A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen⁻¹. This amount corresponds to 10⁴ to 10⁵ CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10⁸ CFU ml⁻¹. The detection limit for food was 3 g of rice containing 10⁶ to 10⁷ CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen⁻¹, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.     

Environment-dependent mechanism of dehalogenation by Rhodococcus chlorophenolicus PCP-1

Cells and cell-free preparations of a soil-bioremediating organism, Rhodococcus chlorophenolicus PCP-1, dehalogenated polychlorophenols both under aerobic and anaerobic conditions. Under aerobic conditions molecular O₂ served as the source of oxygen for the dechlorinating para-hydroxylation reaction. Chlorophenols were dehalogenated and para-hydroxylated also under anaerobic conditions by a cyt P-450 enzyme. Water was used anaerobically as an oxygen source but the reaction required the presence of sulphite ions or iodosobenzene. When the dehalogenating enzyme was given a choice between molecular O₂ and water in the presence of sulphite ions or iodosobenzene, the oxygen was preferably derived from water.Dedicated to the honour of Prof. Dr. Norberto Palleroni for the occasion of his 70th birthday Correspondence to: J. S. Uotila     

Characterization of aromatic dehalogenases of Mycobacterium fortuitum CG-2.

Two different dehalogenation enzymes were found in cell extracts of Mycobacterium fortuitum CG-2. The first enzyme was a halophenol para-hydroxylase, a membrane-associated monooxygenase that required molecular oxygen and catalyzed the para-hydroxylation and dehalogenation of chlorinated, fluorinated, and brominated phenols to the corresponding halogenated hydroquinones. The membrane preparation with this activity was inhibited by cytochrome P-450 inhibitors and also showed an increase in the A448 caused by CO. The second enzyme hydroxylated and reductively dehalogenated tetrahalohydroquinones to 1,2,4-trihydroxybenzene. This halohydroquinone-dehalogenating enzyme was soluble, did not require oxygen, and was not inhibited by cytochrome P-450 inhibitors.     

Effect of molybdate ions on methanation of simulated and natural waste-waters

Summary Sulphate in concentrations of 500 and 1000 mg SO₄-S/l did not inhibit methanation of synthetic waste-water (acetate + methanol + glucose) by sludge from a digester treating neutral spent sulphite process effluents. The role of sulphate reducers in the conversion of those substrates was minor although sulphate-reducing bacteria were present with a viable count similar to that of methane-producing bacteria in the sludge. Neutral spent sulphite liquor was partially converted to methane (40% of chemical oxygen demand) under these conditions.Molybdate (20 mM) inhibited methanation of both synthetic waste-water and neutral spent sulphite liquor. Acetate accumulated in glucose plus molybdate media. Molybdate had a direct inhibitory effect on enriched acetoclastic methane-producing bacteria. Molybdate was bacteriocidic to sulphate-reducing bacteria and bacteriostatic to methane-producing bacteria.     

Comparative study of potential virulence factors in human pathogenic and saprophytic Trichoderma longibrachiatum strains

Potential virulence factors of 9 saprophytic and 12 clinical Trichoderma longibrachiatum strains were examined in the present study, in order to compare their capacity to cause infection in humans. All of the strains were able to grow at temperatures up to 40 degrees C and at pH values ranging from 2.0 to 9.0. Carbon and nitrogen source utilization experiments revealed that all of the strains were able to utilize a series of basic amino acids both as sole carbon and nitrogen sources. The MIC values of the tested antifungal drugs were found to be 0.016-8 microg/ml for amphotericin B, 64-256 microg/ml for fluconazole, 0.5-32 microg/ml for itraconazole and 0.008-1 microg/ml for ketoconazole in the case of the examined isolates. Metabolites of the strains inhibited the growth of different bacteria, furthermore, compounds produced by three clinical isolates reduced the motility of boar spermatozoa, indicating their toxicity to mammalian cells as well. On the whole, there were no significant differences in the examined features between strains derived from clinical or soil samples. The question, however, whether all environmental Trichoderma longibrachiatum strains have the capacity to cause infections or not, remains still unanswered.     

Biological effects of Trichoderma harzianum peptaibols on mammalian cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Immune cell phenotype and functional defects in Netherton syndrome

Background

Netherton syndrome (NS) is a rare life-threatening syndrome caused by SPINK5 mutations leading to a skin barrier defect and a severe atopic diathesis. NS patients are prone to bacterial infections, but the understanding of the underlying immune deficiency is incomplete.

Results

We analyzed blood lymphocyte phenotypes and function in relation to clinical infections in 11 Finnish NS patients, aged 3 to 17?years, and healthy age-matched controls. The proportion of B cells (CD19⁺) and naïve B cells (CD27^?, IgD⁺) were high while memory B cells (CD27⁺) and switched memory B cells (CD27⁺IgM^?IgD^?), crucial for the secondary response to pathogens, was below or in the lowest quartile of the reference values in 8/11 (73%) and 9/11 (82%) patients, respectively. The proportion of activated non-differentiated B cells (CD21^low, CD38l^ow) was below or in the lowest quartile of the reference values in 10/11 (91%) patients. Despite normal T cell counts, the proportion of naïve CD4⁺ T cells was reduced significantly and the proportion of CD8⁺ T central memory significantly elevated. An increased proportion of CD57⁺ CD8⁺ T cells indicated increased differentiation potential of the T cells. The proportion of cytotoxic NK cells was elevated in NS patients in phenotypic analysis based on CD56DIM, CD16⁺ and CD27^? NK cells but in functional analysis, decreased expression of CD107a/b indicated impaired cytotoxicity.The T and NK cell phenotype seen in NS patients also significantly differed from that of age-matched atopic dermatitis (AD) patients, indicating a distinctive profile in NS. The frequency of skin infections correlated with the proportion of CD62L⁺ T cells, naïve CD4⁺ and CD27⁺ CD8⁺ T cells and with activated B cells. Clinically beneficial intravenous immunoglobulin therapy (IVIG) increased naïve T cells and terminal differentiated effector memory CD8⁺ cells and decreased the proportion of activated B cells and plasmablasts in three patients studied.

Conclusions

This study shows novel quantitative and functional aberrations in several lymphocyte subpopulations, which correlate with the frequency of infections in patients with Netherton syndrome. IVIG therapy normalized some dysbalancies and was clinically beneficial.

     

Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-β1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation. Received: 15 September 1997 / Accepted: 6 April 1998     

Structure and Composition of Biological Slimes on Paper and Board Machines

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 μm). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-β-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 × 10¹⁰ to 2.6 × 10¹² (average, 7 × 10¹¹) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 μmol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 × 10¹² cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 × 10¹⁰ to 1.5 × 10¹² (average, 4 × 10¹¹) cells per g (dry weight) of slime was obtained.     

Gene deletions in X-linked muscular dystrophy

Of the approximately 170 families with X-linked muscular dystrophy of the Duchenne (DMD) and Becker (BMD) type in Finland, we have studied 90 unrelated patients for intragenic deletions by using the cDNA probes described by Koenig et al. Forty-five patients (50%) had molecular deletions of one or several of the 65 exon-containing HindIII fragments. In six deletion cases junction fragments of altered size were seen. Thirty-eight (84%) of the 45 deletions were detected using only two (1–2a and 8) of the six cDNA subclones. Using a wheelchair age of 12 years to distinguish between DMD and BMD, we found that the proportions of patients with deletions were similar. Deletions were equally common in familial and sporadic disease. BMD was more commonly caused by deletions in the 5' end of the gene than was DMD. In at least three instances deletions of similar type resulted in diseases of similar severity. Of 14 patients with mental retardation seven had deletions; six of these comprised exons contained in probe 8. We conclude that cDNA hybridization studies provide a powerful diagnostic tool in DMD and BMD and that they promise to produce better insights into molecular-clinical correlations.