Amylosin from Bacillus amyloliquefaciens, a K+ and Na+ channel-forming toxic peptide containing a polyene structure.

Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings produce a 1197 Da toxin, named amylosin. Nuclear magnetic resonance (NMR) data showed that amylosin contains a chromophoric polyene structure and the amino acids leucine/isoleucine, proline, aspartic acid/asparagine, glutamic acid/glutamine and tyrosine. A quantitation method for amylosin was developed using commercially available amphotericin B as a reference compound and a known concentration of amylosin determined by NMR with the electronic reference to access in vivo concentration (ERETIC) method. Purified amylosin inhibited motility of boar sperm cells at an exposure concentration of 135 nM and hyperpolarized their cell membrane and depolarized their mitochondria at exposure to concentration of 33-67 nM for 10 min. In a 3-d exposure time only 27 nM of amylosin was needed to provoke the same toxicity functions. Amylosin was cytotoxic to feline lung cells at concentrations of <170 nM. Purified amylosin provoked adenosine 5'-triphosphate (ATP)-independent cation influx into isolated rat liver mitochondria (RLM), inducing swelling of the mitochondria at concentrations of 200 nM K(+) or >250 nM Na(+) medium. In the K(+)- or Na(+)-containing medium, amylosin uncoupled RLM, causing oxidation of pyridine nucleotides (PN), loss of the mitochondrial membrane potential, and suppressed ATP synthesis. Purified amylosin produced cation channels in black-lipid membranes (BLMs) with a selectivity K(+)>Na(+) at a concentration of 26 nM, i.e. the same concentration at which amylosin was toxic to boar sperm cells. The amylosin cation channels were cholesterol- and ATP-independent and more effective with K(+) than with Na(+). We propose that the toxicity of amylosin may be due its ionophoric properties, representing the first K(+)/Na(+) channel-forming substance reported from B. amyloliquefaciens.

Levels of mycotoxins and sample cytotoxicity of selected organic and conventional grain-based products purchased from Finnish and Italian markets.

The contamination levels of 16 different Fusarium- and Aspergillus-mycotoxins were chemically determined from randomly selected organic and conventional grain-based products purchased from Finnish and Italian markets. The cytotoxicity of the samples was analyzed with an in vitro test using feline fetal lung cells. Overall, the concentrations of the mycotoxins studied were low in all of the samples. Enniatins B and B1 as well as deoxynivalenol were the most predominant mycotoxins in the samples, being present in 97%, 97%, and 90% of the samples, respectively. The geographical origin or the agricultural practice had no influence on the mycotoxin concentrations of the samples. The babyfoods included in the samples had significantly lower concentrations of mycotoxins than the other products with a mean total mycotoxin content of 47 microg/kg compared with 99 microg/kg for the other kinds of food. All the samples evoked toxicity in the in vitro test, but no correlation between cytotoxicity and the mycotoxin concentrations was observed.

Cytolethal distending toxin B gene (cdtB) homologues in taxa 2, 3 and 8 and in six canine isolates of Helicobacter sp. flexispira.

The presence of the cytolethal distending toxin B gene (cdtB) was examined in eight Helicobacter sp. flexispira reference strains, Helicobacter trogontum ATCC 700114(T) and 12 Finnish porcine H. trogontum strains and canine flexispira isolates. Part of the cdtB gene was amplified by PCR with degenerate primers VAT2 and DHF1, cloned and sequenced. The presence/absence of the cdtB gene as determined by PCR was confirmed by Southern hybridization and toxin production by HeLa cell-line experiments. PCR amplification resulted in approximately 700 bp fragments from Helicobacter sp. flexispira taxa 2 (ATCC 49314), 3 (ATCC 49320) and 8 (ATCC 43880, ATCC 49308, ATCC 43879), from six canine isolates as well as from the control strains Helicobacter bilis and Helicobacter hepaticus. The hybridization patterns of HaeIII-, HindIII- and AseI-digested chromosomal DNA confirmed the results of the PCR experiments. The cdtB-positive strains had effects ranging from weak to strong on HeLa cell cultures. PCR amplification from the reference strains Helicobacter sp. flexispira taxa 1 (ATCC 43968), 4 (ATCC 49310) and 5 (ATCC 43966) and H. trogontum (ATCC 700114(T)), and also six of the Finnish strains, was unsuccessful. No toxic effect on HeLa cells was evident when bacterial suspensions of PCR-negative strains were used for toxicity assay. Our results are in accordance with previous observations that the cdtB gene is not present in all Helicobacter species. Further, the presence/absence of the cdtB gene in Helicobacter sp. flexispira strains was in accordance with recent taxonomic analysis of the same strains, which suggests that it could serve as a useful marker in Helicobacter taxonomy.