Thin-layer Chromatographic Mobility Research Articles

A new oxygen modification cyclooctaoxygen binds to nucleic acids as sodium crown complex

BackgroundOxygen exists in two gaseous and six solid allotropic modifications. An additional allotropic modification of oxygen, the cyclooctaoxygen, was predicted to exist in 1990. MethodsCyclooctaoxygen sodium was synthesized in vitro from atmospheric oxygen, or catalase effect-generated oxygen, under catalysis of cytosine nucleosides and either ninhydrin or eukaryotic low-molecular weight RNA. Thin-layer chromatographic mobility shift assays were applied on specific nucleic acids and the cyclooctaoxygen sodium complex. ResultsWe report the first synthesis and characterization of cyclooctaoxygen as its sodium crown complex, isolated in the form of three cytosine nucleoside hydrochloride complexes. The cationic cyclooctaoxygen sodium complex is shown to bind to nucleic acids (RNA and DNA), to associate with single-stranded DNA and spermine phosphate, and to be essentially non-toxic to cultured mammalian cells at 0.1–1.0mM concentration. ConclusionsWe postulate that cyclooctaoxygen is formed in most eukaryotic cells in vivo from dihydrogen peroxide in a catalase reaction catalyzed by cytidine and RNA. A molecular biological model is deduced for a first epigenetic shell of eukaryotic in vivo DNA. This model incorporates an epigenetic explanation for the interactions of the essential micronutrient selenium (as selenite) with eukaryotic in vivo DNA. General significanceSince the sperminium phosphate/cyclooctaoxygen sodium complex is calculated to cover the active regions (2.6%) of bovine lymphocyte interphase genome, and 12.4% of murine enterocyte mitotic chromatin, we propose that the sperminium phosphate/cyclooctaoxygen sodium complex coverage of nucleic acids is essential to eukaryotic gene regulation and promoted proto-eukaryotic evolution.

New thin layer chromatography method for ionizable compounds based on the change in the mobile phase acidity during elution

Thin layer chromatography (TLC), proposed in1938 by Izmailov and Shraiber [1], still remains, as inits initial version, a simple, inexpensive, and fastmethod for the monitoring of reaction mixtures duringthe synthesis of organic compounds and for foodstuffand pharmaceutical quality control [2–4]; it is also usedin test methods of analysis [5]. Varying the compositionof the mobile phase and the nature of the stationaryphase, i.e., the sorbent applied onto the plate, is the keyway for efficient separation of compounds both in clas-sical and in modern instrumental TLC techniques [2]. Ithas been noted that the chromatographic process in pla-nar chromatography is also affected by the gas phaseinside the chromatographic chamber, which consists ofair and vapors of the solvents present in the mobilephase. Due to the uncontrollable modifying influenceof the gas phase on the sorbent activity and on the effi-ciency of the chromatographic process, the presence ofthe gas phase is a key reason for the unsatisfactoryreproducibility of TLC mobility values ( R

Binding of Vibrio anguillarum to neutral glycosphingolipids from intestinal mucosa of rainbow trout (Oncorhynchus mykiss).

To test whether glycosphingolipids (GSLs) on the intestinal mucosa of rainbow trout (Oncorhynchus mykiss) serve as a binding receptor for Vibrio anguillarum, we analyzed neutral GSLs from rainbow trout intestinal mucosa and investigated the binding of bacteria to neutral GSLs. Two kinds of neutral GSLs, designated N-1 and N-2, were identified on high-performance thin-layer chromatography (TLC) plates. In TLC immunostaining tests, V. anguillarum bound only to galactosylceramide (GalCer), lactosylceramide and N-1 having the same TLC mobility as GalCer, but neither to glucosylceramide nor to N-2. These results suggest that N-1 is GalCer (Gal beta 1-1Cer) and also that N-1 (GalCer) on rainbow trout intestinal mucosa act as a receptor for V. anguillarum.

Analysis of gangliosides from carp intestinal mucosa

The gangliosides of carp intestinal mucosa were isolated and analysed by thin-layer chromatography (TLC), TLC immunostaining test, and TLC/secondary ion mass spectrometry (TLC/SIMS). Four species of gangliosides, designated as G-1, G-2, G-3 and G-4, were separated on TLC. The TLC/SIMS analysis of the G-1 ganglioside of carp intestinal mucosa revealed a series of [M-H] −ions from m/z 1061 to m/z 1131 representing the molecular mass range of GM4-like ganglioside with NeuAc. G-2, G-3 and G-4 gangliosides were analysed by the TLC immunostaining test. G-2 ganglioside was recognised by the monoclonal antibody specific for ganglioside GM1 (AGM-1 monoclonal antibody). However, G-3 ganglioside migrating on TLC between GM3 and GM1 ganglioside was not recognised by anti-GM3 monoclonal antibody and by AGM-1 monoclonal antibody. Furthermore, G-4 ganglioside with a similar TLC mobility as GD1a ganglioside did not show the reactivity to the anti-GD1a monoclonal antibody. In addition using the AGM-1 monoclonal antibody, the expression of GM1 ganglioside in the carp intestinal tissue was studied. GM1 ganglioside was detected on the epithelial cell surface of carp intestinal mucosa.

Identification of glycosphingolipid binding sites for SEF21-fimbriated Salmonella enterica serovar Enteritidis in chicken oviductal mucosa

In order to clarify the presence of glycosphingolipids (GSLs) receptors for Salmonella enterica serovar Enteritidis with SEF21 fimbriae, we analyzed neutral GSLs and gangliosides from chicken oviductal mucosa and investigated the binding of bacteria to neutral GSLs and gangliosides. Five types of neutral GSLs, designated as N-1 to N-5, and two types of gangliosides, designated as G-1 and G-2, were identified on the thin-layer chromatography (TLC) plates. In the bacterial binding assay on TLC, the fimbriated bacteria bound only to glucosylceramide (GlcCer) standard, N-1 having the same TLC mobility as GlcCer, GM3 standard and G-1 corresponding to GM3 in TLC mobility, but not to N-2, N-3, N-4, N-5, or G-2. These results suggest the presence of GlcCer (N-1) and ganglioside GM3 (G-1) on the epithelial surface of chicken oviductal tract which act as sites for adherence of SEF21-fimbriated S. Enteritidis.

Thin-layer chromatographic mobility of some tetraphenylporphyrin derivatives

TLC chromatography is a rapid and effective method of monitoring the course of chemical reactions. It is particularly simple in situations in which substrates and products differ substantially in polarity. In such circumstances finding an appropriate chromatographic system is a much easier task than if the hydrophobicity or hydrophilicity of both substrates and products is comparable. Such chromatographic methods have an additional advantage if the substances separated are naturally colored. P. Kuœ, Department of Chemistry, Silesian University, 9, Szkolna Street, 40-006 Katowice, Poland.

Characteristics of the Amylase and its Related Enzymes Produced by Ectomycorrhizal Fungus Tricholoma matsutake

Extracellular amylase properties were examined with the mycelium of Tricholoma matsutake isolated from ectomycorrhizal roots of Pinus densiflora. The molecular weights of α-amylase and glucoamylase were estimated as 34.2 kD and 11.5 kD, respectively, after eluted through Superdex 75 column. The optimum pH of the purified enzyme was found in a range of pH 5.0–6.0, with a peak at pH 5.0. The activities of these enzymes were stable from 4°C to 30°C. The α-amylase of T. matsutake readily hydrolyzed soluble starch and amylose-B, while it weakly hydrolyzed glycogen, dextrin, amylose and amylose-A. The main products of hydrolysis were confirmed to be glucose, maltose and maltotriose on the basis of the similarities in the thin layer chromatographic mobility.

Chromatographic determination of polyiodohalides of organic nitrogen bases

The following biologically active polyiodohalides of organic nitrogen bases were synthesized and studied: trimethylbenzylammonium, triethylbenzylammonium, and N-cetylpyridinium diiodochlorides; tetramethylammonium, tetrabutylammonium, and N-cetylpyridinium diiodobromides; and butylcholinium triiodide. A method for the identification of the above compounds was proposed based on the dependence of their thin-layer chromatographic mobility and IR, UV, and NMR spectrometric properties and on the nature of the organic cation and the structure of the polyiodide anion. Reversed-phase high-performance liquid chromatography in a 1 : 1 methanol–water mixture (pH 6.86) was used for determining the compounds under study. The developed procedure is characterized by high sensitivity (0.09–0.51 μg) at RSD of no worse than 10%.

Inhibition of Vero cell cytotoxic activity in Escherichia coli O157:H7 lysates by globotriaosylceramide, Gb3, from bovine milk.

In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.

Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition.

A virus-host cell-binding assay was developed and used to investigate specific binding between group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. On the basis of these results, enterocytes were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion-exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100 degrees C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.

High-performance thin-layer chromatographic mobility of porphyrins and their copper complexes on different adsorbents

The R F values of twelve synthetic porphyrins including porphine and phenyl- and alkyl-substituted porphines, and also of their copper complexes, were obtained on thin-layer chromatographic plates coated with silica gel, cellulose, NH 2-bonded silica gel and octadecyl-bonded silica gel (ODS) with different developing solvents. On the first three plates, the mobilities ( R F values) of each porphyrin were always smaller than those of the corresponding copper complex, whereas the opposite occurred on the last plate. With an identical solvent, the mobilities of both a porphyrin and its copper complex increase in the following order of polar adsorbents: silica gel < NH 2-silica < cellulose. meso-Tetra(4-pyridyl)porphine was adsorbed so strongly on every polar adsorbent that it hardly migrated from the origin, whereas a large migration with the R F ≈ 0.9 occurred on an ODS plate specifically with N,N-dimethylformamide.

Potential bile acid metabolites. XVIII. Synthesis of stereoisomeric 3,6,12 alpha-trihydroxy-5 beta-cholanoic acids

Two new 6-hydroxylated bile acids, 3 beta, 6 alpha, 12 alpha- and 3 beta, 6 beta, 12 alpha-trihydroxy-5 beta-cholanoic acids, were synthesized from deoxycholic acid. In addition, their C-3 epimers, 3 alpha, 6 alpha, 12 alpha- and 3 alpha, 6 beta, 12 alpha-trihydroxy acids, were prepared by a new route. The principal reactions used were 1) 6 beta-hydroxylation of 3-methoxy-3,5-dienes with m-chloroperbenzoic acid in aqueous dioxane; 2) catalytic hydrogenation of the resulting 6 beta-hydroxy-3-oxo-4-enes to the 6 beta-hydroxy-3-oxo-5 beta compounds with palladium on calcium carbonate catalyst in ethanol; and 3) stereoselective reduction of appropriate 3-oxo derivatives with potassium tri-sec-butylborohydride and tert-butylamine-borane complex. The thin-layer chromatographic, gas-liquid chromatographic, and high performance liquid chromatographic mobilities, and 1H- and 13C-nuclear magnetic resonance spectroscopic data of the four stereoisomers are presented. With this work all the 6-hydroxylated derivatives of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids in the 5 beta series are now known and have been synthesized.

Evidence for a Highly Conserved, Immunoreactive Lipid inMycobacterium tuberculosis

A survey of the fractionated lipids of Mycobacterium tuberculosis H37Rv was conducted using rabbit antiserum raised to homologous and heterologous mycobacteria. One minor, highly apolar lipid was resolved by Florisil column chromatography, which reacted preferentially to anti-M. tuberculosis H37Rv rabbit antibodies. Other chromatographic properties, i.e., thin-layer chromatographic mobility and staining properties, suggested an analog of the phenolic glycolipid of Mycobacterium leprae. Preliminary results in the application of the glycolipid to tuberculous populations in northeast Thailand suggest a usefulness in screening for tuberculosis.

Comparative Persistence and Mobility of Pyridine and Phenoxy Herbicides in Soil

Bioassays with soybeans and lentils were used to compare the persistence of 2,4,5-T, triclopyr, and picloram in soil after applying 0.038, 0.38, and 3.8 kg ae/ha under field conditions. Soil samples were collected from 1 to 269 days after spraying and were kept frozen until growth room bioassays were conducted. Triclopyr was slightly less persistent than 2,4,5-T, but neither herbicide was biologically active during the next season. At least 90% of picloram disappeared within 7 months, but its biological activity was more persistent than that of either triclopyr or 2,4,5-T. Nine months after treatment, neither lentils nor soybeans could be grown in soil treated with picloram at 3.8 kg/ka. Triclopyr and 2,4-D had smiilar soil thin layer chromatographic mobilities in four different scils. Picloram was significantly more mobile than either triclopyr or 2,4,5-T, primarily due to its lower adsorption in the soils examined.

ISOLATION AND IN VITRO CHARACTERIZATION OF A NOVEL IMMUNOSUPPRESSIVE CYCLOSPORINE METABOLITE

A novel metabolite (M-E) was identified by high-performance liquid chromatography in the serum of cyclosporine-treated renal transplant recipients during a second wave of immunosuppressive activity after disappearance of the initial wave due to the direct effect of CsA. M-E was identified in human serum and porcine bile both by HPLC and by a preparative thin-layer chromatography (TLC). It demonstrated homogeneity with characteristic retention times on C8 and C18 column HPLC systems using a variety of elution systems, and distinctive TLC mobility (Rf 0.35). Metabolite E (M-E) was documented to be a CsA metabolite by radioactive tracer studies, by crossreactivity with a polyclonal sheep antibody in radioimmunoassay, and by the presence of a characteristic mass spectrum. Further, in vitro immunosuppressive assays documented effects of M-E similar to those of CsA. The relative activity of M-E versus CsA was quantitated by potency ratios: for inhibition of normal human mixed lymphocyte culture reactions, the ratio was 0.79 +/- 0.23. Interindividual differences were observed in patient susceptibility to MLR inhibition not only by CsA, as previously reported by others, but also by M-E. There was a lesser effect of M-E compared with CsA in inhibiting proliferation of, and IL-2 generation by, C3H murine splenocytes stimulated with concanavalin A: the potency ratios for both systems were about 0.5, possibly reflecting an interspecies variability in generation of or susceptibility to M-E. These studies suggest that heretofore unidentified metabolites--including, but not limited to, M-E--may play an important role in the immunosuppressive effect of CsA in man.

Biosyntheses of galactosyl lipids and polysaccharide in Streptococcus mutans

The syntheses of galactosylphospholipids and a galactose-containing polymer were observed when radiolabeled UDP-galactose was incubated with the membrane enzymes prepared from a strain of Streptococcus mutans, FA-1. The lipids were resolved into two components, lipids-1 and -2, by thin-layer and DEAE-cellulose column chromatographies. In the latter chromatography, lipid-1 was eluted by 0.0075 M and lipid-2 by 0.18 M ammonium acetate. The syntheses of lipids-1 and -2 were strongly inhibited by UDP and UMP, respectively. Both lipids-1 and -2 were degraded by mild acid, but were stable to mild alkaline hydrolysis. These results, together with their mobilities on thin-layer chromatography, suggest that lipid-1 is a galactosylphosphorylundecaprenol, and lipid-2 is a galactosylpyrophosphorylundecaprenol. When UDP-galactose was incubated with radiolabeled undecaprenol and ATP in the presence of membrane enzymes, lipids with thin-layer chromatographic mobilities of lipid-1 and lipid-2 were observed. The phosphate-to-galactose ratios in lipid-1 and lipid-2 were determined to be 1:1 and 2:1, respectively. These results indicated that lipid-1 and lipid-2 formed are galactosylmonophosphorylundecaprenol and galactosylpyrophosphorylundecaprenol, respectively. The polymer formed was eluted from the DEAE-cellulose column with a low concentration of salts (less than 0.1 M), suggesting that it is probably a polysaccharide, but not a lipoteichoic acid or teichoic acid-type polymer. In order to identify the sugars present in the polymer synthesized, the polymer purified by Sephadex G-50 and DEAE-cellulose column chromatographies was subjected to acid hydrolysis followed by NaB 3H 4 reduction and paper chromatographic analysis. [ 3H]Galactitol and a small amount of [ 3H]galactosaminitol were detected. This result suggests that the polymer is a nascent polysaccharide containing mainly galactose and a small amount of galactosamine, which probably derived from N-acetylgalactosamine during acid hydrolysis of the polymer.

Chapter 25 Classification of small cell carcinoma antigens by their association with their association with subcellular components

Abstract The protein components in small cell lung carcinoma (SCLC) were analysed by 2-dimensional gel electrophoresis of 35 Smethionine labelled total cellular extracts, cytoskeleton-associated (detergent insoluble) fractions, cytoskeleton + nuclear fractions, and detergent soluble fractions. Comparisons were made with 2-dimensional gels of 125 I-labelled membrane proteins. The majority of detergent-soluble proteins, particularly those in the cytoplasm, were common to many cell types. However, several proteins characteristic of SCLC were found in the cytoskeleton-associated and membrane fractions. The membrane components were separated further into protein and lipid fractions. Many monoclonal antibodies developed were reactive with carbohydrate epitopes associated with glycoproteins and/or glycolipids. The glycolipid components were separated further into the neutral and ganglioside fractions and analysed by thin layer chromatography (TLC). Several components characteristic of SCLC were found in both the neutral lipid and gangliosides. By analysing changes in TLC mobility after partial digestion with glycosidases, several of these SCLC lipids were found to be fucosylated. Biochemical extraction techniques. 2-D gel electrophoresis of proteins and TLC of lipids can be used to classify SCLC antigens.

Reversed-phase high-performance thin-layer chromatography and column liquid chromatography of metal complexes of pheophorbide a

The thin-layer chromatographic mobilities of pheophorbide a (Phor-a) and six metal-Phor-a complexes were measured on an octadecyl-bonded silica gel (C 18 silica) thin layer with various polar developing solvents, such as methanol, ethanol, acetonitrile and their mixtures with an aqueous solution containing an electrolyte such as sodium chloride. The mobility of the metal complex decreases in the following order of complexed metal ions: Co III > Fe III > Zn II > Phor-a > Ni II ≈ Pd II > Cu II, although the Fe III complex shows an exceptionally smaller mobility than the Zn II complex when pure acetonitrile or the water-acetonitrile containing sodium chloride is used as the developing solvent. The mobility of a metal-Phor-a complex is generally smaller than that of the pheophorbide b complex of the same metal. A successful reversed-phase high-performance liquid chromatographic separation was achieved for a mixture of Phor-a and its Fe III, Co III, Cu II and Zn II complexes by using ethanol-phosphate buffer (pH 3) (85:15, v/v) as the mobile phase.

Isolation and characterization of gangliosides with hybrid neolacto-ganglio-type sugar chains.

We have previously reported the presence of GM2 as the major ganglioside in the roe of striped mullet, Mugil cephalus, (Li, Y.-T., Hirabayashi, Y., DeGasperi, R., Yu, R. K., Ariga, T., Koerner, T. A. W., and Li, S.-C. (1984) J. Biol. Chem. 259, 8980-8985). In addition to GM2, mullet roe also contain a series of gangliosides with thin-layer chromatographic mobilities slower than GM2. Besides enzymatic hydrolysis and NMR spectroscopy, we have employed the thin-layer chromatography overlay technique using a human monoclonal IgM antibody which recognizes the GM2 epitope to study the nature of these gangliosides. Using these methods we have isolated and characterized three novel mullet roe gangliosides with the following structures: (Formula: see text). These three gangliosides all contain neolacto-series sugar chains. However, the unique feature of gangliosides 5 and 10 is that the terminal portion of the sugar chain is of the ganglio-series while the internal portion is of the neolacto series structure. Due to the substitution of a GalNAc on the internal Gal in 9 and 10 in the inner core, these two gangliosides also contain the gangliotriaosyl structure. Thus, the sugar chains in these gangliosides are of novel type and can be considered a hybrid between the two series which can be defined as the neolacto-ganglio series.

Reversed-phase high-performance thin-layer chromatography of hematoporphyrin and its nickel, copper and zinc complexes

The thin-layer chromatographic mobility of hematoporphyrin IX (Hp) and its metal complexes on octyl (C8)- or octadecyl (C18)-bonded silica gel plate is described. The mobility order for those compounds is regular on both kinds of plate with various compositions of methanol-phosphate buffer mixtures as the developing solvents; thus, Cu-complex < Ni-complex < Hp (free acid) < Zn-complex. The combination of a C18-plate with an 85∶15 (vol/vol) mixture of methanol and phosphate buffer (pH 3) is recommended for the successful separation of these four compounds.