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E Bayer

Publications and source records attributed to E Bayer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Coupling of capillary electrochromatography to coordination ion spray mass spectrometry, a novel detection method.

Miniaturized separation techniques such as capillary electrochromatography (CEC), pressurized capillary electrochromatography (pCEC) and capillary high performance liquid chromatography (CHPLC) have been coupled to a new detection technique: coordination ion spray mass spectrometry (CIS-MS). Electrospray ionization (ESI) has found widespread applications in mass spectrometry (MS) for the analysis of polar compounds such as peptides or nucleotides. However, for weakly polar or nonpolar substances, ESI-MS yields poor sensitivity since, in the absence of basic or acidic groups, protonation or deprotonation is not possible. CIS is a universal ionization technique capable of detecting these compounds. Through the addition of a central complexing ion, charged coordination compounds are formed, enabling the detection with good sensitivity. Using the coaxial sheath flow interface commonly employed in CE-MS coupling, we were able to separate and detect various important natural compounds such as unsaturated fatty acid methyl esters, vitamins D2 and D3, and four different estrogens. A central ion solution of 100 microg/mL AgNO3 in water was used as sheath flow liquid, resulting in the formation of positively charged coordination compounds.

Cholecalciferol↗
PubMed1998

Silver-plated vitamins: a method of detecting tocopherols and carotenoids in LC/ESI-MS coupling.

In LC/MS, nonpolar substances in the majority of cases cannot be ionized by standard electrospray ionization (ESI) because they obviously lack a site for protonation or deprotonation. The ionization of carotenoids and tocopherols can be greatly enhanced by the addition of silver ions. The Ag(+)-carotenoid and Ag(+)-tocopherol adducts thus formed render these substances amenable to MS. alpha-, beta-, gamma-, and delta-tocopherol, alpha-tocopherol acetate, and the various isomers of lycopene and beta-carotene were separated by C30 RP-HPLC and could be identified by online ESI-MS. A mixture of six different carotenoids was analyzed by scanning the mass range from m/z 500 to 800. The mass spectra of the peaks revealed that all carotenoids and most tocopherols were partially oxidized to radical cations. The detection limit for canthaxanthin was approximately 500 fmol while that of beta-carotene was below 300 fmol. An increase in sensitivity in the MRM mode can be attained by monitoring ions formed by loss of elemental silver from the adducts in the CID cell. Dichloromethane extracts of tomato, carrot, and vegetable juices, a vitamin drink, and a commercial infant food product were analyzed by LC/MS. After postcolumn argentation, from the mass-selective extracts of the TIC, the carotenoids and tocopherols present could be identified by their masses and their retention times. For all studies, a silver perchlorate solution with an overall concentration of 50 micrograms/mL was used.

Antioxidants↗
PubMed1998

Quantitative analysis of modified antisense oligonucleotides in biological fluids using cationic nanoparticles for solid-phase extraction.

Based on a novel method for solid-phase extraction using cationic polystyrene nanoparticles, the suitability of the extraction procedure for quantitation of terminally and backbone-modified antisense oligonucleotides was investigated. Extractions were carried out from both human plasma and urine. Quantitative analysis of the extracted samples was performed with capillary gel electrophoresis. In accordance with previous results obtained with phosphorothioate oligonucleotides in human plasma, high linearity and accuracy of the assay was demonstrated for an oligodeoxyribonucleotide-palmityl conjugate as well as for a modified oligoribonucleotide. Optimized extraction conditions allow the isolation of oligonucleotides in high yields and purity even for concentrations in the low nanomolar range, down to 5 nM. Comparing the results obtained from human plasma and urine, no significant differences in the absolute recovery rates which reach values up to 95% were observed. However, when the loading capacity of the nanoparticles was exceeded, selective recovery was observed for the coisolation of phosphodiester and phosphorothioate oligonucleotides. This effect can be explained by differences in the attractive forces between PO- and PS-oligonucleotides and the particle surface and appears to be valuable for a modification-dependent enrichment of oligonucleotides out of complex mixtures.

Calibration↗
PubMed1998

Quantitation of phosphorothioate oligonucleotides in human blood plasma using a nanoparticle-based method for solid-phase extraction.

Based on the application of cationic polystyrene nanoparticles, a novel method for solid-phase extraction of phosphorothioate oligonucleotides from human plasma has been developed. A high binding affinity, which is required for an effective isolation out of complex mixtures, is mediated by hydrophobic and multiple electrostatic interactions between the oligonucleotides and the nanoparticles. The principle of the method is based on a pH-controlled adsorption/desorption mechanism. Analysis of the extracted samples was performed by capillary gel electrophoresis. Extraction conditions were optimized, providing the isolation of oligonucleotides (> or = 10 nucleotide units) in high yields and purity even at concentrations in the low-nanomolar range (down to 5 nM). The low salt contamination of the samples allows their direct analysis by electrospray mass spectrometry. The combined linearity and accuracy of the assay together with absolute recovery rates in the range of 60-90% indicate that the developed solid-phase extraction method is generally applicable to quantitation of oligonucleotides in human plasma. Further improvement was achieved with an optimized carrier system of 2-fold enlarged particles which reduces the time consumption of the extraction procedure to approximately 30 min.

Humans↗
PubMed1998

Monitoring of solid phase peptide synthesis by FT-IR spectroscopy.

Aggregation phenomena of growing peptides on the resin have seldom been investigated. We report here how conformations are determined by FT-IR spectroscopy. Therefore the sequence 80-99 of HIV 1-protease was synthesized. After every coupling a resin sample was taken out of the reaction column and a FT-IR spectrum recorded. The results were compared with the UV monitoring obtained from another synthesis of the same peptide.

Amino Acids↗
PubMed1998

Growth inhibition of pancreatic tumor cells by modified antisense oligodeoxynucleotides.

INTRODUCTION: Pancreatic adenocarcinomas are largely resistant to apoptosis. More than 50% of pancreatic tumors reveal mutations in the p53 tumor suppressor gene. METHODS: We investigated the growth of pancreatic tumor cells after downregulation of p53 protein expression by antisense oligodeoxynucleotides. RESULTS: Proliferation and p53 expression of PancTu-I cells overexpressing mutant p53 protein were inhibited by antisense oligodeoxynucleotide treatment. When analyzed, two of three other pancreatic tumor cell lines with mutated p53 were also inhibited in their growth. Two of two wild-type (wt) p53 pancreatic tumor cells were not significantly influenced by p53 expression and were, only to a lesser extent, affected in their proliferation. K562 cells (lacking p53 mRNA) and normal human skin fibroblasts used as a target mismatch control showed no changes in proliferation rates with treatment. The different biological effects in the various cells were not caused by differences in the uptake of the oligodeoxynucleotides as monitored by confocal laser-scanning microscopy. CONCLUSIONS: Truncation and 5'- and 3'-lipophilic modifications of the oligodeoxynucleotides drastically enhanced the growth inhibition of PancTu-I cells, which were resistant to apoptosis-inducing agents. Furthermore, a higher sequence-specificity of the observed effects was achieved with these compounds.

Adenocarcinoma↗
PubMed1998

Directly coupled CZE-NMR and CEC-NMR spectroscopy for metabolite analysis: paracetamol metabolites in human urine.

Direct coupling of NMR spectroscopic detection with both capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) was applied to the separation of metabolites of the drug paracetamol in an extract of human urine. Continuous-flow CZE-NMR and CEC-NMR allowed the detection of the major metabolites, the glucuronide and sulfate conjugates of the drug and the endogenous material hippurate. Identification of these substances was achieved by examination of individual rows of the NMR chromatogram and this also gave estimates of the detection limits. For CEC-NMR, spectra were also obtained in the stopped-flow mode including a two-dimensional TOCSY NMR experiment which afforded confirmatory evidence for paracetamol glucuronide. Characterisation of drug metabolites using NMR spectroscopy is therefore possible with nanolitre sample volumes.

Acetaminophen↗
PubMed1998

Hormonal factors from the mammalian pineal gland interfere with cell development in Hydra.

A partially purified, melatonin-free low-molecular-weight extract from the ovine pineal gland with antitumor activity (YC05R), interferes with terminal differentiation in the interstitial cell line of Hydra. Nematoblasts developed into defective nematocytes that were subject to cell death and the tentacles eventually became devoid of nematocytes. In an attempt to identify the causative components of the extract, several known potential constituents were assayed. Two factors were found to have similar effects, although only in rather high concentrations: 1alpha, 25 dihydroxyvitamin D3 (>150 nM) and pinoline (>5 microM), a natural tryptophan-derived beta-carboline. The proliferative activity in the interstitial cell line was only slightly reduced by these factors. Two other beta-carbolines that occur in the mammalian brain, harmine (10 microM) and n-butyl-beta-carboline-3-carboxylate (beta-CCB), caused the premature death of epithelial cells and thus the development of dwarfish animals which, however, continued to generate new animals by budding. The pineal extract probably contains some more, still unidentified components that interfere more potently with cell development, in Hydra as well as in mammals.

Animals↗
PubMed1998

Low-molecular-weight hormonal factors that affect head formation in Hydra.

Support or inhibition of DAG-induced head formation: Hydra magnipapillata can be caused to form ectopic head structures by periodictreatmentwith PKC activators such as diacylglycerol (DAG). This ectopic head formation is supported by an extract from the ovine pineal gland that contains low-molecular-weight compounds. The frequency of ectopic head formation is also increased when DAG is paired with one of several lipophilic hormonal factors: (1) pinoline, a putative pineal hormone derived from tryptophan, (2) 12-S-HETE, a paracrine derivative of arachidonic acid, or (3) 1alpha, 25 dihydroxyvitamine D3, a hormonal factor also known as calcitriol. Dose-response curves were non-monotonic passing a maximum at low dosages. By contrast, DAG lost its capacity to induce ectopic head structures when it was paired with the provitamin D3, cholecalciferol. Patterning the head: one eicosanoid was found which influences patterning without being combined with DAG: 12-R-HETE caused growth-based elongation of the tentacles and an increase in the number of tentacles without affecting the longitudinal body pattern. Similar effects are brought about by substances that interfere with tyrosine phosphorylation, most potently bythe phosphotyrosine phosphatase inhibitor bisperoxo-(1,10-phenanthroline)-oxovanadate (V). The results speak for the existence of head-inducing hormonal factors, underline the significance of content protein kinases to pattern formation and point to a negative influence of the vitamin D3 content of the food on the capacity of the animals to develop head structures.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1997

Morulin Pm: a modified polypeptide containing TOPA and 6-bromotryptophan from the morula cells of the ascidian, Phallusia mammillata.

A novel polypeptide containing the unusual posttranslationally modified amino acids L-3,4,5-trihydroxyphenylalanine (TOPA) and L-6-bromotryptophan (6-BrW) has been isolated from the morula cells of the vanadium-accumulating ascidian, Phallusia mammillata. The polypeptide, designated Morulin Pm, has a molecular weight of 3825 +/- 0.6 and has a simple amino acid composition consisting mainly of TOPA and 6-BrW as well as Ser, Leu, Phe, and Ala. To our knowledge, this is the first reported example of multiple sites of brominated tryptophan in a polypeptide of this size. Edman degradation revealed the N-terminal sequence to be BrW-Leu-Phe-BrW before sequencing was blocked. While the N-terminal tripeptide could be isolated from chymotrypsin digests of Morulin Pm, the rest of the polypeptide resisted further cleavage by the proteases, a feature common among this class of peptides. However, unlike other ascidian blood cell peptides examined to date, microheterogeneity was minimal. For the first time a detailed NMR investigation could be undertaken on a member of this class of polypeptides. In addition to signals assignable to the constituent amino acids by extensive 2D experiments, resonances were present both in the 13C and 1H spectra not typical of a simple linear peptide. Two proton resonances were identified with a cross peak in the correlation spectrum strongly indicative of a C-terminal decarboxy-delta 2,3-unsaturated TOPA residue as observed in certain tunichromes and clionamide. Chemical degradation experiments were undertaken in an effort to produce identifiable fragments to which these signals could be assigned, including full and partial acid hydrolysis and tryptophan-targeted BNPS-skatole treatment. However, the nature of the modification remains unknown. Possible structures for the modification, which may represent the source of the difficulties encountered in the structural elucidation of this and related peptides, are assessed. Conjecture is made as to the biological relevance of Morulin Pm, based on its localization and chemical characteristics.

Amino Acids↗
PubMed1997

Cationic Polystyrene Nanoparticles: Preparation and Characterization of a Model Drug Carrier System for Antisense Oligonucleotides

Polystyrene nanoparticles were prepared by surfactant-free emulsion polymerization using water-soluble cationic initiators to induce a positive surface charge. Reaction conditions were optimized to obtain monodisperse polymeric particles with mean diameters of 150-200 nm. The nanospheres were characterized by scanning electron microscopy, photocorrelation spectroscopy, conductometric titrations, zeta potential measurements, and investigations of their colloidal stability in different media. Sterically stabilized polymeric latexes, showing a low cytotoxicity in the concentrations required for antisense experiments, are capable of binding negatively charged oligonucleotides. The influence of pH, type of latex, and oligonucleotide modification on the adsorption characteristics was investigated. The results indicate that high attractive forces in terms of multiple electrostatic interactions are predominantly responsible for oligonucleotide binding. Comparing the experimental data with calculations based on the random sequential adsorption (RSA) model, it appears that the surface coverage at the saturation level can be considered as a monolayer of "side-on" adsorbed molecules. Oligonucleotide release can be induced by the addition of anionic surfactants or by increasing the pH of the dispersion medium. Investigations of the enzymatic degradation by micellar electrokinetic capillary chromatography (MECC) indicate a significantly enhanced stability of adsorbed oligonucleotides against nuclease attack. Copyright 1997 Academic Press. Copyright 1997Academic Press

Journal Article↗
PubMed1997

Conformational analysis of neuropeptide Y-[18-36] analogs in hydrophobic environments.

The interactive and conformational behavior of a series of neuropeptide Y-[18-36] (NPY-[18-36]) analogs in hydrophobic environments have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC) and circular dichroism (CD) spectroscopy. The peptides studied comprised a series of 16 analogs of NPY-[18-36], each containing a single D-amino acid substitution. The influence of these single L-->D substitutions on the alpha-helical conformation of the NPY-[18-36] analogs in different solvent environments was determined by CD spectroscopy. Retention parameters related to the hydrophobic contact area and the affinity of interaction were determined with an n-octadecyl (C18) adsorbent. Structural transitions for all peptides were manifested as significant changes in the hydrophobic binding domain and surface affinity between 4 degrees C and 37 degrees C. The results indicated that the central region of NPY-[18-36] (residues 23-33) is important for maintenance of the alpha-helical conformation. Moreover, L-->D amino acid residue substitutions within the N- and C-terminal regions, as well as Asn29 and Leu30, do not appear to affect the secondary structure of the peptide. These studies demonstrate that RP-HPLC provides a powerful adjunct for investigations into the induction of stabilized secondary structure in peptides upon their interaction with hydrophobic surfaces.

Amino Acid Sequence↗
PubMed1997

Use of Fmoc-N-(2-hydroxy-4-methoxybenzyl)amino acids in peptide synthesis.

The use of N,O-bisFmoc-N-(2-hydroxy-4-methoxybenzyl)amino acid derivatives in the synthesis of peptides with difficult sequences has already been described. With these amino acid derivatives the reversible protecting group 2-hydroxy-4-methoxybenzyl (Hmb) for the backbone amide bonds of peptide chains is introduced, and thus the aggregation due to hydrogen-bond interchain association is inhibited. This paper describes the synthesis and use of Fmoc-N-(2-hydroxy-4-methoxybenzyl)amino acid derivatives as an alternative means of introducing Hmb backbone protection. These new monoFmoc derivatives were obtained in higher yield than the bisFmoc derivatives. Coupling yields to the amino peptide resin were the same as those obtained with bisFmoc derivatives, under the TBTU HOBt/DIEA conditions. We also compared different syntheses of a difficult peptide with the Fmoc approach [triple coupling, capping, use of chaotropic agents, backbone protection using monoFmoc (Hmb)Ala] and with optimized Boc chemistry. Both the backbone protection and optimized Boc chemistry approaches gave the desired product in excellent yield and purity.

Amino Acid Sequence↗
PubMed1997

Elucidation of phase I and phase II metabolic pathways of rhein: species differences and their potential relevance.

Because of previously observed species differences in rhein tolerability, with rabbits being very susceptible to kidney disturbances, in vivo and in vitro biotransformation studies were performed to find out whether the differences in the undesired effects of rhein are associated with qualitative, species-dependent differences in its metabolism. First hints on species-dependent biotransformation profiles were obtained from in vivo experiments with 14C-labeled rhein in rat, rabbit, dog, and man. TLC-analysis of urine samples obtained after oral administration of 14C-rhein to rabbits revealed an additional, hydrophilic metabolite fraction in rabbit urine as compared with dog and human urine, all of which contain phenolic monoglucuronide and monosulfate as major metabolites. An investigation of urine samples (obtained from dogs, rabbits, rats, and human volunteers after oral application of unlabeled rhein) was conducted by means of mass spectrometric tandem techniques including on-line HPLC-MS/MS. In vitro experiments with subcellular liver fractions of rats and rabbits revealed the presence of three monohydroxylated metabolites of rhein, their quinoid oxidation products, and a bishydroxylated derivative of rhein. The hydroxylated phase I metabolites were detected as glucuronides in urine samples of all investigated species, whereas the quinoid product was present only in rabbit urine. Moreover, two regioisomeric phenolic glucuronides and sulfates or glucosides of rhein were found as major phase II metabolites in urine of all species. Furthermore, acyl glucuronides of rhein and monohydroxylated rhein and their respective isomeric acyl migration products were identified in human urine. In rabbit urine we discovered different bisglucuronides (bisphenolic glucuronide, mixed ether/ ester glucuronides), whereas in rats only the bisether/ether glucuronide was present. In addition, the investigations of dog and human urine showed the formation of two regioisomeric phenolic glucosides. With respect to a potential reactivity with endogenous macromolecules the quinoid metabolites as well as the bisester/ether glucuronides appear most relevant.

Animals↗
PubMed1995

A dodecapeptide comprising the extended chain-alpha 4 region of the restriction endonuclease EcoRI specifically binds to the EcoRI recognition site.

The restriction endonuclease EcoRI binds and cleaves DNA containing GAATTC sequences with high specificity. According to the crystal structure, most of the specific contacts of the enzyme to the DNA are formed by the extended chain region and the first turn of alpha-helix alpha 4 (amino acids 137-145). Here, we demonstrate that a dodecapeptide (WDGMAAGNAIER), which is identical in the underlined parts of its sequence to EcoRI amino acids 137-145, specifically binds to GAATTC sequences. The peptide inhibits DNA cleavage by EcoRI but not by BamHI, BclI, EcoRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at sites that partially overlap with EcoRI sites. The peptide inhibits cleavage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. It interferes with DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase. It competes with EcoRI for DNA binding. Based on these results, the DNA binding constant of the peptide to GAATTC sequences was calculated to be 3 x 10(4) M-1. DNA binding is not temperature-dependent, suggesting that binding of the peptide is entropy-driven. As the peptide does not show any nonspecific binding to DNA, its DNA binding specificity is similar to that of EcoRI, in spite of the fact that the affinity is much smaller. These results suggest that contacts to the phosphate groups in EcoRI mainly provide binding affinity, whereas the specificity of EcoRI is based to a large extent on sequence-specific base contacts.

Amino Acid Sequence↗
PubMed1995

Chemical phosphorylation of the peptides GGXA (X = S, T, Y): an evaluation of different chemical approaches.

An evaluation was made of the two methods most commonly used for phosphorylation of hydroxyamino acids in peptides, i.e. the tetrazole-catalysed phosphitylation by di-tert-butyl-N,N-diethylphosphoramidite followed by oxidation and the phosphorylation by dibenzylphosphochloridate. As model system the sequence GGXA (X = S, T, Y) was used which represents a random-coil sequence avoiding the influence on the reaction kinetics of secondary structure formation. In the case of serine- and threonine-containing peptides, both synthetic methods gave comparable yields of the desired phosphopeptides. The phosphorylation of tyrosine was achieved more favorably via the phosphoramidite method. However, phosphotyrosine peptides are most easily obtained by peptide synthesis using Fmoc-Tyr(PO3Me2)OH as building block. The dibenzylphosphochloridate method yields the expected phosphopeptides as the only peptide derivative and in addition, a great number of unidentified by-products which can be removed by ion-exchange chromatography. The phosphoramidite method consistently resulted in three peptide derivatives, i.e. the desired phosphopeptide, the phosphitylated peptide and a bridged derivative with two GGXA fragments linked through a phosphodiester bridge. The derivatives were characterised by RP and ion-exchange chromatography, 31P- and 1H-NMR spectroscopy, and ion-spray and electrospray mass spectrometry. Interestingly, even these mild ionisation techniques resulted in partial fragmentation. The observed fragmentation pathways seem to be a diagnostic tool for the identification of phosphorylation sites in peptides. Both the phosphorylated serine and threonine peptide lost phosphoric acid (98 mass units), the tyrosine peptide lost phenyl phosphate (174 mass units).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

N-terminal amino-acid sequence of beta-lactamase from Shigella flexneri UCSF-129.

A beta-lactamase (EC 3.5.2.6 penicillinase, penicillin amino beta-lactam-hydrolase) was purified from Shigella flexneri USCF-129 by an efficient two-stage procedure involving chromatography in Sephadex G-75 and HPLC on a C18-reverse phase column. The homogeneity of the purified enzyme was confirmed by capillary zone electrophoresis (CZE), HPLC electrospray mass spectrometry (LC-ESMS) and amino acid sequence analyses. The highly purified enzyme was a monomeric protein with a molecular mass of 28.903 +/- 2 Da, as determined by LC-ESMS. The amino acid sequence of the first 49 N-terminal residues of this beta-lactamase revealed 100% similarity with the mature forms of the plasmid coded Escherichia coli enzymes (plasmid pBR 322 and R6K) a TEM-type beta-lactamase.

Amino Acid Sequence↗