Interaction of immunologically-active lipopeptides with membranes.

Synthetic tripalmitoyl-S-glycerylcysteinyl (Pam3Cys) peptides are derived from the N-terminal part of bacterial lipoprotein and constitute polyclonal B-lymphocyte and macrophage activators. In order to elucidate the primary events of leukocyte activation, we investigated the biophysical interaction of lipopeptides containing spin labels or fluorescent markers with phosphatidylcholine vesicles or immune cells. Utilizing fluorescence microscopy and FACS analysis we found, that the surface of cells, after incubation with a fluorescein-labelled lipopeptide, was highly fluorescent. In addition, capping and patching was observed. Furthermore, fluorescence quenching experiments and electron paramagnetic resonance studies using vesicles incubated with lipopeptides suggested, that the peptide moiety and other more polar molecules linked to the lipo-amino acid are exposed to the hydrophilic compartment. These results show that in lipopeptide conjugates the Pam3Cys moiety acts as an efficient membrane anchor for molecules covalently coupled to it. The sequestering of the fatty-acid chains of the lipopeptide within the membrane is an early step of interaction, which might induce the uptake of the lipopeptide into the cell and the stimulation of immunocompetent cells.

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.

Structure-function studies of amphiphilic antibacterial peptides.

The synthesis of 11 peptides, ranging in composition from 9 to 17 amino acid residues, by solid-phase methodology was accomplished with the purpose of studying how the amphiphilic and hydrophobic character, the size of the molecule, and the charge distribution modulate the antibacterial activity. It was found that peptides composed of 16 and 17 amino acid residues, with high hydrophobic (mainly due to Trp or Phe) and hydrophilic (due to Lys) character distributed along opposite amphiphilic faces, showed considerable antibacterial activity against clinically isolated bacteria together with Gram positive and Gram negative ATCC bacterial strains. However, the hemolytic capacity of the peptides was also significant. Decreasing the hydrophobic character of the molecule by replacing Trp or Phe with Leu residues while maintaining the basic contribution of Lys drastically reduced the hemolytic activity and only slightly decreased the bioactivity. Peptides composed of 9-10 amino acid residues with high hydrophobic and basic nature possess antibacterial activity but, in general, are less active than the larger counterpart peptides. By replacing all Trp residues of a short peptide by Leu residues, the activity was considerably reduced. Circular dichroism studies and antibacterial assays showed that shorter peptides with very low helical content, and thus deprived of amphiphilic character, still have appreciable bioactivity. This observation, coupled with the fact that due to their small size they cannot span the bacterial outer lipid bilayer, may suggest different mechanisms of action for long-chain vis-a-vis short-chain peptides.

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.

Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis.

For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R-1 and I+1. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A(-4)-E-P-R(-1)- to -A(-4)-E-P-Q(-1)- by site-directed mutagenesis.

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.

Betacyanins from bracts of Bougainvillea glabra.

Betacyanins from the bracts of Bougainvillea glabra were isolated and characterized by a combination of spectroscopic techniques (DAD-HPLC, NMR, LC-MS, GC-MS, electrospray MS, tandem MS) as gomphrenin I (betanidin 6-O-beta-glucoside) and various derivatives of bougainvillein-v (betanidin 6-O-beta-sophoroside), i.e. mono- and diglucosylsophorosides which are acylated with 4-coumaric and caffeic acid (mono- and diesters). Besides the betacyanins, B. glabra bracts accumulated large amounts of flavonols (kaempferol and quercetin conjugates) reaching ratios of flavonol to betacyanin of 1:1.

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.

Anthocyanins from cell suspension cultures of Daucus carota.

Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3-O-lathyroside, cyanidin 3-O-(2''-O-beta-D-xylopyranosyl-6''-O-beta-D-glucopyranosyl-beta-D- galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual 1H NMR chemical shifts and 1H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.

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.

Germicidin, an autoregulative germination inhibitor of Streptomyces viridochromogenes NRRL B-1551.

During germination spores of Streptomyces viridochromogenes NRRL B-1551 excrete a compound, germicidin, which has an inhibitory effect on the germination of its own arthrospores at a concentration as low as 200 pM (40 pg/ml). At higher concentrations germicidin inhibits porcine Na+/K(+)-activated ATPase and retards the germination of the cress Lepidium sativum. Germicidin is the first known autoregulative inhibitor of spore germination in the genus Streptomyces and was isolated from the supernatant of germinated spores, but also from the supernatant of the submerged culture. Spectroscopic analysis and derivatization reactions revealed germicidin to be 6-(2-butyl)-3-ethyl-4-hydroxy-2-pyrone (C11H16O3). Crude isolates of germicidin from the supernatant of submerged culture, but not from the spores, contained a second, structurally very similar compound (C10H14O3), in which in contrast to germicidin a 2-propyl instead of the 2-butyl chain was bound to C-6 and which did not show any activity in the germination and ATPase assay. The germination assay was evaluated as a new screening model for specifically active compounds.

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.

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.

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.

Chrysospermins, new peptaibol antibiotics from Apiocrea chrysosperma Ap101.

Four new members of peptaibol antibiotics, designated as chrysospermins A, B, C, and D, were isolated from the mycelium of Apiocrea chrysosperma Ap101 by solvent extraction, silica gel chromatography and preparative recycling HPLC. Their structures as new nonadecapeptides were settled by detailed spectroscopic analysis and chemical degradation experiments. The chrysospermins display antibacterial and antifungal activity, and induce pigment formation by the fungus Phoma destructiva.

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.

Determination of estrogenic activity by LYES-assay (yeast estrogen screen-assay assisted by enzymatic digestion with lyticase).

In order to enhance the sensitivity and the speed of the yeast estrogen screen (YES)-assay, which has been established in many laboratories for the determination of estrogenic activity of compounds and environmental samples, the LYES-assay, a modified version of the YES-assay including a digestion step with the enzyme lyticase, was developed. With the LYES-assay the estrogenic activities of natural (17beta-estradiol E2 and estrone), synthetic (17alpha-ethinylestradiol EE2) and pharmaceutical estrogens (diethylstilbestrol DES) as well as xenoestrogens (4-nonylphenol NP and five parabens) were determined and compared with the results obtained by other in vitro-assays namely the conventional YES-assay, the E-Screen-assay (MCF-7 breast tumor cell proliferation) and a receptor binding-assay (RB) with human estrogen receptors hER-alpha and hER-beta. In the case of E2 the LYES-assay had a significantly lower limit of quantification (LOQ) than the conventional YES-assay and even two orders of magnitude lower than the RB-assay. Compared to the E-Screen-assay the LOQ of the LYES-assay was almost one order of magnitude higher. The time required to perform the LYES-assay was as little as seven hours compared to three to five days for the conventional YES-assay. Thus, the LYES-assay is a very good alternative to existing estrogenic in vitro-assays, since it has a good sensitivity, is cheap and much faster than the other assays.

Substances with estrogenic activity in effluents of sewage treatment plants in southwestern Germany. 1. Chemical analysis.

A gas chromatography/mass spectrometry method for the simultaneous quantitative determination of natural and synthetic estrogens (17beta-estradiol, estrone, 17alpha-ethinylestradiol, and mestranol), phytoestrogens (genistein and beta-sitosterol), and xenoestrogens (benzyl butyl phthalate, dibutyl phthalate, bisphenol A, 4-nonylphenol [NP], 4-nonylphenoxyacetic acid [NP1EC], 4-nonylphenol diethoxylate [NP2EO], and alpha-endosulfan) in effluents of sewage treatment plants (STPs) was developed. Identification and quantification were carried out with the standard addition method using analyte-specific and, in some cases, deuterium-labeled internal standards. The effluents of 18 STPs were investigated. Apart from alpha-endosulfan and mestranol, all selected substances were detected in the majority of samples. The median concentrations of steroidal estrogens were between 0.4 ng/L (17alpha-ethinylestradiol) and 1.6 ng/L (17beta-estradiol). The metabolites of the nonylphenol polyethoxylates, NP, NPIEC, and NP2EO were found in concentrations ranging from the upper-ng/L-range (NP) to the lower-microg/L range (NP1EC). For all substances except mestranol and alpha-endosulfan, median values were calculated and compared to the results of other investigations in Europe and the United States. Possible dependencies of measured concentrations on the geographical location, the capacity, the influent composition, and the technical fitting of the STPs are discussed.

Substances with estrogenic activity in effluents of sewage treatment plants in southwestern Germany. 2. Biological analysis.

The proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-Screen assay) was applied for quantitative determination of total estrogenic activity in 24-h composite effluent samples from 16 municipal and two industrial sewage treatment plants (STPs) in the state of Baden-Württemberg, southwestern Germany. The estrogenic efficacy relative to the positive control, 17beta-estradiol, was between 26 and 74% (median, 48%) for the 16 municipal STPs. Estradiol equivalent concentrations (EEQs) were between 0.2 and 7.8 ng/L (median, 1.6 ng/L) and, thereby, were lower than those found in a pilot study, which revealed EEQs of greater than 10 ng/L in the effluents of two other STPs. The EEQs in 14 of the 16 effluent samples were very similar (0.9-3.3 ng/L), indicating a rather constant input of estrogenic substances via STPs into rivers. Additional activated charcoal filtration turned out to be very efficient in further eliminating estrogenic activity from effluents. The EEQs of the E-Screen assay and those calculated from the results of extensive chemical analysis using the estradiol equivalency factors determined for 13 natural and synthetic estrogenic substances were comparable for most of the effluent samples. 17beta-Estradiol, 17alpha-ethinylestradiol, and, to a lesser extent, estrone contributed to 90% or more of the EEQ value.

Isolation and analysis of moenomycin and its biosynthetic intermediates from Streptomyces ghanaensis (ATCC 14672) wildtype and selected mutants.

Streptomyces ghanaensis (ATCC 14672) produces the phosphoglycolipid antibiotic moenomycin consisting of several components. A solid phase extraction procedure was developed which allowed a rapid isolation of both moenomycin and its biosynthetic intermediates from culture filtrates. Semi-preparative high performance liquid chromatography followed by high performance liquid chromatography-mass spectrometry provided structural data on the different moenomycin components. In order to obtain initial information on the biosynthetic pathway, moenomycin non-producing mutants were isolated. They were shown to release intermediates with shorter lipid chains suggesting that the lipid chain synthesis probably takes place at a later stage of the moenomycin biosynthesis. Based on the biological activity and the analytical data, we assume that a modification and in particular a shorter lipid portion drastically influences the inhibitory activity of this antibiotic.