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Biomedical subjects

J W Metzger

Publications and source records attributed to J W Metzger.

At least 19 recordsLinked to original sources
PubMed2000

Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation.

Recently, a bacterium capable to oxidize ammonium anaerobically at a high rate was identified as novel member of the Planctomycetales (Strous, M., Fuersi, J. A., Kramer, E. H. M., Logemann, S., Muyzer, G., van de Pas-Schoonen, K. T., Webb, R. I., Kufnen, J. G., and Jetten, M. S. M.: Nature 400, 446-449, 1999). Here we investigated the microbial community structure of a trickling filter biofilm with a high anaerobic ammonium oxidation activity. Fluorescence in situ hybridization (FISH) with a set of nine probes designed for specific identification of the recently described anaerobic ammonium oxidizer demonstrated that only one probe hybridized to bacteria within the biofilm. For phylogenetic characterization of putative biofilm anaerobic ammonium oxidizers a full-cycle 16S rDNA approach was performed by using a Planctomycetales-specific forward primer for PCR amplification. Of the twenty-five 16S rDNA fragments (1364 bp in length) amplified from the biofilm, nine were affiliated to the Planctomycetales. Comparative analysis showed that these sequences were more than 98.9% similar to each other but only distantly related to the previously recognized anaerobic ammonium oxidizer (below 91% similarity) and all other organisms represented in public 16S rRNA databases (similarities of below 79%). The retrieved sequences and the previously recognized anaerobic ammonium oxidizer represent two well-separated groups of a deep-branching lineage within the Planctomycetales. Quantitative FISH analysis with a newly designed specific probe showed that the novel bacterium, provisionally classified as "Candidatus Kuenenia stuttgartiensis" constituted the dominant fraction of the biofilm bacteria. In situ probing revealed that ammonia-oxidizing bacteria of the beta-subclass of Proteobacteria were also present, albeit in significant smaller amounts, within the anoxic biofilm. Comparative sequence analysis of a stretch of the gene encoding ammonia-monooxygenase (amoA) demonstrated the occurrence of the DNA of at least three different populations of beta-subclass ammonia oxidizers within the biofilm.

Anaerobiosis↗
PubMed1999

Tryptic digest mapping by gradient capillary electrochromatography.

A high performance liquid chromatography (HPLC) system complemented with T-split, capillary detection cell and a high voltage power supply was used for peptide mapping by gradient electrochromatography and nanoliquid chromatography (nano-LC). With capillary columns of 100 microm ID, 6 cm packed with octadecylated 1.5 microm silica particles, the typical analysis time was approximately 10-15 min. The resolution of a tryptic digest of cytochrome c obtained by electrochromatography at 100 kV/m was superior compared to the analysis by nano-LC. Bubble formation caused by Joule heating at currents up to 100 microA was successfully suppressed by using a resistor capillary of 25 microm ID connected to the outlet of the packed column.

Chromatography, Liquid↗
PubMed1999

Disposable sensor for measuring the biochemical oxygen demand for nitrification and inhibition of nitrification in wastewater.

A disposable-type microbial sensor was developed for the determination of both the biochemical oxygen demand for nitrification (N-BOD) and inhibiting effects on nitrifying bacteria. The sensor was based on the respiratory activity of nitrifying bacteria immobilized on a miniature oxygen electrode. Typical response times for measuring N-BOD of ammonium standard solutions as well as of wastewater samples were in the range of 6-12 min. A dynamic evaluation of the signals after a measuring time of 120 s also resulted in good reproducibility and sensitivity. A daily profile of a municipal sewage plant was recorded, comparing the biosensor data with two standard methods. For the measurement of nitrification-inhibiting effects a 120-s dynamic signal evaluation was preferred to a steady-state method because of the long recovery times resulting from extended exposure to inhibitors. However, steady-state measurement techniques allowed allylthiourea detection with a ten times higher sensitivity. Because of the advantages of this miniaturized electrode, e.g. short response time, simple measuring procedure and low costs of production, this sensor system is considered to be suitable for commercial application in environmental analysis.

Bacteria↗
PubMed1998

A microbial sensor for detecting inhibitors of nitrification in wastewater.

A biosensor for rapid and reproducible measurements of inhibitors of nitrification in environmental samples has been developed. The biosensor is mainly designed to be used for wastewater control and consists of a Clark oxygen probe as a transducer and an immobilised mixed nitrifying culture as the microbial component. The measuring principle is based on the direct determination of bacterial metabolic activity by measuring the oxygen consumption rate of the microbial immobilisate. Both the prototype of a laboratory device and a field device have been realised. The laboratory device can be used to determine the nitrification inhibiting effect of individual chemical compounds as well as of environmental samples. The field device was constructed for on-line monitoring of inhibitors in sewage systems.

Bacteria↗
PubMed1998

Spirofungin, a new antifungal antibiotic from Streptomyces violaceusniger Tü 4113.

A new secondary metabolite was detected in the culture filtrate and extracts of Streptomyces violaceusniger Tü 4113 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The compound named spirofungin has a polyketide-spiroketal structure and shows various antifungal activities, particularly against yeasts.

Antifungal Agents↗
PubMed1997

Composition and purity of combinatorial aryl ether collections analyzed by electrospray mass spectrometry.

Electrospray mass spectrometry (ESI-MS), tandem mass spectrometry and on-line RP-HPLC-ESI-MS were used to evaluate the composition and purity of three different aryl ether mixtures consisting of 10 and 45 aryl ethers synthesized on solid support by Williamson etherification. The libraries feature two potential pharmacophores connected with three different spacers and serve as models for a detailed component analysis. Individual members of the library and by-products were identified rapidly and conveniently by product ion scans. Compound collections obtained by two different synthetic methods, the split/combine approach and the premix method, showed different mass distributions in the ESI-MS spectra. Some components were not detected in direct ESI-MS measurements, but were found by MS/MS experiments. Precursor ion and constant neutral loss scans allowed the identification of components with common structural features.

Chromatography, High Pressure Liquid↗
PubMed1996

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.

Alanine↗
PubMed1996

Betacyanins from plants and cell cultures of Phytolacca americana.

Betacyanins from cell cultures of Phytolacca americana were characterized and compared with those of the stems and ripening fruits of the plant. Whereas in fruits prebetanin (betanin 6'-O-sulphate) and its isoform predominate, in the stem and cell cultures feruloylated derivatives occur as the major components. These were rigorously identified by various spectroscopic techniques (DAD-HPLC, NMR, LC-MS and electrospray MS-MS) and carbohydrate analyses as betanidin 5-O-[(5"-O-E-feruloyl)-2'-O-beta-D-apiofuranosyl] -beta-D-glucopyranoside, a new betacyanin of higher plants, and betanidin 5-O-(6'-O-E-feruloyl)-beta-D-glucopyranoside (lampranthin II), together with their isoforms.

Betacyanins↗
PubMed1996

Kanchanamycins, new polyol macrolide antibiotics produced by Streptomyces olivaceus Tü 4018. I. Taxonomy, fermentation, isolation and biological activities.

The kanchanamycins, a group of novel 36-membered polyol macrolide antibiotics were detected in the culture filtrate and mycelium of Streptomyces olivaceus Tü 4018 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The compounds show antibacterial and antifungal activities, and are especially effective against Pseudomonas fluorescens. Besides the kanchanamycin complex, strain Tü 4018 produces the 42-membered macrolactones, oasomycin A and desertomycin A, as well as tryptophan-dehydrobutyrine diketopiperazine and daidzein.

Anti-Bacterial Agents↗
PubMed1996

Kanchanamycins, new polyol macrolide antibiotics produced by Streptomyces olivaceus Tii 4018. II. Structure elucidation.

Kanchanamycins are a new group of polyol macrolide antibiotics isolated from Streptomyces olivaceus Tü 4018. They all share a common bicyclic carbon skeleton formed by a 36-membered lactone ring and a 6-membered hemiacetal ring. A feature unusual for that class of macrolides is the terminal urea moiety observed in kanchanamycin A. The structures of the kanchanamycins were determined by electrospray MS and modern 2D NMR techniques. Due to substantial overlap of the signals intensive use of inverse detected heteronuclear correlation experiments (HSQC, HMBC, 2D-HSQC-TOCSY) was made.

Anti-Bacterial Agents↗
PubMed1996

Neurokinetic and myokinetic effects of the peptide xenin on the motility of the small and large intestine of guinea pig.

The effects of xenin 25, a peptide of the neurotensin/xenopsin family, were examined on the motility of the guinea pig jejunum and colon in vitro. In the jejunum, xenin induced a biphasic response: first a small relaxation and then a large contraction. In the colon, xenin induced relaxation. The tenia coli was contracted. Deletion or amidation of the C-terminal leucine inactivated xenin. A peptide sequence of 16 C-terminal amino acids was necessary to elicit a full response in the jejunum, whereas in the colon, the potencies of all fragments of xenin 25, including the 6 C-terminal amino acid sequence (xenin 6), were not significantly different from that of xenin 25. In the jejunum, contraction was abolished or reduced by tetrodotoxin, by atropine, by met-enkephalin, by antagonists to the tachykinin receptors NK1 and NK2 and by the P2-purinoceptor antagonist suramin. L-NNA, phentolamine, methysergide, hexamethonium and apamin had no effect. In the colon, all actions of xenin were tetrodotoxin-resistant; the potency of xenin to relax was reduced by apamin and suramin. The actions of xenin on small and large bowel were attenuated by the neurotensin receptor antagonist SR 48692. The xenin analog neurotensin was significantly less potent than xenin 25 in contracting the jejunum and more potent than xenin 25 in relaxing the colon. We conclude that xenin exerts excitatory effects on a neuronal subtype receptor in the jejunum, with participation of muscarinic, purinergic and tachykinin-related mechanisms. Xenin exerts relaxing effects on the colon by interaction with a myokinetic subtype receptor involving Ca(++)-dependent K+ channels and the P2-purinoceptor.

Animals↗
PubMed1995

beta-Glucosylarginine: a new glucose-protein bond in a self-glucosylating protein from sweet corn.

In the search for a protein primer for starch synthesis, an autocatalytic self-glucosylating protein has been isolated from sweet corn. Several tryptic peptides were obtained from the [14C]glucosylated protein and were sequenced, corresponding to over 40% of the estimated total sequence (molecular mass 42 kDa). There is no homology with the amino acid sequence of the autocatalytic glycogen primer, glycogenin, nor in respect of the nature of the union between the autocatalytically added glucose and the protein, which, in the case of the corn protein, now named amylogenin, is a novel glucose-protein bond, a single beta-glucose residue joined to an arginine residue.

Amino Acid Sequence↗
PubMed1995

The tetracyclic lantibiotic actagardine. 1H-NMR and 13C-NMR assignments and revised primary structure.

The primary structure of the peptide lantibiotic actagardine was determined in (2H3)acetonitrile/H2O by homonuclear two- and three-dimensional NMR spectroscopy as well as 2D 1H(13C) correlation spectra at natural abundance. Actagardine was found to be a tetracyclic 19-residue peptide containing one lanthionine and three overlapping beta-methyllanthionine bridges. Sequential resonance assignment and location of the four thioether rings was accomplished by 2D NOESY, 3D NOESY-TOCSY and gradient-enhanced 1H(13C)-HMBC spectra. The C-terminal thioether bridge was shown to be oxidized to a sulfoxide. The NMR data were additionally confirmed by mass spectrometry and Edman degradation after chemical modification, which allowed sequencing of lanthionine and beta-methyllanthionine residues. Our studies clearly show, that the structure of actagardine as previously published by Kettenring et al. (1990) J. Antibiot. 43, 1082-1088 is not correct.

Alanine↗
PubMed1995

Sequence analysis by NMR spectroscopy of the peptide lantibiotic epilancin K7 from Staphylococcus epidermidis K7.

The amino acid sequence of the novel lantibiotic epilancin K7 from Staphylococcus epidermidis K7 was determined by NMR spectroscopy. NMR spectroscopy was used because sequencing by conventional Edman degradation techniques was prohibited by internal sequence blocks owing to the presence of modified residues. Epilancin K7 consists of 31 residues, including two alpha,beta-didehydroalanine (one-letter code U) and two alpha,beta-didehydrobutyrine (O) residues, one lanthionine (A-S-A), two beta-methyllanthionines (A*-S-A), and six lysines. Epilancin K7 has a molecular mass of 3032 +/- 1.5 Da. The amino acid sequence of epilancin K7 was derived from both through-space dipolar proton-proton interactions and through-bond scalar proton-carbon interactions as detected by two-dimensional 1H-NOESY, 1H-ROESY and three-dimensional 1H-TOCSY-NOESY, and by two-dimensional 1H,13C-heteronuclear multiple-bond correlation spectroscopy, respectively. The sequence is as follows: [sequence: see text] The N-terminal residue X partly resembles an alanine but its exact nature is unclear. The organization of the sulfide-bridge-containing (beta-methyl-)lanthionines was revealed by 1H-NMR and 1H,13C-NMR spectroscopy. Epilancin K7 has a linear structure and a high positive net charge, and therefore is classified as a type-A lantibiotic. NMR analysis of a degraded though still active form of epilancin K7 showed that two N-terminal residues of epilancin K7 were missing, owing to decomposition at the alpha,beta-didehydro alanine at position 3; it was called the epilancin K7-(3-31)-peptide (peptide fragment of epilancin K7 consisting of positions 3-31). The usefulness of three-dimensional 1H-TOCSY-NOESY, and two-dimensional 1H,13C-heteronuclear multiple-bond correlation spectroscopy at natural abundance for the study of (modified) polypeptides is demonstrated.

Amino Acid Sequence↗
PubMed1995

Proximity mapping of the Tet repressor-tetracycline-Fe2+ complex by hydrogen peroxide mediated protein cleavage.

We demonstrate in a quantitative in vitro induction assay that tetracycline-Fe2+ is a more than 1000-fold stronger inducer of Tet repressor compared to tetracycline-Mg2+. Oxidative cleavage of the Tet repressor-tetracycline-Fe2+ complex with H2O2 and ascorbate results in an Fe(2+)-dependent specific fragmentation of the protein. The maximal yield of about 15% and a reaction time of less than 30 s are only observed in the presence of the drug, whereas about 1% cleavage is obtained after 30 min in the presence of Fe2+ without tetracycline. Cleavage is not inhibited by several radical scavengers, suggesting a highly localized reactivity of the redox-active oxo intermediates in the proximity of the Fe(2+)-tc chelater where they are generated. The products can be separated by HPLC only after denaturation, indicating that the complex is not disrupted by cleavage. Residues at which the cleavage takes place are identified using the masses of the fragments determined by electrospray mass spectrometry and their N-terminal sequences. The major cleavage site maps to residues 104 and 105 of Tet repressor. Less efficient cleavages occur at residues 56 and 136, and the least efficiently cleaved sites are around residues 144 and 147. The cleavage efficiencies correlate to the distances and orientations of the respective peptide bonds to Mg2+ in the crystal structure of the Tet repressor-tetracycline-Mg2+ complex. We discuss potential reaction mechanisms leading to protein cleavage.

Amino Acid Sequence↗
PubMed1995

Distribution, formation, and molecular forms of the peptide xenin in various mammals.

In the present investigation we isolated the recently discovered pentacosapeptide xenin from gastric mucosa of man, dog, pig, guinea pig, rat, and rabbit. HPLC, mass spectrometry, and amino acid sequence analysis showed xenin-25 in concentrations of 54-144 pmol/g tissue in gastric mucosa of each species. Extraction with 2% TFA followed by analytical C18 HPLC revealed 0.02-84 pmol/g xenin-25 also in hypothalamus, lung, liver, heart, kidney, adrenal gland, pancreas, testicle, skin, and duodenal, jejunal, ileal, and colonic mucosa of dog and man, respectively. Digestion of these acid extracts with pepsin liberated xenin-25 in concentrations from 2 up to 166 pmol/g tissue. Gel chromatography revealed a large molecular weight precursor of xenin-25 and evidence for an endogenous acid protease coeluting with pepsinogen capable of releasing xenin-25 from its precursor. Maximal concentrations of xenin-25 were obtained when canine gastric mucosa was incubated with 2% TFA at room temperature for 2 h. Longer incubation times led to a decline of xenin-25 concentration and to formation of xenin-16 and xenin-9, both C-terminal fragments of xenin-25. We conclude that xenin-25 is present not only in human gastric mucosa but also in the stomach of various other mammals. Xenin-25 is further present in low concentrations in many other organs where a pepsin-like protease generates xenin-25 from a large precursor and processes it to smaller fragments.

Amino Acid Sequence↗
PubMed1995

Biosynthetic capacities of actinomycetes. 4. Echinoserine, a new member of the quinoxaline group, produced by Streptomyces tendae.

A new member of the quinoxaline group antibiotics has been detected by HPLC-diode-array screening. The main compound produced by Streptomyces tendae strain Tü 4031 showed a high degree of similarity in the UV-visible spectral region with echinomycin and their structural similarity was confirmed by structure elucidation using electron tandem mass spectrometry and 2D nuclear magnetic resonance. The new compound, named echinoserine, is a non-cyclic form of echinomycin, but it is not a biosynthetic precursor. Echinoserine is less antibiotically active than echinomycin.

Antibiotics, Antineoplastic↗