, 0.31; new fuchsin, 0.19.     

Fractionation of simian virus 40 DNA fragments by RPC-5 column chromatography

This report describes several modifications of the original radioenzymatic assay for serotonin (4) which increase the sensitivity of the assay 10-fold as well as enhance its reliability. Serotonin is converted to [³H]melatonin, in two steps. First, serotonin is acetylated to N-acetylserotonin by acetic anhydride. The N-acetylserotonin is then incubated with hydroxyindole-O-methyltransferase and S-[methyl-³H]adenosyl methionine and is converted to [³H]melatonin. The radioactive melatonin is extracted with toluene-isoamyl alcohol (7:3), dried, reconstituted, isolated by one-dimensional, silica gel, thin-layer chromatography, and counted in a liquid scintillation counter. The assay is specific and sensitive to approximately 5 pg of serotonin and thus can be used to measure serotonin levels in single brain nuclei or microliter quantities of biological fluids. The assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography

A simple, precise and rapid high-performance thin-layer chromatographic method has been developed for the estimation of phyllanthin (1) and hypophyllanthin (2), the important lignans of Phyllanthus species, especially Phyllanthus amarus. Separation of 1 and 2 was carried out on silica gel 60 F254 layers eluted with hexane:acetone:ethyl acetate (74:12:8), and the analytes were visualised through colour development with vanillin in concentrated sulphuric acid and ethanol. Scanning and quantification of spots was performed at 580 nm. Recoveries of 1 and 2 were 98.7 and 97.3%, respectively. The method was validated and the peak purities and limits of detection and quantification were determined.     

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography

[¹⁴C]-labelled thromboxane B₂ and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-¹⁴C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E₂, A₂, D₂ and F_2α. Chromatographic properties in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-¹⁴C]-arachidonic acid to thromboxane B₂ and prostaglandin E₂ by human lung fibroblasts in tissue culture.     

Simple identification of the neutral chlorinated auxin in pea by thin layer chromatography

One of the neutral chlorinated auxins of immature pea seeds was readily identified by thin layer procedures simple enough to serve in student's laboratory courses. 4-Chloroindole-3-acetic acid methyl ester was extracted from 50 g of commercial, frozen peas by either water or acetone, concentrated to small volumes and chromatographed in CHCl₃ or CCl₄ solvent systems separating the chlorinated auxin from indoleacetonitrile and the methyl or ethyl esters of indoleacetic acid. Colour reaction was carried out with some of the Salkowski FeCl₃ sprays of which Ehmann's FeCl₃/dimethylaminobenzaldehyde modification gave the most stable blue colour.