ABSTRACT
The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.
Subject(s)
Calcitonin/analogs & derivatives , Calcitonin/analysis , Chromatography, High Pressure Liquid/methods , Glycopeptides/analysis , Protein Precursors/analysis , Amidine-Lyases/antagonists & inhibitors , Calcitonin/biosynthesis , Calcitonin/metabolism , Carboxypeptidase H/antagonists & inhibitors , Cell Line, Tumor , Fatty Acids, Monounsaturated/pharmacology , Glycopeptides/biosynthesis , Glycopeptides/chemistry , Glycopeptides/metabolism , Glycosylation , Humans , Mixed Function Oxygenases/antagonists & inhibitors , Protein Precursors/biosynthesis , Protein Precursors/chemistry , Protein Precursors/metabolism , Solid Phase Extraction/methods , Succinates/pharmacologyABSTRACT
OBJECTIVE: To study the chemical constituents of Gentiana striata. METHODS: The constituents were isolated from the whole herb of Gentiana striata by recrystalization, silica gel column chromatography, polyamide column chromatography and Sephadex LH-20,etc. Their structures were elucidated through MS, 1H-NMR, 13C-NMR. RESULTS: -8 compounds were isolated and identified as: Desoxyloganin (1), Gmephiloside (2), 5,7,4'-trihydroxy-3'-methoxyflavone (3), (+)-8-hydroxypinoresinol (4) 3S,5R, 6R, 9S-tetra-hydroxymegastigmane (5), Quercetin-3-O-beta-D-glucoside (6), Ferulic acid (7) and Ursolic acid (8). CONCLUSION: All the compounds are isolated for the first time from Gentiana striata.
Subject(s)
Coumaric Acids/isolation & purification , Flavonoids/isolation & purification , Gentiana/chemistry , Lignans/isolation & purification , Coumaric Acids/chemistry , Flavonoids/chemistry , Glucosides , Lignans/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Quercetin/analogs & derivatives , Triterpenes/chemistry , Triterpenes/isolation & purification , Ursolic AcidABSTRACT
The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibitors with human peptidylglycine α-amidating monooxygenase (PAM) have been investigated. The substrates and inhibitors are the prohormones of calcitonin and oxytocin and their analogues. PAM both secreted into the medium by and extracted from DMS53 small lung carcinoma cells has been studied. The results show that recognition of the prooxytocin and procalcitonin peptide sequences by the enzyme extends more than four and five amino acid residues, respectively, from their C-termini. This substrate sequence recognition is mirrored by increased inhibitor potency with increased peptide length in the glycolate peptidomimetics. Substitution of the C-terminal penultimate glycine and proline residues of prooxytocin and procalcitonin and their analogues with phenylalanine increases the enzyme binding affinity. However, this changes the binding mode from one that depends on peptide sequence recognition to another primarily determined by the phenylalanine moiety, for both the substrates and analogous glycolate inhibitors.
Subject(s)
Enzyme Inhibitors/metabolism , Glycolates/metabolism , Mixed Function Oxygenases/metabolism , Multienzyme Complexes/metabolism , Peptides/metabolism , Amino Acid Sequence , Calcitonin/chemistry , Calcitonin/metabolism , Calcitonin Gene-Related Peptide , Enzyme Inhibitors/chemistry , Glycolates/chemistry , Humans , Kinetics , Mixed Function Oxygenases/chemistry , Multienzyme Complexes/chemistry , Oxytocin/chemistry , Oxytocin/metabolism , Peptides/chemistry , Peptidomimetics , Protein Binding , Protein Precursors/chemistry , Protein Precursors/metabolism , Substrate SpecificityABSTRACT
The anti-inflammatory activity and the mechanism of action of Gentiana striata Maxim. has been investigated. The most active phase, the ethyl acetate extract of Gentiana striata Maxim. (EGS), displayed potent inhibitory activity on feet oedema of rheumatoid arthritis (RA) inflicted rats. This anti-inflammatory activity might be partly based on the notable reduction of prostaglandin E2 (PGE2) and nitric oxide (NO) levels. Six further compounds isolated from EGS have previously been reported as having anti-inflammatory activity.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Gentiana/chemistry , Plant Extracts/pharmacology , Animals , Body Weight/drug effects , Dinoprostone/metabolism , Nitric Oxide/metabolism , RatsABSTRACT
OBJECTIVE: To study the chemical constituents of leaves of Paulownia fortunei (Seem.) Hemsl. METHODS: The constituents were isolated by column chromatography and their structures were elucidated through spectroscopic analysis. RESULTS: The compounds were identified as mimulone (I), apigenin (II), luteolin (III), 2alpha, 3beta, 19beta-trihydroxyurs-28-O-beta-D-galactonopyranos ylester (anserinoside, IV), 3alpha-hydroxyl-ursolicacid (V), ursolicacid (VI), daucosterol (VII), beta-sitosterol (VIII). CONCLUSION: The compounds I - V are obtained from leaves of Paulownia fortunei (Seem.) Hemsl for the first time.
