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PubMed1993

A novel ganglioside with a free amino group in bovine brain.

A novel ganglioside which binds cholera-toxin B-subunit was purified from bovine brain by an h.p.l.c. system using an Aquasil column subsequent to Q-Sepharose column chromatography. T.l.c./immunostaining showed that the isolated ganglioside had about 60% of the binding reactivity of the authentic ganglioside GM1 for cholera-toxin B-subunit. On h.p.l.c., this ganglioside migrated between ganglioside GD1a and GD1b, and was found to give positive reactions with ninhydrin and fluorescamine reagents which specifically react with amino groups. The presence of a free amino group was further confirmed by chemical re-N-acetylation. The N-acetylated product had an identical RF value on h.p.l.c. and similar reactivity with cholera-toxin B-subunit as the authentic GM1. H.p.t.l.c., t.l.c./immunostaining, negative-ion fast-atom-bombardment (f.a.b.)-m.s., and 1H-n.m.r. spectroscopy of the novel ganglioside unequivocally demonstrated that it has the basal structure of GM1 with de-N-acetylated neuraminic acid instead of N-acetylneuraminic acid. In the present study we report for the first time that a ganglioside derivative containing de-N-acetylated neuraminic acid, de-N-acetylated GM1, exists in natural brain tissues.

Acetylation↗
PubMed1998

Isolation and partial characterization of antifungal metabolites of Bacillus pumilus.

Antifungal metabolites produced by Bacillus pumilus in Potato Dextrose Broth (PDB) were isolated from culture supernatant fluid by precipitation with ammonium sulphate. The antifungal metabolites inhibited mycelial growth of many species of Aspergillus, Penicillium and Fusarium. They also inhibited production of aflatoxins, cyclopiazonic acid, ochratoxin A and patulin. The metabolites were heat-stable and remained active after sterilization at 121 degrees C for 15 min. Their activity was stable over a wide range of pH (2-10). The metabolites were resistant to hydrolysis by various proteases, peptidases and other enzymes. They were also resistant to denaturation by many protein-denaturing detergents except Nonidet P-40. The metabolites were soluble in water and relatively polar organic solvents. Chromatographic bioassay revealed that a crude precipitate of the metabolites contained only one compound with antifungal activity. The active compound did not form a fluorescent derivative with fluorescamine suggesting that the compound is either a cyclic polypeptide or a non-peptidic compound.

Adolescent↗
PubMed2002

Selective peroxidation and externalization of phosphatidylserine in normal human epidermal keratinocytes during oxidative stress induced by cumene hydroperoxide.

Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar oxidative stress in vivo would under go phosphatidylserine oxidation/translocation. This would make them targets for macrophage recognition and phagocytosis, and thus limit their potential to invoke inflammation or give rise to neoplastic transformations.

Adult↗
PubMed1987

Binding properties of human thrombospondin: interaction with mucopolysaccharides.

The interaction of human thrombospondin with mucopolysaccharides has been measured quantitatively. Binding of thrombospondin to heparin was examined utilizing an assay employing an 125I-labeled LMW heparin glycosaminoglycan (Mr = 8500). By this means, a class of binding sites was detected that bound approximately 2 moles of LMW heparin per mole of thrombospondin with a Kd of 2.4 nM. The binding stoichiometry of LMW heparin to thrombospondin was confirmed by fluorescence polarization experiments in which thrombospondin bound 3 moles of fluorescamine-labeled LMW-heparin mole protein. A variety of mucopolysaccharides were able to inhibit the interaction of thrombospondin with 125I-LMW heparin, the most effective being heparin sulfate and dermatan sulfate. However, PF4 was found to be an even more potent inhibitor, approximating unfractionated heparin in this respect. The ability of mucopolysaccharides to interact with purified thrombospondin suggests a role for such molecules in the regulation of the biologic activity of thrombospondin, possibly in interactions with connective tissue, such as the subendothelium. Given that there are three binding sites per molecule and that thrombospondin appears to form polymeric aggregates with itself, significant binding energies could be developed.

Animals↗
PubMed1974

Studies on the extended active sites of acid proteinases.

The kinetics of the hydrolysis of a series of peptide substrates at a single peptide bond (between L-phenylalanyl and L-phenylalanyl) by the acid proteinases gastric pepsin A (EC 3.4.23.1), Rhizopus pepsin (EC 3.4.23.9), and beef-spleen cathepsin D (EC 3.4.23.5) have been determined by use of the fluorescamine assay method. The results indicate that the extended active site of pepsin can accommodate a sequence of at least seven amino-acid residues. Although the other two acid proteinases appear to act at the sensitive L-phenylalanyl-L-phenylalanyl bond by a mechanism similar to that of pepsin, the influence of structural changes on either side of the sensitive dipeptidyl unit on the kinetic parameters is different from that for pepsin. These data give further evidence for the importance of secondary interactions in determining the catalytic efficiency of enzymes that act on oligomeric substrates.

Amino Acid Sequence↗
PubMed1974

A hyperglycemic peptide from pituitary growth hormone: preparation with pepsin and assay in ob-ob mice.

A fragment that induces glucose intolerance in hereditarily obese mice has been prepared from pituitary growth hormone(sheep and human) by controlled digestion with pepsin in 0.05 M sodium acetate buffer, pH 3.7. The digest was purified by column chromatography on Bio-Gel P-6, followed by chromatography on Bio-Rex 70 and Bio-Gel P-2; in each successive step peptide was quantitated with the fluorescamine procedure. The fragment from sheep growth hormone has these characteristics: (1) It induces glucose intolerance in fasted ob/ob female mice when injected subcutaneously in a divided dose 15 min before, and concurrently with, glucose. (2) It is a basie peptide, as judged from its elution behavior on Bio-Rex 70, H(+) form. (3) It does not crossreact with antiserum to human growth hormone in radioimmunoassay. Further purification and initial sequence determinations have been undertaken.

Animals↗
PubMed1976

Fluorometric assay of vasopressin and oxytocin: a general approach to the assay of peptides in tissues.

A fluorometric method for the quantitative assay of vasopressin and oxytocin in individual rat pituitaries has been developed. Acid extracts of pituitaries are freed of amino acids and polyamines by passage over a copper-Sephadex column, and the peptides fraction is then labeled by reaction with fluorescamine. The resulting peptide fluorophors are separated by chromatography on a reverse-phase bonded column. Specificity of the procedure was ascertained by several criteria, including bioassay and amino-acid analysis of the eluted peptide fluorophors. The procedure serves as a model system for the assay of tissue peptides in the picomole range.

Amino Acids↗
PubMed1989

Immunoregulatory activity of peptides related to platelet factor 4.

Platelet factor 4 (PF4), a secreted platelet protein, alleviates concanavalin A-induced immunosuppression in mice. We now find that activity also resides in (i) the C-terminal tridecapeptide of PF4 (P13S), (ii) an analog of this in which arginine replaces the lysine residues and in which the last two amino acids are absent, (iii) the C-terminal 18 amino acids of low-affinity platelet factor 4, which is very similar to P13S, and (iv) peptide fragments of P13S that contain only 5-9 amino acids. P13S treated with fluorescamine to derivatize the free amino groups retained immunoregulatory activity but did not bind to heparin-agarose. The N-terminal and middle portions of PF4, polylysine, protamine, and three unrelated peptides were inactive in this assay.

Amino Acid Sequence↗
PubMed2000

Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light.

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 degrees C following UV irradiation of collagen, compared with 39 degrees C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.

Animals↗
PubMed2002

Autolytic processing at Glu586-Ser587 within the cysteine-rich domain of human adamalysin 19/disintegrin-metalloproteinase 19 is necessary for its proteolytic activity.

We investigated the regulation of the proteolytic activity of human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19). It was processed at Glu(586)(P1)-Ser(587)(P1') site in the cysteine-rich domain as shown by protein N-terminal sequencing. This truncation was autolytic as illustrated by its R199A/R200A or E346A mutation that prevented the zymogen activation by furin or abolished the catalytic activity. Reagents that block furin-mediated activation of pro-hADAM19, decRVKR-CMK, and brefeldin A abrogated this processing. The sizes of the side chains of the P1 and P1' residues are critical for the processing of hADAM19. The amount of processing product in the E586Q or S587A mutant with a side chain almost the same size as that in the wild type was almost equal. Conversely, very little processing was observed when the size of the side chain was changed significantly, such as in the E586A, E586G, or S587F mutants. Two mutants with presumably subtle structural distinctions from wild type hADAM19, E586D and S587T, displayed rare or little processing and had very low capacities to cleave alpha2-macroglobulin and a peptide substrate. Therefore, this processing is necessary for hADAM19 to exert its proteolytic activities. Moreover, a new peptide substrate, Ac-RPLE-SNAV, which is identical to the processing site sequence, was cleaved at the E-S bond by soluble hADAM19 containing the catalytic and disintegrin domains. This enzyme cleaved the substrate with K(m), k(cat), and k(cat)/K(m) of 2.0 mm, 2.4/min, and 1200 m(-1) min(-1), respectively, using a fluorescamine assay. Preliminary studies showed that a protein kinase C activator, phorbol 12-myristate 13-acetate, promoted the cellular processing of hADAM19; however, three calmodulin antagonists, trifluoperazine, W7, and calmidazolium, impaired this cleavage, indicating complex signal pathways may be involved in the processing.

ADAM Proteins↗
PubMed2004

Pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases.

The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from beta-[14C]casein and studied in HeLa cell extracts the degradation of the 9-17 residue fraction and also of synthetic deca- and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30-50%. Pure TOP failed to degrade proteasome products 18-24 residues long but degraded the 9-17 residue fraction to peptides of 6-9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9-17 residue proteasome products were also converted to peptides of 6-9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9-17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9-17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6-9 residue fragments are further digested to amino acids by aminopeptidases.

Aminopeptidases↗
PubMedDate not supplied

Protein leakage in the lungs of mice exposed to 0.5 ppm nitrogen dioxide.

Forty-four mice were continuously exposed to 0.47 ppm nitrogen dioxide for ten, 12, and 14 days. The protein content of homogenized lung tissue was assayed fluorometrically after the animals had received intravenous injections of fluorescamine, a new reagent for protein assay. The mean protein value of all exposed animals was higher than that of the control animals (P less than .025). No correlation was noted between serum fluorophor levels and those in the lung homogenate.

Animals↗
PubMed1987

Quantification and gel electrophoretic analysis of proteins extracted from vascular endothelial cells.

The inner walls of tubular organs and body cavities are lined with a sheet composed of cells which control the passage through the walls and thus are of considerable physiological and biochemical interest. They are difficult to prepare but their content can be extracted and analysed, using laminar extraction and sensitive bioluminescence methods. This has been shown for the endothelial cells which line the interior of blood vessels. Although it is dealing with small amounts of material, only a minor part of it is consumed in the analyses. The bioluminescence measurements can thus be combined with other analytical procedures such as protein assay and electrophoresis. The possibilities of extending the biochemical information is of interest in the efforts to clarify the pathogenesis of vascular diseases. Laminar elution of protein from endothelial cells of the rat aorta was traced by the simultaneous efflux of adenine nucleotides. These were determined in a bioluminescence assay after conversion to ATP, while the fluorescamine reaction was used for the quantification of protein. The elution patterns obtained by these two methods showed an initial peak with a common maximum. The new possibilities of detecting relevant biochemical changes were evidenced by the finding of a marked protein loss in the fractions containing the outflux from the endothelial cells of diabetic rats. Electrophoresis of proteins eluted from endothelial cells resulted in separation into a large number of bands, but no differences were detected in the electropherograms at comparisons between normal and diabetic rats. It remains to be clarified, whether the protein depletion of the endothelial sheet is due to a concomitant loss of cells or to a cytoplasmatic loss without profound changes of the cytoplasmatic composition.

Adenine Nucleotides↗
PubMed1984

A rapid thin-layer chromatographic screening method for five sulfonamides in animal tissues.

A procedure is described for the detection of five sulfonamides in muscle and kidney of swine, chicken and cattle. The method consists of an extraction with dichloromethane, cleaning up of the extracts on a Sep-Pak silica disposable column, and analysis by thin-layer chromatography on a silica plate with chloroform/n-butanol as an eluent. The sulfonamides were visualized by spraying with a fluorescamine solution. In spiked tissues the presence of the sulfonamides at concentrations of 0.05 mg/kg and higher is easily demonstrated. Direct detection of sulfonamides in tissue extracts of kidneys and lean tissues under the same chromatographic conditions is also described. This method, in which the Sep-Pak cleaning up procedure is omitted, has been successfully applied to spiked swine and cattle kidneys, chicken liver and breast tissue.

Animals↗
PubMed2000

A method for determining fumonisin B1 in corn using immunoaffinity column clean-up and thin layer chromatography/densitometry.

A method for determining fumonisin B1 (FB1) in corn was developed and the clean-up optimized in order to give an extract suitable for one-dimensional thin layer chromatographic (TLC) analysis. FB1 was extracted with a solution of methanol:water (80:20, v/v), purified through an immunoaffinity column and separated on a C18 reversed phase TLC plate. The FB1 was visualized with 0.1 mol/l sodium tetraborate, 0.40 mg/ml fluorescamine in acetonitrile and 0.01 mol/l boric acid:acetonitrile (2:3, v/v) for fluorescence detection, and quantified by densitometric analysis. Water, acetonitrile:water (1:1 v/v) and acetonitrile:water (4:1 v/v) were evaluated as TLC solvents for running both standards and samples together with derivatization procedures aimed at improving separation, resolution, sensitivity and linearity. The mean recovery for FB1 for spiked samples was found to be 85% and the linear equation of standard calibration curve by densitometric analysis gave an r2 value higher than 0.99. The maximum coefficient of variation for replicate analysis of spiked samples was 19%. The absolute amount of FB1 standard detectable on a TLC plate was 2 ng, giving a detection limit for the method of 0.1 mg/kg. The method has been shown to be robust in the application of FB1 monitoring in corn (214 samples) collected in different regions of the country. FB1 was detected in 99% of these samples in the range of 0.2 to 6 mg/kg.

Carboxylic Acids↗
PubMed2004

Determination of the mycotoxin fumonisin B1 in maize by reversed-phase thin-layer chromatography: a collaborative study.

A simple and cost-effective method using thin-layer chromatography for the determination of the mycotoxin fumonisin B1 in maize is described. The analytical method consisted of the extraction of ground maize by shaking with methanol/water (75:25) for 60 min and clean-up of the resultant extract by means of strong anion exchange solid-phase extraction. The purified residue, formed by evaporation of the elution solvent, was reacted with fluorescamine and the fumonisin B1-derivative was separated by reversed-phase thin-layer chromatography using a developing solution of methanol/aqueous 4% potassium chloride (70:30). The derivatized FB1 was readily visualized as a greenish-yellow spot under long wavelength ultraviolet light and quantified by visual comparison with a set of similarly derivatized standards in the range 20-300 ng FB1 spotted on plate. Based on visual comparison, levels down to 0.5 mg kg(-1) were successfully estimated. The method was collaboratively studied in 14 laboratories using four duplicate maize meal samples (including a blank) and a spiked sample for determination of recovery. No significant difference was observed between mean FB1 levels by high-performance liquid chromatography or thin-layer chromatography. Based on within-laboratory relative standard deviations of 27.1-41.7% and between-laboratory relative standard deviations of 35.0-63.3%, the method can be considered semiquantitative. The mean recovery achieved by participants at a spiking level of 2.00 mg kg(-1) was 74.5%.

Carcinogens↗
PubMed1979

Fluorescence detection and determination of patulin by thin-layer chromatography of its aniline imine.

A sensitive and simple procedure is descrobed for the analysis of the mycotoxin patulin. The method involves extraction of patulin from the sample, silica gel column chromatographic clean-up, preparation of the aniline imine, thin-layer chromatographic separation on silica gel, hydrogen chloride hydrolysis of the imine, and fluorophore formation from the liberated aniline with fluorescamine. A linear calibration curve was obtained for the 10-100 ng range and the limit of detection was 5 ng. Recoveries of patulin from samples of apple juice spiked at 50-500 ppb were quantitative.

Aniline Compounds↗
PubMedDate not supplied

Utilizing a D-amino acid as a drug carrier for antineoplastic nitrogen mustard groups.

The evaluation of an alkylating nitrogen mustard agent that utilizes D-alanine as a drug carrier for three chloroethyl substituents (CICH2CH2-) is shown. Various important pharmacological properties were determined including polar surface area, partition coefficient, molar volume, polarizability, numbers of -OH and -NH2 groups, and aqueous solubility. The synthetic approach utilizes 1,2-dichloroethane reaction with the primary amine of D-alanine resulting in chloroethyl (CICH2CH2-) substituents. A nitrogen mustard group results, and this agent showed alkylation activity in aqueous solution directed toward a nucleophilic primary amine group. Kinetics of alkylation activity is determined by placing p-chloroaniline and the nitrogen mustard agent in sodium bicarbonate buffered aqueous solution at physiological pH 7.4 and 37 degrees C. Samples taken from the test solution are injected with fluorescamine that reacts specifically with primary amine functional groups. Absorbance measurements obtained at 400 nm by UV-Vis spectrometer indicates the relative amounts of nonalkylated p-chloroaniline in the test solution. The D-alanine nitrogen mustard agent effectively alkylated the nucleophilic primary amine of p-chloroaniline with zero-order kinetics. The rate constant was determined to be 7.445E-04 mol/l/min. Formula weight, polar surface area, Log P, molar volume, violations of Rule of 5 for D-alanine mustard agent are 276.59, 29.543 angstroms2, 1.605, 222.3 cm3, and zero violations, respectively. Cluster analysis of molecular properties showed D-alanine mustard agent to be quite similar to cyclophosphamide. This agent showed good druglikeness and zero violations of the Rule of 5, indicating good bioavailability. Molecular properties calculated for the mustard agent are numerically comparable to some clinical anticancer drugs.

Alanine↗