Biomedical subjects

E Pittenauer

Publications and source records attributed to E Pittenauer.

13 recordsLinked to original sources

PubMed1999

Peptidoglycan fine structure of the radiotolerant bacterium Deinococcus radiodurans Sark.

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-(gamma)-L-Orn-[(delta)Gly-Gly]-D-Ala-D-Ala. Cross-linkage was mediated by (Gly)2 bridges, and glycan strands were terminated in (1-->6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.

Amino Acid Sequence↗

PubMed1998

Complete structural elucidation of triacylglycerols by tandem sector mass spectrometry.

We developed a method to elucidate the complete structure of triacylglycerols by means of high-energy collisional activation tandem mass spectrometry (MS/MS). Both ESI- and FAB-produced [M + NH4]+ and [M + met.]+ ions (where met. = Li, Na, and Cs) of triacylglycerols undergo charge-remote and charge-driven fragmentations. We emphasize the study of fragment ions from ESI-produced [M + NH4]+ and [M + Na]+ ions and FAB-produced [M + Na]+ ions. ESI-produced [M + NH4]+ ions fragment to produce four types of ions, [M + NH4 - RnCOONH4]+, [RnCO + 128]+, [RnCO + 74]+, and RnCO+ ions, from which the carbon number and the degree of unsaturation of each acyl group are obtained. In addition, three series of ions are produced by charge-remote fragmentations (CRFs), and analysis of their patterns gives the position and the number of double bonds on the acyl groups. Information about the position of each acyl group on the glycerol backbone, however, is not provided by collisionally activated dissociation of [M + NH4]+ ions. On the other hand, ESI- and FAB-produced [M + Na]+ ions fragment to form eight types of ions (named A-J ions) that, like those produced by CRF, are highly structurally informative. The absence of certain series members also carries useful structural information. Interpretation of these patterns enables one to obtain the number of carbons, degrees of unsaturation, and location of double bonds, as well as the positions of acyl groups on the glycerol backbone.

Acylation↗

PubMed1998

Improved high-performance liquid chromatographic separation of peptidoglycan isolated from various Staphylococcus aureus strains for mass spectrometric characterization.

Reversed-phase high-performance liquid chromatography (RP-HPLC) of muropeptides, obtained by muramidase digestion of peptidoglycan in combination with amino acid analysis and plasma desorption time-of-flight mass spectrometry is today by far the best tool to analyze the fine structure of the peptidoglycans. Here we report further improvements of the RP-HPLC separation of muropeptides for analyzing the peptidoglycans of various methicillin-resistant strains of Staphylococcus aureus, with emphasis on a more detailed characterization of the interpeptide bridge of the peptidoglycans of this species.

Chromatography, High Pressure Liquid↗

PubMed1998

Metabolism of the anticancer drug flavopiridol, a new inhibitor of cyclin dependent kinases, in rat liver.

Flavopiridol (FLAP) is a promising novel chemotherapeutic agent currently undergoing clinical phase I trials. To examine hepatic metabolism and biliary disposition of FLAP we applied the isolated perfused rat liver system. Besides FLAP two metabolites were detected by high performance liquid chromatography in bile and perfusate. Twenty-five min after FLAP (30 microM) addition to the perfusion medium, biliary secretion of metabolite 1 and 2 reached a maximum of 1.04 +/- 0.52 and 11.34 +/- 4.72 nmol/g.liver.min, respectively. Biliary excretion of parent FLAP, however, continuously increased for 60 min up to 406 +/- 134 pmol/g liver.min. In the perfusate, metabolite 1 was below detection limit and release of metabolite 2 was low (2.8 +/- 0.7 pmol/g liver.min after 60 min). Enzymatic hydrolysis with beta-glucuronidase, mass spectroscopy and electron absorption spectroscopy revealed that both metabolites are monoglucuronides with the glucuronide in position 5 and 7 of the flavonoid core, respectively. The amount of FLAP, metabolite 1 and metabolite 2 excreted into bile during the 60 min of perfusion was 1.94 +/- 0.91, 5.15 +/- 1.95 and 57.29 +/- 23.60% of FLAP cleared from the perfusate during 60 min, respectively. In contrast to the structurally similar flavonoids genistein and daidzein, no inhibition of UDP-glucuronyltransferase with methylumbelliferone as a substrate was observed indicating that different UDP-glucuronyltransferase isoforms are involved in FLAP metabolism. In conclusion, we find that glucuronidation is the major mechanism of hepatic FLAP biotransformation. Metabolites are mainly excreted into bile but also released into systemic circulation. The pharmacological and toxicological effects of these metabolites remain to be elucidated.

Animals↗

PubMed1998

Charge-remote fragmentations are energy-dependent processes.

Fast atom bombardment-produced [M + Na]+ ions of tristearoylglycerol and [M - H]- ions of stearic or nervonic acid undergo charge-remote fragmentations (CRFs) to produce one series of product ions reflecting CnH2n + 2, losses, whereas electrospray ionization-produced ions fragment to give two series of product ions reflecting CnH2n + 2 and CnH2n + 1 losses. These results and those from previous studies show that the mechanisms and energetics of CRFs are complex and unsettled. We demonstrate that several pathways are simultaneously involved in CRFs, and the preference for certain pathways (by CnH2n + 1 and CnH2n + 2 losses) is determined by the internal energy of the compound itself and the ionization and activation energies that are applied to it.

Chemical Phenomena↗

PubMed1998

Metabolism of roxithromycin in the isolated perfused rat liver.

Roxithromycin is a macrolide antibiotic with high clinical potency. N-Demethylation is considered to be one of the main pathways of roxithromycin metabolism in rats. We have studied the hepatic metabolism of roxithromycin in the isolated perfused rat liver. After addition of roxithromycin (30 microM) to the perfusion medium the parent compound and one major metabolite were detected in bile by high-performance liquid chromatography. The metabolite was identified as monodesmethylated roxithromycin by mass spectrometric analysis. Onset of biliary excretion of native roxithromycin was fast, reaching a maximum (130.52 +/- 43.88 pmol g(-1) min(-1)) after only 10 min, whereas excretion of the metabolite was delayed (maximum 75.83 +/- 11.92 pmol g(-1) min(-1) at 30 min). The cumulative excretion of roxithromycin and its metabolite into bile during the 60 min of application amounted to only 1.09 +/- 0.30 and 0.64 +/- 0.22% of the roxithromycin cleared from the perfusate during the same time. The liver content was 0.48 micromol (g liver)(-1), indicating high retention within the organ. No release of the metabolite into the perfusate was detected. In conclusion, this study has demonstrated the importance of phase-I metabolism for the biliary excretion of roxithromycin in rat liver. These findings might be predictive of roxithromycin biotransformation and biliary excretion in man.

Animals↗

PubMed1996

N,N'-Bisformyl dityrosine is an in vivo precursor of the yeast ascospore wall.

The amino acid, dityrosine, is a major component of the spore wall surface of the yeast Saccharomyces cerevisiae, where it is part of a highly cross-linked macromolecular network of yet unknown chemical structure, consisting mostly of glucosamine, dityrosine and few other amino acids. Biosynthesis of the dityrosine moiety of this network consists of several steps, including the chemical modification of free L-tyrosine and the subsequent oxidative cross-linking of the modified tyrosine residues (catalyzed by a cytochrome P-450), leading to soluble dityrosine-containing spore wall precursors. We isolated, purified and characterized the dityrosine-containing precursor that appears late in spore wall synthesis and that is thought to be directly incorporated into the maturing spore wall. Chemical and spectroscopic analyses showed that this precursor is N,N'-bisformyl dityrosine. In addition, we identified a tyrosine-containing spore wall precursor as N-formyl tyrosine. The elucidation of the chemical structure of soluble spore wall precursors is crucial for the characterization of the function of the enzymes involved in maturation of the spore surface, e.g. by in vitro systems. A dityrosine-containing fragment, which was solubilized from mature spore walls by partial hydrolysis, was identified as N-formyl dityrosine. Mature spore walls contain significant amounts of N-formyl dityrosine and N,N'-bisformyl dityrosine. This supports the assumption that the dityrosine-containing macromolecular network on the spore surface has an unusual, non-peptidic structure.

Cell Wall↗

PubMed1996

Primary structure of cyanelle peptidoglycan of Cyanophora paradoxa: a prokaryotic cell wall as part of an organelle envelope.

The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E. Pittenauer, E. R. Schmid, G. Allmaier, B. Pfanzagl, W. Löffelhardt, C. Quintela, M. A. de Pedro, and W. Stanek, Biol. Mass Spectrom. 22:524-536, 1993). In addition to the 4 monomers already known, 8 dimers, 11 trimers, and 6 tetramers were characterized. An average glycan chain length of 51 disaccharide units was determined by the transfer of [U-14C]galactose to the terminal N-acetylglucosamine residues of cyanelle peptidoglycan. The muropeptide pattern is discussed with respect to peptidoglycan biosynthesis and processing.

Amino Acid Sequence↗

PubMed1995

Isoforms of Bet v 1, the major birch pollen allergen, analyzed by liquid chromatography, mass spectrometry, and cDNA cloning.

Bet v 1, the major allergen of birch pollen, displays a considerable degree of heterogeneity. Several charge variants have been detected by two-dimensional IgE immunoblots and isoelectric focusing techniques. This heterogeneity has been attributed to glycosylation (or other post-translational modifications) or to isogenes coding for Bet v 1 isoforms and/or allelic variants. However, until now, only limited structural data for Bet v 1 have been published. Recently, we described the expression, purification, and immunological properties of recombinant Bet v 1 (rBet v 1) produced in Escherichia coli as a non-fusion protein (Ferreira, F. D., Hoffmann-Sommergruber, K., Breiteneder, H., Pettenburger, K., Ebner, C., Sommergruber, W., Steiner, R., Bohle, B., Sperr, W. R., Valent, P., Kungl, A. J., Breitenbach, M., Kraft, D., and Scheiner, O. (1993) J. Biol. Chem. 268, 19574-19580). Here, we present a more detailed structural characterization of Bet v 1 by both cDNA cloning and mass spectrometry. Thirteen different cDNA clones coding for Bet v 1 isoforms were obtained by polymerase chain reaction amplification of birch pollen cDNA with a sequence-specific 5'-terminal primer and a nonspecific 3'-terminal primer or by immunological screening of a birch pollen cDNA library. These isoforms are referred to as Bet v 1b to Bet v 1n, whereas the previously isolated Bet v 1 cDNA (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935-1938) is now referred to as Bet v 1a. High performance liquid chromatography and plasma desorption mass spectrometry of proteolytic fragments of purified natural Bet v 1 (nBet v 1) and rBet v 1a were used to (i) confirm the primary structure of all Bet v 1 isoforms and (ii) to investigate any possible postsynthetic modifications on rBet v 1a or on the natural mixture of isoallergens obtained from birch pollen. Except for the cleavage of initiating methionine, no postsynthetic modifications were found in either nBet v 1 or rBet v 1a.

Allergens↗

PubMed1995

Structure of peptidoglycan from Thermus thermophilus HB8.

The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented. The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported. The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide. In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid. This is the first time phenylacetic acid is described as a component of bacterial murein. Possible implications for murein physiology and biosynthesis are discussed. Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges. Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues. Neither reducing sugars nor murein-bound macromolecules were detected. Murein from T. thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms. The murein composition and peptide cross-bridges of T. thermophilus are typical for a gram-positive bacterium. However, the murein content, degree of cross-linkage, and glycan chain length for T. thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp.

Acetylglucosamine↗

PubMed1994

252Cf-plasma desorption mass spectrometry. II--A perspective of new directions.

The 252Cf-PDMS method is a 'mature' technique, having survived 20 years of research, development and application. Recent advances in the application of the method have led to the use of the technique in clinical quantitation studies, the development of a microscopic method for measuring chemical homogeneity, in situ applications where serial modifications can be made and studied in a sequential fashion and the utilization of the primary fragmentation patterns to correlate with primary structure. Future directions in research, development and application will focus more attention on the chemistry of the 252Cf-PDMS process and the understanding and control of gas-phase reactions that occur in the ejection plume. Predictions are that the application of the in situ modification technique could lead to a new approach to pharmacokinetic studies as well as cell surface interactions where 252Cf-PDMS is effectively used to expand the capabilities of in vitro studies of biological processes.

Californium↗

PubMed1993

Molecular weight-determination of biosynthetically modified monomeric and oligomeric muropeptides from Escherichia coli by plasma desorption-mass spectrometry.

The presence of certain D-amino acids in the growth media of Escherichia coli results in the accumulation of 2 major and 3-5 minor new muropeptides in the murein sacculus. Preliminary data suggested that the major muropeptides correspond to a monomer and a cross-linked dimer with one residue of D-amino acid per molecule. We have analyzed several D-amino acid-modified muropeptides by plasma desorption-mass spectrometry. Our results confirmed that the general structures of the major modified muropeptides are: GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-X, and GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala; GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-X, being X a residue of the D-amino acid. These results corroborate the usefulness of this technique for the structural analysis of muropeptides.

Amino Acids↗

PubMed1993

Structural characterization of the cyanelle peptidoglycan of Cyanophora paradoxa by 252Cf plasma desorption mass spectrometry and fast atom bombardment/tandem mass spectrometry.

A strategy for the structural characterization of the four major NaBH4-reduced peptidoglycan monomers derived from muramidase-digested peptidoglycan from the cyanelles of the flagellate Cyanophora paradoxa Korschikoff is described. Initial molecular weight determination of these glycopeptides was performed by positive and negative ion plasma desorption mass spectrometry. Due to the presence of two pairs of disaccharide tripeptide and disaccharide tetrapeptide monomers differing in mass by 112 units, respectively, an as yet unknown peptidoglycan modification either at the carbohydrate or at the peptide moiety was assumed. beta-Elimination of the disaccharide unit from the unreduced peptidoglycan monomers yielded the corresponding (modified) N1-lactyltripeptides and -tetrapeptides, respectively. These peptides, N-terminally blocked with lactic acid, unambiguously showed the modification to be located on the peptide moiety. By positive ion fast atom bombardment/hybrid tandem mass spectrometry of the reduced peptidoglycan monomers as well as of the corresponding deglycosylated monomers (= N1-lactylpeptides) the modification was determined to be linked to the glutamic acid moiety. Based on combined data from plasma desorption mass spectrometry, tandem mass spectrometry, accurate mass measurement and amino acid analysis of the acid hydrolysate after derivatization with o-phthaldialdehyde by high-performance liquid chromatography we could establish the structure of the modification as N-acetylputrescine. Finally, the confirmation of the linkage of the glutamic acid to diaminopimelic acid via the gamma-COOH was based on the presence of a-type peptide backbone fragment ions in the positive ion plasma desorption mass spectra of the modified N1-lactylpeptides.

Amino Acid Sequence↗