Bafilomycin A1 is a potassium ionophore that impairs mitochondrial functions

Novel activities of bafilomycin A1, a macrolide antibiotic known as an inhibitor of V-ATPases, were discovered. Bafilomycin A1 induced uptake of potassium ions by energized mitochondria and caused mitochondrial swelling, loss of membrane potential, uncoupling of oxidative phosphorylation, inhibition of the maximal respiration rates, and induced pyridine nucleotide oxidation. The mitochondrial effects provoked by nanomolar concentrations of bafilomycin A1 were connected to its activity as a potent, K⁺-specific ionophore. The K⁺ ionophoric activity of bafilomycin A1 was observed also in black lipid membranes, indicating that it was an inherent property of the bafilomycin A1 molecule. It was found that bafilomycin A1 is a K⁺ carrier but not a channel former. Bafilomycin A1 is the first and currently unique macrolide antibiotic with K⁺ ionophoric properties. The novel properties of bafilomycin A1 may explain some of the biological effects of this plecomacrolide antibiotic, independent of V-ATPase inhibition.     

K(ATP)-dependent neurotransmitter release in the neuronal network of the rat caudate nucleus

K(ATP) channels can couple the bioenergetic metabolism of the cell to membrane excitability. Here, we show gamma-aminobutyric acid (GABA) mediated inhibition of dopamine outflow from slices of the rat caudate nucleus that is regulated by extracellular glucose via high- and low-affinity K(ATP) channels. During glucose reduction, a biphasic dopamine effect could be observed with first a dopamine increase followed by a decline at low glucose concentrations. Both phases were inhibited by glibenclamide. Pinacidil decreased DA outflow without an effect of glucose reduction implying an overall activation of K(ATP) channels. The first phase with dopamine increase was related to reduced GABAergic activity and could be blocked by bicuculline. Our results may be explained by different types of K(ATP) channels with low affinity of ATP and glibenclamide on inhibitory GABAergic and high-affinity on excitatory DAergic neurons. This led us to suggest a biological principle through which neuronal networks are functioning.     

Gastrointestinal calcium absorption in sheep is mostly insensitive to an alimentary induced challenge of calcium homeostasis

For ruminants, marked differences to monogastric species have been described concerning the localisation and vitamin D sensitivity of gastrointestinal calcium absorption, particularly with respect to the forestomach compartment. Therefore, we investigated gastrointestinal calcium transport of sheep as influenced by a dietary calcium restriction and/or a supraphysiological dosage of exogenous calcitriol. Using the Ussing chamber technique, we determined calcium and mannitol flux rates to differentiate between para- and transcellular calcium transport in rumen, duodenum, jejunum and colon. Expression of epithelial calcium channels, calbindin-D(9K), and basolateral extrusion mechanisms was determined by quantitative RT-PCR and Western blot analysis. Active calcium transport could be demonstrated in jejunum and rumen. A significant stimulation of jejunal calcium absorption was only observed in animals treated with calcitriol. The alimentary calcium restriction alone did not result in significant effects indicating a less effective intestinal adaptation to alimentary calcium restriction than observed in monogastric animals. The observed ruminal calcium transport was not affected at all, neither by the diet nor the calcitriol treatment. Furthermore, no significant expression of epithelial calcium channels or calbindin-D(9K) could be detected in the rumen; therefore it is concluded that calcium transport in the forestomachs is probably mediated by a different, so far unknown mechanism.     

Metabolism of chloroguaiacols by Rhodococcus chlorophenolicus

Summary A polychlorophenol degrader, Rhodococcus chlorophenolicus, was shown to metabolize five different chlorinated guaiacols, namely tetrachloroguaiacol, 3,4,6-trichloroguaiacol, 3,5,6-trichloroguaiacol, 3,5-dichloroguaiacol and 3,6-dichloroguaiacol. Seven different intermediate metabolites, each with three hydroxyl or methoxyl groups, were identified. Four of these metabolites were also dehalogenation products, three carrying one chlorine atom less than the parent compound, and one metabolite from tetrachloroguaiacol where two chlorine atoms had been removed. Tetrachloroguaiacol was shown to undergo reductive dehalogenation. Demethylation of guaiacol to catechol was observed with the dichloroguaiacols, but not with polychloroguaiacols.Abbreviations DCG dichloroguaiacol - TCG trichloroguaiacol - TeCG tetrachloroguaiacol - DCC dichlorocatechol - TCC trichlorocatechol - TeCC tetrachlorocatechol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol. An example of numeration - 346-TCG 3,4,6-trichloroguaiacol - GLC gas liquid chromatography     

Cereulide-producing strains of <Emphasis Type="Italic">Bacillus cereus</Emphasis> show diversity

Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24 strains of B. cereus including two high cereulide producers (600–1,800 ng cereulide mg⁻¹ wet weight biomass), seven average producers (180–600 ng cereulide mg⁻¹ wet weight biomass), four low cereulide producers (20–160 ng cereulide mg⁻¹ wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The 13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234–02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought.     

Application of cation-exchange membranes for characterisation and imaging ammonia-oxidising bacteria in soils

A new approach, in which ammonia-oxidizing bacteria (AOB) are entrapped from soil onto cation-exchange membranes, was applied to identify terrestrial AOB by fluorescence in situ hybridization (FISH). An experimental hot spot of ammonia oxidation was developed by establishing a gradient of ammonium substrate (200 to <20 mg NH4+-N l(-1)) diffused through the cation-exchange membranes incubated in soil for 6 months. By this approach we were able to characterise and image indigenous AOB populations growing in heavily oil-polluted soil using FISH and sequence analysis of PCR-amplified 16S rRNA genes, respectively. The FISH results revealed that Nitrosospira-like AOB were dominant on the ammonium-enriched membranes incubated in the soil. Fourteen unique Nitrosospira-like 16S rRNA gene sequences belonging to clusters 2 and 3 were recovered from the soil-incubated membranes and from the soil, suggesting the importance of Nitrosospira-like AOB in the oil-polluted landfarming soil.     

Differentiated Paju cells have increased resistance to toxic effects of potassium ionophores

In this study we have investigated the impact of differentiation of neuronal cells on their sensitivity to microbial toxins. We used the human neural crest-derived tumor cell line Paju, which can be induced to differentiation in vitro by treatment with phorbol 12-myristate 13-acetate. Addition of the highly toxic potassium ionophores cereulide (4.5 and 9.0 ng/ml) or valinomycin (20 ng/ml), to cultures of undifferentiated Paju cells caused collapse of the mitochondrial membrane potential - measured with the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazole carbocyanine iodide (JC-1) followed by detachment of the cells and their apoptotic death. After induced differentiation of the Paju cells, their mitochondria retained the membrane potential upon exposure to the toxins and the cells displayed increased resistance to apoptosis as compared with undifferentiated cells. This effect may be caused by an elevated expression of the anti-apoptotic protein Bcl-2 and of the neuroprotective factor, stanniocalcin, in differentiated cells.     

Psychrotolerant Paenibacillus tundrae isolates from barley grains produce new cereulide-like depsipeptides (paenilide and homopaenilide) that are highly toxic to mammalian cells

Paenilide is a novel, heat-stable peptide toxin from Paenibacillus tundrae, which colonizes barley. P. tundrae produced 20 to 50 ng of the toxin mg(-1) of cells (wet weight) throughout a range of growth temperatures from +5°C to +28°C. Paenilide consisted of two substances of 1,152 Da and 1,166 Da, with masses and tandem mass spectra identical to those of cereulide and a cereulide homolog, respectively, produced by Bacillus cereus NS-58. The two components of paenilide were separated from those of cereulide by high-performance liquid chromatography (HPLC), showing a structural difference suggesting the replacement of O-Leu (cereulide) by O-Ile (paenilide). The exposure of porcine spermatozoa and kidney tubular epithelial (PK-15) cells to subnanomolar concentrations of paenilide resulted in inhibited motility, the depolarization of mitochondria, excessive glucose consumption, and metabolic acidosis. Paenilide was similar to cereulide in eight different toxicity endpoints with porcine and murine cells. In isolated rat liver mitochondria, nanomolar concentrations of paenilide collapsed respiratory control, zeroed the mitochondrial membrane potential, and induced swelling. The toxic effect of paenilide depended on its high lipophilicity and activity as a high-affinity potassium ion carrier. Similar to cereulide, paenilide formed lipocations, i.e., lipophilic cationic compounds, with K(+) ions already at 4 mM [K(+)], rendering lipid membranes electroconductive. Paenilide-producing P. tundrae was negative in a PCR assay with primers specific for the cesB gene, indicating that paenilide was not a product of plasmid pCER270, encoding the biosynthesis of cereulide in B. cereus. Paenilide represents the first potassium ionophoric compound described for Paenibacillus. The findings in this paper indicate that paenilide from P. tundrae is a potential food-poisoning agent.     

Regulation of 17β-hydroxysteroid dehydrogenases in cancer: regulating steroid receptor at pre-receptor stage

Increased intracelullar hormone concentration levels have been shown to be the cause of several endocrine-related cancers including breast, prostate, endometrial, ovarian, cervix, testicular, thyroid, and osteosarcoma. Deregulated expression of steroidogenic enzymes in these tumors seems to be the source of a positive balance in active steroids that bind to the corresponding nuclear receptor, thus ultimately stimulating cell proliferation. Among these enzymes, 17β-hydroxysteroid dehydrogenases catalyze the interconversion between 17-ketosteroids and 17-hydroxysteroids on the last steps of sex hormones biosynthesis and metabolism. To date, 14 isoforms have been identified in vertebrates although only 13 are present in humans. Development and clinical evaluation of specific inhibitors to block their activity is currently under progress especially against the best characterized members 1 to 5. Selectivity and potency of these drugs constitute the main challenge in this new approach to cancer and steroid-dependent diseases treatment at the "pre-receptor level". Here we review the current state of knowledge regarding the deregulation of the expression of some of these enzymes in endocrine-related tumors.     

Chlamydia pneumoniae binds to the lectin-like oxidized LDL receptor for infection of endothelial cells

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis.