Biomedical subjects

G Winkelmann

Publications and source records attributed to G Winkelmann.

At least 19 recordsLinked to original sources

PubMed2006

Basidiochrome -- a novel siderophore of the Orchidaceous Mycorrhizal Fungi Ceratobasidium and Rhizoctonia spp.

A novel trishydroxamate siderophore, named basidiochrome, was isolated as the principal siderophore from low-iron culture filtrates of Ceratobasidium and Rhizoctonia species which are known as mycorrhizal fungi associated with orchid roots. Ion-exchange chromatography and preparative HPLC yielded a pure compound which contained two components according to GC-MS analysis: L: -N(5)-hydroxy-ornithine and 3-methyl-2-cis-pentenedioic acid (3-methyl-cis-glutaconic acid). FTICR-ESI-MS of both the iron-free and ferric form indicated an elemental composition of C(33)H(47)N(6)O(16)Fe (MW = 839) for the ferric form of basidiochrome. The connectivity was further elucidated by 2D-NMR techniques (HSQC, HMBC, COSY, NOESY) indicating that basidiochrome is a novel linear tripeptide consisting of three L: -N(5)-hydroxy-ornithines each linked to 3-methyl-2-cis-pentenedioic acid residues.

Basidiomycota↗

PubMed2004

Tyramine functions as a toxin in honey bee larvae during Varroa-transmitted infection by Melissococcus pluton.

From wounds of honey bee pupae, caused by the mite Varroa destructor, coccoid bacteria were isolated and identified as Melissococcus pluton. The bacterial isolate was grown anaerobically in sorbitol medium to produce a toxic compound that was purified on XAD columns, gelfiltration and preparative HPLC. The toxic agent was identified by GC-MS and FTICR-MS as tyramine. The toxicity of the isolated tyramine was tested by a novel mobility test using the protozoon Stylonychia lemnae. A concentration of 0.2 mg/ml led to immediate inhibition of mobility. In addition the toxicity was studied on honey bee larvae by feeding tyramine/water mixtures added to the larval jelly. The lethal dosis of tyramine on 4-5 days old bee larvae was determined as 0.3 mg/larvae when added as a volume of 20 microl to the larval food in brood cells. Several other biogenic amines, such as phenylethylamine, histamine, spermine, cadaverine, putrescine and trimethylamine, were tested as their hydrochloric salts for comparison and were found to be inhibitory in the Stylonychia mobility test at similar concentrations. A quantitative hemolysis test with human red blood cells revealed that tyramine and histamine showed the highest membranolytic activity, followed by the phenylethylamine, trimethylamine and spermine, while the linear diamines, cadaverine and putrescine, showed a significantly lower hemolysis when calculated on a molar amine basis. The results indicate that tyramine which is a characteristic amine produced by M. pluton in culture, is the causative agent of the observed toxic symptoms in bee larvae. Thus this disease, known as European foulbrood, is possibly an infection transmitted by the Varroa destructor mite.

Animals↗

PubMed2004

Liver transplantation in pigs with small-for-size grafts: effect of portocaval shunt.

Small-for-size livers are associated with graft dysfunction, probably due to portal hyperperfusion. Modulation of the recipient portal inflow is a new option in these cases. This article sought to analyze the effect of portocaval shunt in small-for-size liver grafts in pigs. Twelve liver transplants were performed in white pigs. The donors' mean weight was 10 kg and the recipient's mean weight was 34 kg. In all cases a standard technique was utilized. A portocaval shunt was added on the back-table in six cases. One hour after the procedure, the livers were sent for histologic examination. In all six cases without a portocaval shunt, the livers showed hemorrhagic necrosis, which was not observed in any of the six livers with a portocaval shunt. In small-for-size liver grafts in pigs, a portocaval shunt prevents hemorrhagic necrosis.

Animals↗

PubMed2003

Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN.

Members of a family of catecholate siderophores, called salmochelins, were isolated by reversed-phase HPLC from Salmonella enterica serotype Typhimurium and structurally characterized by Fourier transform ion cyclotron resonance-MSMS and GC-MS. The tentative structure of salmochelin 1 contained two 2,3- dihydroxybenzoylserine moieties bridged by a glucose residue, bound to the serine hydroxyl group of one moiety and the carboxylate of the second moiety. Salmochelin 2 contained in addition a second glucose residue linked to a third 2,3-dihydroxybenzoylserine moiety. Salmochelins were not produced by an iroBC mutant, which indicated that the IroB protein might be responsible for the glucosyl transfer predicted by sequence similarities to known glycosyltransferases. Uptake experiments with radiolabeled (55)Fe-salmochelin and growth promotion tests with salmochelins showed that the IroN outer membrane receptor, encoded in the iroA locus of S. enterica and uropathogenic Escherichia coli strains, was the main receptor for ferric salmochelin transport.

Cell Membrane↗

PubMed2002

Ferricrocin--an ectomycorrhizal siderophore of Cenococcum geophilum.

The ectomycorrhizal fungus Cenococcum geophilum was grown in low-iron medium and the excreted siderophores were extracted, purified and analyzed by HPLC. The principal hydroxamate siderophore produced, was identified as ferricrocin as confirmed by analytical HPLC, FAB-mass spectrometry and 1H- and 13C-NMR spectra. Although the occurrence of ferricrocin has been shown earlier to occur in the ericoid mycorrhizal fungi, this is the first report of ferricrocin in a true ectomycorrhizal fungus which is taxonomically related to the ascomycetes.

Ascomycota↗

PubMed2002

Microbial siderophore-mediated transport.

Microbial iron chelates, called siderophores, are synthesized by bacteria and fungi in response to low iron availability in the environment. The present review summarizes structural details of siderophore ligands with respect to their transport properties. This presentation is largely centred on the occurrence and function of siderophores in the various bacterial and fungal genera.

Bacteria↗

PubMed2001

Penicillin V production by Penicillium chrysogenum in the presence of Fe3+ and in low-iron culture medium.

Late-exponential-phase Penicillium chrysogenum mycelia grown in a complex medium possessed an intracellular iron concentration of 650 mumol/L (2.2 +/- 0.6 mumol per g mycelial dry mass). This iron reserve was sufficient to ensure growth and antibiotic production after transferring mycelia into a defined low-iron minimal medium. Although the addition of Fe3+ to the Fe-limited cultures increased significantly the intracellular iron levels the surplus iron did not influence the production of penicillin V. Supplements of purified major P. chrysogenum siderophores (coprogen and ferrichrome) into the fermentation media did not affect the beta-lactam production and intracellular iron level. Neither 150 nor 300 mumol/L extracellular Fe3+ concentrations disturbed the glutathione metabolism of the fungus, and increased the oxidative stress caused by 700 mmol/L H2O2. Nevertheless, when iron was applied in the FeII oxidation state the oxidative cell injuries caused by the peroxide were significantly enhanced.

Culture Media↗

PubMed2001

Heterobactins: A new class of siderophores from Rhodococcus erythropolis IGTS8 containing both hydroxamate and catecholate donor groups.

We report here on a new class of siderophores isolated from Rhodococcus erythropolis IGTS8, the first structurally characterized from any species of Rhodococcus and for which we suggest the name heterobactins. These siderophores consist of a tripeptide of sequence (N-OH)-L-Orn-Gly-D-Orn-(delta-N-dihydroyxbenzoate). The alpha amino group of the D-Orn is derivatized either as a 2-hydroxybenzoxazolate in heterobactin A or remains free in heterobactin B. The structures were determined by a combination of amino acid analysis, mass spectrometry and NMR methods. The two new compounds are true siderophores in that they relieve iron limited growth in the producing strain. The heterobactins are also transported by other non-producing bacteria. Growth promotion tests using various transport mutants revealed that in E. coli heterobactin A is only recognized by the catecholate receptor Cir while heterobactin B is taken up in both E.coli and A. flavescens JG9 via a hydroxamate transport system.

Bacterial Proteins↗

PubMed2001

In vitro activities and postantifungal effects of the potent dolastatin 10 derivative auristatin PHE.

The pentapeptide dolavaline-valine-dolaisoleuine-dolaproine-phenylalanine-methyl ester (auristatin PHE) is a derivative of the anticancer drug dolastatin 10 (dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine). Broth microdilution assays with a wide variety of yeast and filamentous fungal species demonstrated the specificity of auristatin PHE for Cryptococcus neoformans and several species of Trichosporon. The duration of the postantifungal effect (PAFE) for C. neoformans was determined for exposure times ranging from 30 min to 2 h. For the derivative, a PAFE was detectable after 45 min of exposure. The effect plateaued after 1 h of exposure, with a PAFE of approximately 6.5 h at four or eight times the auristatin PHE MIC. In contrast, there was no measurable PAFE after 1 h of exposure to dolastatin 10. Human serum greatly prolonged the PAFE of auristatin PHE at eight times the MIC. Auristatin PHE arrested C. neoformans in the budding stage, possibly due to a tubulin-inhibitory action. Auristatin PHE has potential as a narrow-spectrum fungicidal agent and as a probe that can be used to study cryptococcal cell division.

Antifungal Agents↗

PubMed2000

Identification and substrate specificity of a ferrichrome-type siderophore transporter (Arn1p) in Saccharomyces cerevisiae.

Genes encoding transporters for heterologous siderophores have been identified in Saccharomyces cerevisiae, of which SIT1, TAF1, and ENB1 encode the transporters for ferrioxamines, ferric triacetylfusarinine C and ferric enterobactin, respectively. In the present communication we have shown that a further gene encoding a member of the major facilitator superfamily, ARN1 (YHL040c), is involved in the transport of a specific class of ferrichromes, possessing anhydromevalonyl residues linked to N(delta)-ornithine (ARN). Ferrirubin and ferrirhodin, which both are produced by filamentous fungi, are the most common representatives of this class of ferrichromes. A strain possessing a disruption in the ARN1 gene was unable to transport ferrirubin, ferrirhodin and also ferrichrome A, indicating that the encoded transporter recognizes anhydromevalonyl and the structurally-related methylglutaconyl side-chains surrounding the iron center. Ferrichromes possessing short-chain ornithine-N(delta)-acetyl residues such as ferrichrome, ferricrocin and ferrichrysin, were excluded by the Arn1 transporter. Substitution of the iron-surrounding N-acyl chains of ferrichromes by propionyl residues had no effect, whereas substitution by butyryl residues led to recognition by the Arn1 transporter. This would indicate that a chain length of four C-atoms is sufficient to allow binding. Using different asperchromes (B1, D1) we also found that a minimal number of two anhydromevalonyl residues is sufficient for recognition by Arn1p. Contrary to the iron-surrounding N-acyl residues, the peptide backbone of ferrichromes was not an important determinant for the Arn1 transporter.

Carrier Proteins↗

PubMed2000

Immunostimulation by bacterial components: II. Efficacy studies and meta-analysis of the bacterial extract OM-89.

The bacterial extract OM-89 (Uro-Vaxom) consisting of immunostimulating components derived from 18 Escherichia coli strains is used for the treatment of recurrent urinary tract infections. We investigated in the mouse the immunogenicity of the bacterial extract after oral administration. After repeated administration of OM-89, a specific serum IgG and IgA response against a number of bacterial strains was obtained. Supernatants of cell cultures prepared from the urogenital tract of immunized mice also contained increased levels of strain specific IgG and IgA. We could show a bias towards a Th1 type immune response as indicated by increased IgG2a levels in sera, and increased IFNgamma levels in supernatants of spleen cells. These findings may contribute to an understanding of the therapeutic effect of Uro-Vaxom: the metaanalysis of several clinical studies confirmed that Uro-Vaxom constitutes an effective prophylaxis for urinary tract infections.

Adjuvants, Immunologic↗

PubMed2000

Fusarinines and dimerum acid, mono- and dihydroxamate siderophores from Penicillium chrysogenum, improve iron utilization by strategy I and strategy II plants.

Cucumber, as a strategy I plant, and Maize as a strategy II plant, were cultivated in hydroponic culture in the presence of a ferrated siderophore mixture (1 microM) from a culture of Penicillium chrysogenum isolated from soil. The siderophore mixture significantly improved the iron status of these plants as measured by chlorophyll concentration to the same degree as a 100-fold higher FeEDTA supply. Analysis of the siderophore mixture from P. chrysogenum by HPLC and electrospray mass spectrometry revealed that besides the trihydroxamates, coprogen and ferricrocin, large amounts of dimerum acid and fusarinines were present which represent precursor siderophores or breakdown products of coprogen. In order to prove the iron donor properties of dimerum acid and fusarinines for plants, purified coprogen was hydrolyzed with ammonia and the hydrolysis products consisting of dimerum acid and fusarinine were used for iron uptake by cucumber and maize. In short term experiments radioactive iron uptake and translocation rates were determined using ferrioxamine B, coprogen and hydrolysis products of coprogen. While the trihydroxamates revealed negligible or intermediate iron uptake rates by both plant species, the fungal siderophore mixture and the ammoniacal hydrolysis products of coprogen showed high iron uptake, suggesting that dimerum acid and fusarinines are very efficient iron sources for plants. Iron reduction assays using cucumber roots or ascorbic acid also showed that iron bound to hydrolysis products of coprogen was more easily reduced compared to iron bound to trihydroxamates. Ligand exchange studies with epi-hydroxymugineic acid and EDTA showed that iron was easily exchanged between coprogen hydrolysis products and phytosiderophores or EDTA. The results indicate that coprogen hydrolysis products are an excellent source for Fe nutrition of plants.

Biological Transport, Active↗

PubMed2000

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae.

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3delta background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using 55Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.

Bacterial Outer Membrane Proteins↗

PubMed1999

Molecular recognition of synthetic siderophore analogues: a study with receptor-deficient and fhu(A-B) deletion mutants of Escherichia coli.

The biological activity of six synthetic siderophore analogues (two dihydroxamates, two trihydroxamates, one tetrahydroxamate and one 3-hydroxy-4(1H)pyridinone) has been studied in Escherichia coli, Morganella morganii 13 and Proteus mirabilis 8993 strains by using growth promotion tests. Various transport-deficient mutants of E. coli were used to study the route of entry into gram-negative bacteria. The results indicated that the synthetic hydroxamate compounds are transported via Fhu-mediated transport systems, although receptor specificity was low. This could be proven by using a delta (fhuA-B) E. coli mutant as a control in which growth promotion by natural hydroxamates was completely abolished, suggesting that a periplasmic binding-protein-dependent transport system (FhuB, C, D) is required for the transport of all synthetic ferric hydroxamate complexes. Although utilization of the synthetic hydroxamates was generally lower than that of the natural siderophores, differences in growth promotion could be detected. Highest activity was observed with the dihydroxamate DOCYDHAMA ligand which supported growth at concentrations < 1 mM. In comparison with other polyamino-polyhydroxamate ligands studied, this dihydroxamate ligand has an extra diamide backbone that could be important for the interaction with the receptors or with FhuD. The synthetic trihydroxamate and tetrahydroxamate ligands showed a relatively low siderophore activity. Studies with Proteus and Morganella in the presence of increasing bipyridyl concentrations showed a decreased growth promotion with the synthetic ferric hydroxamates, suggesting the involvement of a reduction step during iron mobilization or an increased toxicity of bipyridyl. This was not observed in the case of the 3-hydroxy-4(1H)pyridinone where bipyridyl had no effect.

Bacterial Outer Membrane Proteins↗

PubMed1999

Degradation of desferrioxamines by Azospirillum irakense: assignment of metabolites by HPLC/electrospray mass spectrometry.

Based on a recent finding that an Azospirillum isolate ASP-1 possessing high 16S rDNA similarity to Azospirillum irakense was able to degrade desferrioxamine type siderophores (Winkelmann et al. BioMetals 9, 78-83, 1996), various members of the genus Azospirillum were analyzed for their ability to degrade desferrioxamines. While the desferrioxamine-degrading activity was absent or scarcely detectable in strains of A. lipoferum, A. brasilense, A. amazonense, degradation activity seemed to be confined to the species A. irakense (KBC-1, KA3). Also the identity of strain ASP-1 as A. irakense could be confirmed by species-specific oligonucleotide hybridization, Inter-LINE PCR fingerprinting and carbon source utilization pattern (BIOLOG) analysis. Products of desferrioxamine B degradation were analyzed by analytical HPLC and HPLC/electrospray mass spectrometry. Using whole cells and purified enzyme it was shown that the trihydroxamate desferrioxamine B (561 amu) is split at the N-terminal amide bond yielding a monohydroxamate (MH1, 219 amu) and a dihydroxamate (DH1, 361 amu) metabolite. A second monohydroxamate (MH2, 319 amu) resulted from DH1 after splitting the acetylhydroxamate bond. Minor amounts of a further dihydroxamate (DH2, 419 amu) originated from splitting the second amide bond in desferrioxamine B. In addition to desferrioxamine B, several other linear and cyclic desferrioxamines and derivatives were degraded, whereas desferricoprogen and desferri-ferrichrome were not degraded, indicating high substrate specificity of the desferrioxamine hydrolase in A. irakense species. A simple microtiter plate assay was developed which can be used to phenotypically discriminate and identify species of A. irakense from other Azospirillum species by their characteristic feature of desferrioxamine degradation.

Azospirillum↗

PubMed1999

Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in Saccharomyces cerevisiae as a member of the major facilitator superfamily.

Transport proteins of microorganisms may either belong to the ATP-binding cassette (ABC) superfamily or to the major facilitator (MFS)-superfamily. MFS transporters are single-polypeptide membrane transporters that transport small molecules via uniport, symport or antiport mechanisms in response to a chemiosmotic gradient. Although Saccharomyces cerevisiae is a non-siderophore producer, various bacterial and fungal siderophores can be utilized as an iron source. From yeast genome sequencing data six genes of the unknown major facilitator (UMF) family were known of which YEL065w Sce was recently identified as a transporter for the bacterial siderophore ferrioxamine B (Sit1p). The present investigation shows that another UMF gene, YHL047c Sce, encodes a transporter for the fungal siderophore triacetylfusarinine C. The gene YHL047c Sce (designated TAF1) was disrupted using the kanMX disruption module in a fet3 background (strain DEY 1394 delta fet3), possessing a defect in the high affinity ferrous iron transport. Growth promotion assays and transport experiments with 55Fe-labelled triacetylfusarinine C showed a complete loss of iron utilization and uptake in the disrupted strain, indicating that TAF1 is the gene for the fungal triacetylfusarinine transport in Saccharomyces cerevisiae and possibly in other siderophore producing fungi.

ATP-Binding Cassette Transporters↗

PubMed1999

Transport and utilization of rhizoferrin bound iron in Mycobacterium smegmatis.

Transport and metabolization of iron bound to the fungal siderophore rhizoferrin was analyzed by transport kinetics, Mössbauer and EPR spectroscopy. Saturation kinetics (vmax = 24.4 pmol/(mg min), K(m) = 64.4 microM) and energy dependence excluded diffusion and provided evidence for a rhizoferrin transport system in M. smegmatis. Based on the spectroscopic techniques indications for intracellular presence of the ferric rhizoferrin complex were found. This feature could be of practical importance in the search of novel drugs for the treatment of mycobacterial infections. EPR and Mössbauer spectroscopy revealed different ferritin mineral cores depending on the siderophore iron source. This finding was interpreted in terms of different protein shells, i.e. two types of ferritins.

Electron Spin Resonance Spectroscopy↗

PubMed1998

Cognitive frontal lobe dysfunction in obsessive-compulsive disorder.

BACKGROUND: There is evidence that dysfunction within associative frontostriatal circuits represents a feature of obsessive-compulsive disorder (OCD). Previous neuropsychologic studies have yielded diverging results, which may in part be explained by differences in the selection of subjects and methods. The present study focused on the question of cognitive frontal lobe performance in OCD. METHODS: Twenty-nine unmedicated OCD patients were compared to a double-size control group of normal subjects matched individually for age, sex, and intelligence. A series of 12 neuropsychologic tests was applied, most of which are thought to be sensitive to different aspects of cognitive frontal lobe function. RESULTS: OCD patients were unimpaired at tests of abstraction, problem solving, set-shifting, response inhibition, active memory search, and choice reaction speed. Deficits of approximately one standard deviation were observed at timed tests of verbal and nonverbal fluency, attentional processing, and weight sorting. CONCLUSIONS: OCD patients exhibited selective deficits in tasks involving controlled attentional processing and self-guided, spontaneous behavior. We discuss a link between this neuropsychologic profile and dysfunctioning within the anterior cingulate, but not the dorsolateral prefrontal circuit.

Adolescent↗