Chemical chain termination resolves the timing of ketoreduction in a partially reducing iterative type I polyketide synthase.

Synthetic chain terminators were used to capture the biosynthetic intermediates from a partially reducing iterative type I polyketide synthase, which is integrated into a multimodular biosynthesis enzyme. The off-loaded metabolites clarified the timing of ketoreduction and aromatization in the assembly of the antibiotic micacocidin.

Precursor-directed biosynthesis of micacocidin derivatives with activity against Mycoplasma pneumoniae.

Micacocidin is a promising natural product for the treatment of Mycoplasma pneumoniae infections. In the biosynthesis of this antibiotic, a fatty acid-AMP ligase (FAAL) activates the starter unit hexanoic acid as acyl-adenylate and forwards it to an iteratively acting polyketide synthase. Biochemical analysis of the FAAL revealed an extended substrate tolerance, thereby opening the door for the modification of a micacocidin residue that is barely accessible via semisynthesis. A total of six new analogues were generated by precursor-directed biosynthesis in this study and profiled against M. pneumoniae.

An iterative type I polyketide synthase initiates the biosynthesis of the antimycoplasma agent micacocidin.

Micacocidin is a thiazoline-containing natural product from the bacterium Ralstonia solanacearum that shows significant activity against Mycoplasma pneumoniae. The presence of a pentylphenol moiety distinguishes micacocidin from the structurally related siderophore yersiniabactin, and this residue also contributes to the potent antimycoplasma effects. The biosynthesis of the pentylphenol moiety, as deduced from bioinformatic analysis and stable isotope feeding experiments, involves an iterative type I polyketide synthase (iPKS), which generates a linear tetraketide intermediate from acyl carrier protein-tethered hexanoic acid by three consecutive, decarboxylative Claisen condensations with malonyl-coenzyme A. The final conversion into 6-pentylsalicylic acid depends on a ketoreductase domain within the iPKS, as demonstrated by heterologous expression in E. coli and subsequent site-directed mutagenesis experiments. Our results unveil the early steps in micacocidin biosynthesis and illuminate a bacterial enzyme that functionally resembles fungal polyketide synthases.

Characterization of a genotoxic impact compound in Alternaria alternata infested rice as Altertoxin II.

Toxicity-guided fractionation was used to identify DNA strand breaking impact compounds in extracts obtained from rice heavily infested with the Alternaria alternata strains DSM 62006 and DSM 62010. The major genotoxic potential measured in the comet assay using human colon carcinoma cells (HT29) could be attributed to three unknown peaks, whereas the fractions containing alternariol, its monomethylether or tenuazonic acid showed no significant DNA damaging effects. According to (1)H and (13)C-NMR spectroscopy, one genotoxic impact compound was identified as Altertoxin II (ATXII). ATXII showed potent DNA damaging properties in HT29 cells with substantial induction of formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. However, no effect was observed with respect to the cellular redox status, measured in the DCF assay and as total glutathione. The induction of apoptosis could be excluded as a potential reason for enhanced DNA damage. After 24 h of incubation with 1 µM ATX II, a significant increase of cells in the G(0)/G(1) phase was observed together with an inhibition of cell proliferation in the sulforhodamine B assay. Taken together, ATX II was found to contribute substantially to the genotoxic effects of complex extracts obtained from Alternaria alternata infested rice. The results demonstrate the high genotoxic potency of ATX II in human cells, underlining the necessity for further studies on the occurrence in food and its relevance for food safety.

Genomics-driven discovery of taiwachelin, a lipopeptide siderophore from Cupriavidus taiwanensis.

A genome mining study led to the identification of a previously unrecognised siderophore biosynthesis gene cluster in the nitrogen-fixing bacterium Cupriavidus taiwanensis LMG19424. Based upon predicted structural residues, a convenient strategy for an NMR-assisted isolation of the associated metabolite was designed. The structure of the purified siderophore, taiwachelin, was fully characterized by spectroscopic methods and chemical derivatisation.

Minor contribution of alternariol, alternariol monomethyl ether and tenuazonic acid to the genotoxic properties of extracts from Alternaria alternata infested rice.

Alternaria spp. are known to form a spectrum of secondary metabolites with alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tenuazonic acid (TA) as the major mycotoxins with respect to quantity. In the present study we investigated the contribution of these compounds for the DNA damaging properties of complex extracts of Alternaria spp. infested rice. Five different Alternaria strains were cultured on rice and analyzed for their production of AOH, AME, ALT and TA. The extracts of two strains with distinctly different toxin profiles were selected for further toxicological analysis. An extract from A. alternata DSM 1102 infested rice, found to contain predominantly TA, exhibited substantial DNA strand breaking properties in cultured human colon carcinoma cells in the comet assay, whereas TA as a single compound did not affect DNA integrity up to 200µM. An extract of A. alternata DSM 12633 infested rice, containing in comparable proportions AOH, AME and TA, exceeded by far the DNA damaging properties of the single compounds. In contrast to AOH, AME and TA, both selected extracts induced an increase of DNA modifications sensitive to the bacterial repair enzyme formamidopyrimidine DNA glycosylase (FPG) in the comet assay, indicative for oxidative DNA damage. Toxicity-guided fractionation of the DSM 12633 extract confirmed that these effects were not caused by AOH, AME or TA. Taken together, the mycotoxins AOH, AME and TA, representing the major mycotoxins with respect to quantity in A. alternata infested food, play only a subordinate role for the genotoxic properties of complex extracts and appear not to be involved in the induction of FPG sensitive sites.

Structure and biosynthetic assembly of cupriachelin, a photoreactive siderophore from the bioplastic producer Cupriavidus necator H16.

The bacterium Cupriavidus necator H16 produces a family of linear lipopeptides when grown under low iron conditions. The structural composition of these molecules, exemplified by the main metabolite cupriachelin, is reminiscent of siderophores that are excreted by marine bacteria. Comparable to marine siderophores, the ferric form of cupriachelin exhibits photoreactive properties. Exposure to UV light induces an oxidation of its peptidic backbone and a concomitant reduction of the coordinated Fe(III). Here, we report the genomics-inspired isolation and structural characterization of cupriachelin as well as its encoding gene cluster, which was identified by insertional mutagenesis. Based upon the functional characterization of adenylation domain specificity, a model for cupriachelin biosynthesis is proposed.

Weight maintenance through behaviour modification with a cooking course or neurolinguistic programming.

We compared the effect on weight regain of behaviour modification consisting of either a gourmet cooking course or neurolinguistic programming (NLP) therapy. Fifty-six overweight and obese subjects participated. The first step was a 12-week weight loss program. Participants achieving at least 8% weight loss were randomized to five months of either NLP therapy or a course in gourmet cooking. Follow-up occurred after two and three years. Forty-nine participants lost at least 8% of their initial body weight and were randomized to the next step. The NLP group lost an additional 1.8 kg and the cooking group lost 0.2 kg during the five months of weight maintenance (NS). The dropout rate in the cooking group was 4%, compared with 26% in the NLP group (p=0.04). There was no difference in weight maintenance after two and three years of follow-up. In conclusion, weight loss in overweight and obese participants was maintained equally efficiently with a healthy cooking course or NLP therapy, but the dropout rate was lower during the active cooking treatment.

Biosynthesis of a complex yersiniabactin-like natural product via the mic locus in phytopathogen Ralstonia solanacearum.

A genome mining study in the plant pathogenic bacterium Ralstonia solanacearum GMI1000 unveiled a polyketide synthase/nonribosomal peptide synthetase gene cluster putatively involved in siderophore biosynthesis. Insertional mutagenesis confirmed the respective locus to be operational under iron-deficient conditions and spurred the isolation of the associated natural product. Bioinformatic analyses of the gene cluster facilitated the structural characterization of this compound, which was subsequently identified as the antimycoplasma agent micacocidin. The metal-chelating properties of micacocidin were evaluated in competition experiments, and the cellular uptake of gallium-micacocidin complexes was demonstrated in R. solanacearum GMI1000, indicating a possible siderophore role. Comparative genomics revealed a conservation of the micacocidin gene cluster in defined, but globally dispersed phylotypes of R. solanacearum.

Patent coronary artery and myocardial infarction in the era of primary angioplasty: assessment of an old problem in a new setting with data from the Swedish Coronary Angiography and Angioplasty Registry (SCAAR).

AIMS: The purpose of this study was to evaluate factors that contribute to a patent IRA (infarct - related artery) and the prognostic impact of a patent IRA in patients with ST-elevation myocardial infarction. METHODS AND RESULTS: Using the Swedish angiography and angioplasty registry (SCAAR) we included all patients with STEMI and one-vessel coronary artery disease who underwent primary PCI of the culprit lesion only from May 2005 to December 2007. A patent IRA was found in 1,104 of 3,284 patients. Patients with an occluded IRA had significantly increased 7-day mortality (HR, 3.03, 95% CI 1.68-5.46, P<0.001). The incidence of an occluded IRA increased with higher age, in patients over 80 years of age (OR, 1.23, 95% CI; 0.92-1.64), lower in patients on lipid-lowering drugs (OR, 0.68, 95% CI; 0.54-0.86) and lower in patients pre-treated with heparin (OR 0.71, 95% CI; 0.60-0.83) or GPIIb/IIIa receptor blockade (OR 0.77, 95% CI; 0.61-0.97). Treatment with acetylsalicylic acid or clopidogrel had no effect on IRA patency. CONCLUSIONS: IRA patency was associated with a lower 7-day mortality. Older STEMI patients and patients not taking lipid-lowering drugs or pre-treated with heparin or GPIIb/IIIa receptor blockers seem to constitute risk groups for having an occluded IRA.

Fat mass measured by DXA varies with scan velocity.

OBJECTIVE: To study the influence of scan velocities of DXA on the measured size of fat mass, lean body mass, bone mineral content and density, and total body weight. RESEARCH METHODS AND PROCEDURES: The subjects were 71 healthy white adults, 38 women and 33 men. The mean age was 41.7 +/- 13.5 years and body mass index was 28.6 +/- 5.6 kg/m(2). The subjects were scanned consecutively in slow, medium, and fast scan mode by a Lunar DPX-IQ DXA scanner. RESULTS: Throughout the body mass index and sagittal height ranges, scanned lean body mass significantly decreased with higher scan velocity and lean body mass was 2.7% lower in fast than in medium mode (p < 0.0001). In contrast, fat mass, percentage of body fat, and bone mineral contents were higher with increasing scan velocity. Areas not analyzed by the scanner, so called blue spots, increased with scan velocity and sagittal height, and their presence significantly enhanced the error. Body weight estimated by DXA in slow mode was -0.8% lower than scale weight in the women (p < 0.001) and -0.2% in men (not significant), and the difference was greater with increasing scan velocity. DISCUSSION: Scan velocity significantly influences the measured fat mass size, lean body mass, bone mineral content, and body weight. To obtain the most accurate results, slow mode is preferable and fast scans should be avoided. Future studies should report and take scan velocity into consideration.