Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing.

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

Cisapride in functional dyspepsia in general practice. A placebo-controlled, randomized, double-blind study.

BACKGROUND: Functional dyspepsia is recognized as a common disorder in clinical practice. The aim of this study was to determine the efficacy and adverse effects of cisapride compared to a placebo in patients from a general practice with functional dyspepsia (FD). Secondly we investigated whether Helicobacter pylori-positive FD patients present with specific symptoms and determined the efficacy of cisapride for FD patients with H. pylori. METHODS: In a placebo-controlled double-blind study, patients were randomized to receive fixed doses of either cisapride (10 mg three times daily) or placebo. Symptoms were evaluated after 2 and 4 weeks of treatment. The selection of FD patients,collection of data, and evaluation of symptoms as well as adverse effects were performed by general practitioners. Dyspeptic patients were referred to the Gastroenterology Department in order to exclude ulcers, oesophagitis, pancreatitis and gallstones. Biopsies of gastric mucosa were taken for histological examination and H. pylori culture. PATIENTS: 121 patients entered this study (61 took cisapride, 60 placebo). There were 113 patients (56 cisapride, 57 placebo) available for analysis of the efficacy and 120 patients (61 cisapride, 59 placebo) for evaluation of adverse effects.In total 102 biopsies were tested for the presence of gastritis by histological examination. There were 30 H. pylori-positive cultures among 111 patients. RESULTS: After 4 weeks a statistically significant reduction in symptoms was found, but it was similar in the two groups. No symptoms specific for H. pylori-positive patients were found. There was not a significant difference in the response to cisapride between H. pylori-positive and H. pylori-negative patients. The difference in overall (63%) response in the cisapride group and the 44% response in the placebo group did not reach statistical significance. CONCLUSIONS: No significant difference was found between placebo and cisapride in the treatment of FD in general practice. H. pylori-positive patients did not present with specific symptoms nor did they exhibit a different response to cisapride.

Structure and biological activity of chemically modified nisin A species.

Nisin, a 34-residue peptide bacteriocin, contains the less common amino acids lanthionine, beta-methyl-lanthionine, dehydroalanine (Dha), and dehydrobutyrine (Dhb). Several chemically modified nisin A species were purified by reverse-phase HPLC and characterized by two-dimensional NMR and electrospray mass spectrometry. Five constituents, [2-hydroxy-Ala5]nisin, [Ile4-amide,pyruvyl-Leu6]des-Dha5-nisin, [Met(O)21]nisin, [Ser33]nisin, and nisin-(1-32)-peptide amide, were found in a commercial nisin sample. A further species, [2-hydroxy-Ala5]nisin-(1-32)-peptide amide, was obtained by freeze drying an acidic nisin solution. These compounds are formed by chemical modification of nisin: the addition of a water molecule to the dehydroalanine residues, which can lead to the cleavage of the polypeptide chain, or the oxidation of methionine residues. The 2-hydroxyalanine-containing products have a limited stability; they are spontaneously converted into the corresponding des-dehydroalanine derivatives. The growth-inhibiting activity of the modified nisins towards different bacteria was determined. The 2-hydroxyalanine-containing species and the des-dehydroalanine derivative show a strong reduction in biological activity as compared to native nisin. [Met(O)21]nisin and [Ser33]nisin show moderate or no reduction in biological activity.

Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering.

Nisin is a 3.4-kDa antimicrobial peptide that, as a result of posttranslational modifications, contains unsaturated amino acids and lanthionine residues. It is applied as a preservative in various food products. The solubility and stability of nisin and nisin mutants have been studied. It is demonstrated that nisin mutants can be produced with improved functional properties. The solubility of nisin A is highest at low pH values and gradually decreases by almost 2 orders of magnitude when the pH of the solution exceeds a value of 7. At low pH, nisin Z exhibits a decreased solubility relative to that of nisin A; at neutral and higher pH values, the solubilities of both variants are comparable. Two mutants of nisin Z, which contain lysyl residues at positions 27 and 31, respectively, instead of Asn-27 and His-31, were produced with the aim of reaching higher solubility at neutral pH. Both mutants were purified to homogeneity, and their structures were confirmed by one- and two-dimensional 1H nuclear magnetic resonance. Their antimicrobial activities were found to be similar to that of nisin Z, whereas their solubilities at pH 7 increased by factors of 4 and 7, respectively. The chemical stability of nisin A was studied in the pH range of 2 to 8 and at a 20, 37, and 75 degrees C. Optimal stability was observed at pH 3.0. Nisin Z showed a behavior similar to that of nisin A. A mutant containing dehydrobutyrine at position 5 instead of dehydroalanine had lower activity but was significantly more resistant to acid-catalyzed chemical degradation than wild-type nisin Z.

Pharmacokinetic studies of hematoporphyrin derivatives on rat hepatocytes. Protoporphyrin dimethyl ester hematoporphyrin ether versus dihematoporphyrin ether-free hematoporphyrin derivative.

Rat liver cells incorporate monomeric as well as dimeric hematoporphyrin derivatives. Time-dependent incubation assays gave evidence that monomeric compounds are more efficiently incorporated compared to protoporphyrin dimethyl ester hematoporphyrin ether. HL60 cells take up dimeric porphyrins in substantially higher quantities than hepatocytes do. These results allow the conclusion that physiological versus tumor cells behave differently with respect to porphyrin uptake: Whereas physiological cells prefer monomeric porphyrins, tumor cells preferentially incorporate dimeric porphyrins.

[Scintigraphy using I 131 iodobenzylguanidine and malignant melanomas]. / Scintigrafie met behulp van joodbenzylguanidine I 131 en het maligne melanoom.

The successful use of iodine-131-meta-iodobenzylguanidine (I-131-MIBG) scintigraphy for diagnosis of phaeochromocytoma and neuroblastoma stimulated investigation into its diagnostic and therapeutic usefulness in other neural crest tumours. It appears that there is a difference in capacity to absorb I-131-MIBG between the different tumour types. In I-131-MIBG scintigraphy of carcinoids there are more false negative results in comparison with phaeochromocytomas and neuroblastomas. Melanoma, also a neural crest tumor, turned out to be false negative in 100% of the cases reported until 1989. The authors present a case of a malignant melanoma with metastases in liver and stomach, concentrating I-131-MIBG. Biochemical examination demonstrated that this particular tumour was metabolically very active. It is suggested that the I-131-MIBG-positive scintigram of the melanoma may be related to the level of metabolic activity. By biochemical screening in proven cases of malignant melanoma it may be possible to select cases in which I-131-MIBG scintigraphy is worthwhile with a view to therapeutic application of I-131-MIBG.

Pharmacokinetic and -dynamic investigations on HL60 cells with a new protoporphyrin dimethyl ester hematoporphyrin dimer.

Pharmacokinetic and -dynamic studies using a novel porphyrin dimer were performed in human line HL60 promyelocytic leukemia cells. Uptake of the dimer into leukemic cells was observed to occur at substantially lower concentrations in comparison to a previously described monomeric dihematoporphyrin ether-free hematoporphyrin derivative. Both dimer and monomer derivatives could be demonstrated to inhibit cell growth, with no remarkable quantitative differences being found in cell proliferation studies regarding [3H]-thymidine incorporation and assay for colony formation. Structurally, the new compound represents an unsymmetrically substituted diethyl ether having protoporphyrin dimethyl ester and hematoporphyrin as substituents. For this reason the compound was designated as protoporphyrin dimethyl ester hematoporphyrin ether.

[Rotor syndrome: relevance of the determination of coproporphyrin isomers in the urine in comparison with intrahepatic (alcohol-induced) cholestasis]. / Rotor-Syndrom: Relevanz der Koproporphyrin-Isomerenbestimmung im Urin im Vergleich zur intrahepatischen (alkoholtoxischen) Cholestase.

Porphyrin isomer examinations have been performed in two patients with Rotor syndrome (RS), one patient with Gilbert-Meulengracht syndrome and 12 patients with alcohol toxic cholestasis. Under both conditions, cholestasis and RS, total urinary coproporphyrin excretion as well as coproporphyrin isomer I was relatively and absolutely increased. Despite the different degree of the increase of coproporphyrin isomer I excretion between RS (69 vs. 72%) and cholestasis (47% on average), there are single cases with a coproporphyrin isomer I portion around 60%. In such cases, the differential diagnosis is quite difficult, so that the diagnosis "Rotor syndrome" should never be gained by one distinct examination; it is a diagnosis performed by exclusion of other diseases.

On the preparation of dihematoporphyrin ether-free hematoporphyrin derivative.

A dihematoporphyrin ether-free hematoporphyrin derivative has been prepared by a base-catalysed dehydration of hematoporphyrin with sodium hydroxide. The identification was performed by HPLC and mass spectroscopy (FD-MS). The reaction of hematoporphyrin with 1 M sodium hydroxide for 24 h yields more than 90% of the monomeric porphyrins.

Purification and some physicochemical properties of bovine kappa-casein.

1. A description is given of the fractionation of kappa-casein on DEAE-cellulose using a pH gradient. With this method an improved separation of the kappa-casein components with a higher negative charge is obtained. 2. It is shown that at least one of the kappa-casein fractions has a second phosphate ester group. The heterogeneity of kappa-casein therefore is not exclusively caused by a varying N-acetylneuraminic acid content. 3. Ultracentrifuge experiments and exclusion gel chromatography show that the purified kappa-casein fraction having the lowest electrophoretic mobility exhibits a monomer-polymer association equilibrium. The free energy of association per mol monomer in 0.2 M NaCl is approximately --36 kJ-mol-1.