Experiences of the Czech toxicological information centre with ethylene glycol poisoning

The objective was to evaluate the severity of ethylene glycol (EG) intoxications in a 3-year retrospective study of the calls to the Toxicological Information Centre (TIC). Data about clinical course of patients with EG poisoning reported to the TIC in the years 2000-2002 were analysed. They were completed by the data from discharge records from the hospitals and by toxicological analyses. The chi-square test, Student's t-test, Fisher's test and the calculation of linear correlation coefficient were used for statistical analysis. The significance level was set at 0.05. TIC received total 188 calls concerning EG, from which 33 discharge reports were gained. There were 30 males (age 5-74 years) and 3 females (age 10-54 years). The patients ingested 252 ml on average (30-1000 ml); lethal dose (100 ml) was exceeded in 14 patients. Mean time interval from ingestion to admission was 3 hours (3-24 hours), mean length of hospitalisation 6 days (1-76 days). Fourteen patients developed metabolic acidosis, nine unconsciousness, thirteen signs of nephrotoxicity and nine signs of hepatotoxicity. Three patients died. Antidote ethanol was given in 30 patients. Other treatment included haemodialysis (20 cases) and B vitamins (23 cases). Ingested dose and the time interval between ingestion and admission correlated with severity of kidney damage. These data confirm that EG poisoning could seriously threaten the life. Renal parameters were abnormal in 30 % of patients who were discharged from the hospital. Those patients will be followed to evaluate the reversibility of EG toxic kidney damage.     

Antioxidant activity of new ruthenium compounds

Many biological properties have been attributed to ruthenium complexes including anti-tumor activity and the attenuation of reperfusion damage and infarct size. In this work, we characterize the antioxidant activity of trans-[RuCl2(nic)4] where nic is 3-pyridinecarboxylic acid and trans-[RuCl2(i-nic)4] where i-nic is 4-pyridinecarboxylic acid by (i) evaluation of total antioxidant potential (TRAP); (ii) prevention of DNA damage induced by hydrogen peroxide using the alkaline comet assay; and (iii) the prevention of lipid peroxidation and cell death induced by iron in liver slices. Our results suggest that nic has stronger antioxidant potential when compared to the i-nic. Higher doses (above 200 microM) of these compounds gave genotoxic effects, but the antioxidant potential could be obtained with the use lower doses (0.1-10 microM).     

The PIN auxin efflux facilitators: evolutionary and functional perspectives

It is widely believed that the PIN proteins are crucial for proper cellular coordination. Since the analysis of the Arabidopsis pin-formed mutant in 1991, and the subsequent cloning of AtPIN1, a further seven members of the family have been discovered. Here, we present an overview of this family of auxin efflux facilitators in monocot and dicot plants, summarizing their evolutionary history, expression profiles and, where appropriate, relating them to protein function.     

Similar mating and sperm displacement patterns in two highly divergent D. simulans populations from Africa and Europe

The frequency of remating in Drosophila melanogaster is affected by both genetic and ecological factors. We studied the remating behaviour in one European (Italy) and one African (Uganda) Drosophila simulans population using six highly polymorphic microsatellite markers. Despite that the populations were genetically distinct (F(ST) = 0.18) and originated from very dissimilar ecological settings with different population densities, we inferred a very similar mating pattern. The remating parameter alpha was similar in both populations (a = 1.3-1.4). No more than two distinct paternal genotypes per family were detected in each population.     

Response to cadmium of Daucus carota hairy roots dual cultures with Glomus intraradices or Gigaspora margarita

Ri T-DNA-transformed carrot roots were cultivated in two experiments either non-inoculated or inoculated with the arbuscular mycorrhizal (AM) fungi Glomus intraradices or Gigaspora margarita. The influence of two concentrations of cadmium (Cd) in the medium (2 mg l^–1, 4 mg l^–1) on both root and mycelium growth was tested. Both parameters were estimated at 10-day intervals for 70 or 100 days for G. intraradices and Gi. margarita, respectively. In the first experiment, G. intraradices showed a rapid spread of extraradical mycelium (ERM) and reached average densities per treatment of about 90 cm cm^–2 agar medium after 70 days. At the higher Cd level, the growth of ERM was delayed in comparison to the treatment without Cd addition. Root growth was inhibited by both Cd levels; the inhibition was, however, significantly lower in the treatments inoculated with G. intraradices compared to the non-inoculated control. In the second experiment, the ERM of Gi. margarita started to grow after a period of 50 days and reached average densities per treatment of only up to 27 cm cm^–2 by the end of the cultivation. The growth of Gi. margarita mycelium was not inhibited by Cd. No differences in root growth were observed between the Gi. margarita inoculated and non-inoculated treatments. The inhibitory effect of Cd on root growth differed between the non-inoculated treatments in both experiments. The study has shown that the AM fungus Glomus intraradices can alleviate Cd-induced growth inhibition to carrot hairy roots. The potential and limits of the monoxenic system in studying the interaction between AM fungi and heavy metals are discussed.     

The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7

We recently described that in the metastasizing rat pancreatic carcinoma line BSp73ASML the cell-cell adhesion molecule EpCAM, CD44 variant isoforms and the tetraspanins D6.1A and CD9 form a complex that is located in glycolipid-enriched membrane microdomains. This complex contains, in addition, an undefined 20 kDa protein. As such complex formation influenced cell-cell adhesion and apoptosis resistance, it became of interest to identify the 20 kDa polypeptide. This 20 kDa protein, which co-precipitated with EpCAM in BSp73ASML lysates, was identified as the tight junction protein claudin-7. Correspondingly, an association between EpCAM and claudin-7 was noted in rat and human tumors and in non-transformed tissues of the gastrointestinal tract. Co-localization of the two molecules was most pronounced at basolateral membranes, but was also observed in tight junctions. Evidence for direct protein-protein interactions between EpCAM and claudin-7 was obtained by co-immunoprecipitation after treatment of tumor cells with a membrane-permeable chemical cross-linker. The complex, which is located in glycolipid-enriched membrane microdomains, is not disrupted by partial cholesterol depletion, but claudin-7 phosphorylation is restricted to the localization in glycolipid-enriched membrane microdomains. This is the first report on an association between EpCAM and claudins in both non-transformed tissues and metastasizing tumor cell lines.     

Molecular dynamics simulations indicate a possible role of parallel beta-helices in seeded aggregation of poly-Gln

The molecular structures of amyloid fibers characterizing neurodegenerative diseases such as Huntington's or transmissible spongiform encephalopathies are unknown. Recently, x-ray diffraction patterns of poly-Gln fibers and electron microscopy images of two-dimensional crystals formed from building blocks of prion rods have suggested that the corresponding amyloid fibers are generated by the aggregation of parallel beta-helices. To explore this intriguing concept, we study the stability of small beta-helices in aqueous solution by molecular dynamics simulations. In particular, for the Huntington aggregation nucleus, which is thought to be formed of poly-Gln polymers, we show that three-coiled beta-helices are unstable at the suggested circular geometries and stable at a triangular shape with 18 residues per coil. Moreover, we demonstrate that individually unstable two-coiled triangular poly-Gln beta-helices become stabilized upon dimerization, suggesting that seeded aggregation of Huntington amyloids requires dimers of at least 36 Gln repeats (or monomers of approximately 54 Gln) for the formation of sufficiently stable aggregation nuclei. An analysis of our results and of sequences occurring in native beta-helices leads us to the proposal of a revised model for the PrP(Sc) aggregation nucleus.     

Bacterial dynamics in spring water of alpine karst aquifers indicates the presence of stable autochthonous microbial endokarst communities

Spring water of two alpine karst aquifers differing in hydrogeology but of nearby catchments were investigated for their bacterial population dynamics. Dolomite karst aquifer spring 1 (DKAS 1) represents a dolomitic-limestone karst aquifer spring showing high average water residence time and relative constant flow. Limestone karst aquifer spring 2 (LKAS 2) constitutes a typical limestone karst aquifer spring with a dynamic hydrological regime and discharge. Dolomite karst aquifer spring 1 yielded constantly lower cell counts and biomasses (median of 15 x 10(6) cells l(-1) and 0.22 microg C l(-1)) as the LKAS 2 (median of 63 x 10(6) cells l(-1) and 1.1 microg C l(-1)) and distribution of morphotypes and mean cell volumes was also different between the considered systems, indicating the influence of hydrogeology on microbial spring water quality. Molecular bacterial V3 16S-rDNA profiles revealed remarkable constancy within each spring water throughout the investigation period. Time course analysis of a flood event in LKAS 2 further supported the trend of the temporal constancy of the microbial community. Except for one case, retrieval of partial and full length 16S rDNA gene sequences from the relative constant DKAS 1 revealed similarities to presently known sequences between 80% to 96%, supporting the discreteness of the microbial populations. The gathered results provide first evidence for the presence of autochthonous microbial endokarst communities (AMEC). Recovery of AMEC may be considered of relevance for the understanding of alpine karst aquifer biogeochemistry and ecology, which is of interest as many alpine and mountainous karst springs are important water resources throughout the world.     

Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases

Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.