Chromatographic Evidence Research Articles

Gastrin-releasing peptide from the intestine of the elasmobranch fish, Scyliorhinus canicula (common dogfish)

The concentration of gastrin-releasing peptide in the intestine of the elasmobranchian fish, Scyliorhinus canicula, measured with an antiserum directed against the COOH-terminal region of porcine gastrin-releasing peptide, was higher than the concentrations measured in mammalian intestines. The immunoreactivity was resolved by gel permeation chromatography into two peaks with the approximate elution volumes of porcine gastrin-releasing peptide and bombesin/neuromedin C. The primary structure of the larger peptide was established as Ala Pro Val Glu Asn Gln Gly Ser Phe Pro Lys Met Phe Pro Arg Ser His (Trp) Ala Val Gly (His Leu Met · NH 2). Residues in parentheses are only tentatively assigned. Chromatographic evidence and the presence of the arginyl residue at position 15 in the peptide suggest that the smaller molecular form of gastrin-releasing peptide may be identical to mammalian neuromedin C. Amphibian bombesin was not identified in the dogfish gut.

Caffeine-splitting of bilirubin/albumin complex: its relevance to the spectrophotometry of bilirubin in serum.

By means of gel-filtration of bilirubin/albumin mixtures, it is shown that unconjugated bilirubin remains completely linked to albumin (both human and bovine) in tetraborate buffer (pH 9.3), protein-free bilirubin appearing only when the bilirubin/albumin molar ratio exceeds two. On the other hand, bilirubin is completely set free from its protein link in the caffeine reagent. Additional chromatographic and spectrophotometric evidence is reported indicating the formation of a low-affinity complex between bilirubin and caffeine. These data explain why the spectrophotometric properties of bilirubin/albumin mixtures are matrix-dependent if measured in the tetraborate buffer but are no longer so when measured in the caffeine reagent. The relevance of these findings to the spectrophotometric "direct" assay of bilirubin in serum is discussed with reference to the occurrence of "delta-bilirubin" in pathological sera: this tightly protein-bound bilirubin fraction does not split in the presence of caffeine.

32P-postlabeling analysis of DNA adduction in mice by synthetic metabolites of the environmental carcinogen, 7H-dibenzo[c,g]carbazole: chromatographic evidence for 3-hydroxy-7H-dibenzo[c,g]carbazole being a proximate genotoxicant in liver but not skin.

The DNA adduction by the environmental carcinogen 7H-dibenzo[c,g]carbazole (DBC) and chemically synthesized 2-OH, 3-OH, and 4-OH metabolites of DBC was investigated in liver and skin of female CD-1 mice. After topical application to the skin of 37 mumol/kg of DBC or the phenolic metabolites, DNA adducts were measured by a 32P-post-labeling assay employing carrier-free [gamma-32P]ATP and ATP-deficient conditions. In liver, DBC produced four major and several minor chromatographically distinct adducts of as yet undetermined chemical structure. The adduct pattern elicited by 3-OH-DBC was qualitatively similar to the DBC adduct pattern, while this was not the case for 2-OH-DBC and 4-OH-DBC. On the basis of co-chromatography experiments under various conditions, the DBC and 3-OH-DBC adducts appeared identical, and the total of adduction elicited by these compounds in liver was substantial. Similar results were observed when DBC or 3-OH-DBC were administered i.p. As a major difference between the two compounds, one 3-OH-DBC adduct (no. 3) was 4.4- and 7.0-fold lower than the corresponding DBC adduct after i.p. and topical dosing, respectively. In skin, DBC produced two major adduct fractions after topical application, one of which could be chromatographically resolved into three subcomponents. Prominent adducts produced in skin DNA by each of the three metabolites were different from those elicited by DBC, and the level of adduction by the metabolites was significantly lower than that by DBC. Comparison of the skin and liver DBC-DNA adduct patterns after topical application of DBC showed that only one of the four major chromatographically resolved skin adducts corresponded to a major liver adduct (no. 3), and that total adduction in liver was 13.5-fold higher than in skin. These results suggested that activation of DBC to DNA-binding compounds in liver occurs through at least two pathways with 3-OH-DBC being a proximate carcinogen involved in the formation of most of the adducts; 3-OH-DBC and the other two phenolic metabolites investigated play a minor role, if any, in the formation of DBC-DNA adducts in skin; metabolic activation of DBC to DNA-binding compounds in liver and skin appears to follow pathways that are different in terms of both the chemical nature and the amount of the adducts formed; and DBC and 3-OH-DBC exhibit a strong preference for liver versus skin DNA.

Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin.

The single disulfide bond in Escherichia coli thioredoxin was reduced by reaction with a 20-fold excess of reduced dithiothreitol at neutral pH and 25 degrees C. For some measurements, reduced thioredoxin was further reacted with iodoacetamide to alkylate the cysteinyl residues. The denaturation transitions of oxidized, reduced, and reduced alkylated thioredoxin were observed by using far-ultraviolet circular dichroic and exclusion chromatographic measurements. Cleavage of the disulfide bond lowers the stability of the native thioredoxin to denaturation by about 2.4 kcal/mol, and subsequent alkylation lowers the stability by a further 1.6 kcal/mol. The kinetics of the conformational change of reduced thioredoxin in guanidine hydrochloride were observed by using exclusion chromatography at moderate pressure and 2 degrees C. Analyses of single and multimixing protocols are consistent with a predominant nonnative configuration in the denatured state and the transient accumulation of a compact nativelike intermediate during refolding. The intermediate can incorporate the nonnative configuration and can accommodate its isomerization. No compelling chromatographic evidence was found for a conformation having an elution time different from that characteristic for either the native or the denatured protein.

RECENT DEVELOPMENTS IN THE MOLECULAR STRUCTURE OF CIGUATOXIN

Crystalline ciguatoxin isolated from moray eel (Lycodontis ≡ Gymnothorax javanicus) viscera has an LD50 of 0.45 g/kg (i.p., mice). It has a molecular weight of 1111.7 ± 0.2 daltons. 1H NMR studies have shown that it is a polar and highly oxygenated molecule belonging to the class of polyethers. On basic alumina ciguatoxin is reversibly converted to a chromatographically distinct less polar form, which is equally toxic and elicits typical ciguatoxin symptoms in mice.From parrotfish (Scarus sordidus), which originated on a ciguateric reef on Tarawa atoll (Kiribati), we have isolated two toxins that evoke ciguatera symptoms in mice at approximately equal levels. Chromatographic evidence suggests that the two toxins are identical with the two ciguatoxins of different polarity and that the less polar form is the previously described scaritoxin.

O-acetylethanolamine a natural product from the leguminosae

O-Acetylethanolamine, studied as its dns derivative throughout, was isolated from Lens culinaris and identified by spectroscopy/synthesis. Chromatographic evidence indicated its presence in 12 other legumes.

The metabolism of [14C]N-ethoxycarbonyl-3-morpholinosydnonimine (molsidomine) in man.

[14C]-N-Ethoxycarbonyl-3-morpholinosydnonimine (molsidomine, Corvaton) was administered orally at a dose of 2 mg per subject to eight healthy male volunteers. Maximal plasma concentrations of total radioactivity of 32.4 +/- 6.4 ng equiv./ml (mean +/- S.D.) were detected compared with maximum plasma concentrations of 14.1 +/- 5.9 ng/ml (mean +/- S.D.) of molsidomine. In both cases these were attained at 0.5 h after dosing. From the peak, concentrations of parent drug fell rapidly with a half-life of 1.25 +/- 0.38 h (mean +/- S.D.). In contrast, total radioactivity declined more slowly with a terminal half-life of 138 +/- 42.7 h (mean +/- S.D.). The bulk of the radiolabel was rapidly excreted as metabolites in the urine, with over 85% of the dose recovered in the first 24 h. The main urinary radiolabelled metabolites appeared, from chromatographic evidence, to be similar to those previously identified in animals, namely N-morpholinosydnonimine, N-cyanomethylamino-N-(2'-hydroxyethyl)glycine and (N-cyanomethylenamino-2-aminoethoxy)-acetic acid.

Imidazole, a new natural product from the leguminosae

Imidazole was isolated from Lens culinaris seeds as its dansyl derivative and shown to be identical to a synthetic standard by co-chromatography, 1H NMR and EIMS. Chromatographic evidence indicated that it was also present in the seeds of 11 other legumes, 30% of those tested.

Chromatographic evidence for high-molecular-mass galanin immunoreactivity in pig and cat adrenal glands

Galanin was measured by radioimmunoassay in extracts of pig, cat and rat adrenals using non-C- and mid to C-terminally directed antibodies. The extracts were fractioned by gel chromatography and HPLC. The non-C-terminal galanin immunoreactivity in pig was 92.8 ± 11.7 pmol g , in cat 9.1 ± 0.9 pmol g and in rat < 1 pmol g . Two higher molecular forms of galanin have been identified in both pig and cat adrenal. One major large form behaves as if it was N-terminally extended ( K av, pig 0.58, cat 0.48) and the other, a very high-molecular-mass form ( K av pig 0.10, 0.24, cat 0.10), as if it had both N- and C-terminal extensions. Galanin Molecular form Adrenal gland Radioimmunoassay Chromatography

Contact allergy to 3-methylol phenol, 2,4-dimethylol phenol and 2,6-dimethylol phenol

Thirteen patients with contact allergy to phenol-formaldehyde resins (P-F-R) were patch tested with 3-methylol phenol, 2,4-dimethylol phenol and 2,6-dimethylol phenol. Nine patients reacted to at least 1 compound, all giving positive test responses to 2,4-dimethylol phenol. Seven patients reacted simultaneously to 2,6-dimethylol phenol while only 1 patient reacted to 3-methylol phenol. Negative test responses were noted in 20 controls. Chemical investigation by high pressure liquid chromatography indicated that the compounds tested were pure and separable. The 3 reported sensitizers may, theoretically, be generated during the manufacture of P-F-R. 2,4-Dimethylol phenol and 2,6-dimethylol phenol have been demonstrated and there has been chromatographic evidence of 3-methylol phenol in the P-F-R used in the routine test series at the department.

Antibiotics in sclerotia and mycelium of Rhizoctonia species

The sclerotia and mycelium of a number of species of Rhizoctonia were found to contain materials having antibiotic properties. Those of R. cerealis, R. oryzae-sativae and R. tuliparum were particularly effective. Most antibiotic-producing species were binucleate. Chromatographic evidence indicated that the antibiotics produced by R. cerealis, R. oryzae-sativae and R. tuliparum were different but probably chemically related.

Differences and similarities in cardenolide contents of queen and monarch butterflies in florida and their ecological and evolutionary implications

Florida queen butterflies are highly variable in cardenolide content and, in three populations studied, contained less cardenolide than did a sample of sympatric Florida monarchs. The possibility that queens stored a more potent set of cardenolides from their host plants (and therefore were as well protected as monarchs, even at lower concentrations) is refuted by Chromatographic analysis of wild butterflies, as well as controlled laboratory rearings. It therefore appears that, with respect to cardenolides, monarchs are better defended than are queens. Consequently, cardenolides are unlikely to explain the apparent shift in Florida viceroy mimicry away from resemblance of the monarch, toward mimicry of the queen. Other hypotheses to explain this mimetic phenomenon are suggested. Adult monarchs exhibit significant negative correlations between the concentration of cardenolide stored in their tissues and both body size and weight, whereas queens show no such correlations. The implications of these results for the study of "metabolic costs" of allelochemic storage are discussed. Chromatographic evidence is provided that monarchs do breed in south Florida during the winter months and that the likely host plant employed by the population studied wasAsclepias curassavica. This represents the first practical application of cardenolide "fingerprinting" to identify the larval host plants of wild danaid butterflies.

Irreversible and reversible phase transitions of alkyl monolayers. Gas chromatographic evidence of polar solvent influence on the adsorbing properties of the layer

Further to Gilpin's observations in liquid chromatography of the irreversible transformation of alkyl grafts, following the replacement of an organic mobile phase by an aqueous one, we describe some gas chromatographic experiments which lead to similar (log Vs, I/T) plots, as a consequence of the same solvent release process. We use paraffin films on siliceous supports or hydroxylated liquid substrates or C22-alkyl bonded silica. These experiments demonstrate that this irreversible transition and the melting-like reversible transition are completely different phenomena. They confirm the existence of at least three kinds of monomeric alkyl films.

Leakage of Dhurrin and p-Hydroxybenzaldehyde from Young Sorghum Shoots Immersed in Various Solvents

Spectral scanning was used to provide estimates of the leakage of the cyanogenic glucoside, dhurrin (p-hydroxy-[S]-mandelonitrile-beta-d-glucoside), and its metabolite, p-hydroxybenzaldehyde (p-HB), from young light-grown shoots of Atlas sorghum (Sorghum bicolor [L.] Moench) when these shoots were immersed in water, toluene, chloroform or mixtures of water and toluene or water and chloroform. Minimal leakage of dhurrin and virtually no leakage of p-HB occurred with water as the solvent. The 0.5% concentration (v/v) of both toluene and chloroform was more effective than either the 1.0 or 2.0% concentrations in effecting leakage of the two solutes. With either 0.5% toluene or 0.5% chloroform as the solvent, 80 to 90% of the total dhurrin was extracted from shoots in a 3-hour period. Breakdown of dhurrin during extraction was much more extensive with 0.5% chloroform than with 0.5% toluene. Some loss of p-HB occurred during 3- or 6-hour extractions in the water-organic solvent mixtures; spectral and chromatographic evidence suggested partial conversion of p-HB to p-hydroxybenzoic acid. With undiluted toluene or chloroform as solvents, extracts contained appreciable amounts of free p-HB but essentially no dhurrin. These solvents were less effective than the water-organic solvent mixtures in extracting the solutes from the shoot issue.

Size Exclusion Chromatography of Poly(vinylpyrrolidone): I. The Chromatographic Method

Abstract The optimum size exclusion chromatographic (SEC) method for poly-(vinylpyrrolidone) (PVP) was found to be based upon a stationary phase of diol derivatized silica of pore sizes 3000, 500, and 75A and a mobile phase of 50:50 (v/v) MeOH/H2O containing 0.1M-LiNO3. Sample recovery under identical conditions varied for the commercial packings investigated and was found to be inversely related to molecular weight. The latter phenomenon was rationalized on the basis of a limited number of active substrate sites available for binding. Methanol was found to be a more effective mobile phase modifier than either dimethyl formamide or acetonitrile apparently due to its ability to function as a proton donor in hydrogen bonding with PVP. Chromatographic evidence for the existence of semipolyampholyte character in PVP is presented. A procedure for the construction of a column set log-linear in calibration and of extended dynamic range is described and is based upon hydrodynamic volume theory.

Gel chromatographic evidence for the participation of the higher polymers in the self-association system of a flavoenzyme D-amino acid oxidase.

The self-association of subunits of D-amino acid oxidase holoenzyme was studied by high-speed gel filtration with a short column of TSK-GEL G3000 SW in 0.1 M sodium pyrophosphate (pH 8.3) at 25 degrees C. Over the range of the peak concentrations of 0.009-4.45 mg/ml in the presence of FAD the apparent Stokes radii increased with an increase of the concentrations and did not level off. The largest value obtained in this study was 61.5 A. This would correspond to that calculated for the hexamer with linear subunit arrangement which has the largest Stokes radius among the various arrangements. These results provide the first gel chromatographic evidence that the higher polymers greater than the dimer participate in the self-associating system of the enzyme.

Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.

La guattégaumérine, nouvel alcaloïde bisbenzylisoquinoléinique de Guatteria gaumeri

The aqueous-alcoholic extract of the bark of GUATTERIA GAUMERI (Mexican Annonaceae) is used for the treatment of cholelithiasis and hypercholesterolemia. Therefore it seems very interesting to perform a phytochemical study of this drug in order to know its composition and to determine, if possible, all the active principles. We describe now the isolation of the main alkaloid named guattegaumerine. This new bisbenzylisoquinoline alkaloid has been assigned structure 1, on the basis of spectroscopic, chemical and chromatographic evidence.

Metabolism of 25-hydroxycholecalciferol by human bone.

Although there is abundant evidence from animal studies that 1,25-dihydroxychole-calciferol [1,25-(OH)2D3] is synthesized in the kidney, it is still unknown whether human bone might also be involved in the formation of this metabolite. To investigate whether 1,25-(OH)2D3 can be synthesized by human bone, 25-hydroxycholecalciferol (25-OH D3) metabolism was assessed in vitro by incubation with human trabecular bone. The bone tissue, obtained from the femoral heads of 34 patients undergoing hip replacement surgery, was ground and thoroughly cleaned of blood and bone marrow. Five hundred mg of spongiosa were incubated with (3H)-25-OH D3 at a concentration of 1.67 nM in 2 ml Eagle’s minimum essential medium. The metabolites formed were separated either by LH 20 column chromatography followed by high performance liquid chromatography (HPLC) or by HPLC alone and the elution profiles compared to either commercially available or biologically synthesized tritiated 1,25-(OH)2D3 and 24,25-(OH)2D3. The formation of a metabolite which was chromatographically similar to 1,25-(OH)2D3 was observed in 15, and a metabolite with the chromatographic features of 24,25-(OH)2D3 in 13 of 21 bone samples. In 4 samples an additional metabolite more polar than 1,25-(OH)2D3 was observed. I n 6 bone samples no chromatographic evidence for the production of any metabolite of 25-OH D3 could be found. Ten per cent isopropanol, 1 ml of uremic serum or replacement of NADPH by NADH in the incubation medium completely suppressed the production of all derivatives. The demonstration that human bone is able to metabolize 25-OH D3 may be important for the understanding of bone physiology.

Biotransformation of Cannabidiol to Cannabielsoin by Suspension Cultures of Cannabis sativa and Saccharum officinarum

Suspension cultures of CANNABIS SATIVA L. and SACCHARUM OFFICINARUM L. have been shown to biotransform cannabidiol to Cannabielsoin under normal growth conditions. Both Cannabielsoin C-1 stereoisomers were produced. Mass spectral and chromatographic evidence were used in this identification. This is the first reported biologically catalyzed synthesis of Cannabielsoin as well as the first biotransformation of any cannabinoid by plant tissue culture systems. Possible mechanisms and significance of this conversion are discussed.