Induction of the multixenobiotic/multidrug resistance system in various cell lines in response to perfluorinated carboxylic acids

The multixenobiotic resistance (closely related to multidrug resistance) system controls transport across the plasma membrane as a defense against toxic molecules. Multixenobiotic resistance system consists of an efflux pump, ABCB1 (also named P-glycoprotein, P-gp), and/or a molecule of the ABCC family (also named multiple resistance associated protein, MRP). ABCB1 is able to increase efflux of many low-molecular foreign molecules. Measuring system induction may be used as a biomarker of cell/organism exposure to foreign substances. Various established cell lines were tested for constitutive and induced multixenobiotic resistance proteins by Western blotting immunodetection. The pumping function was indirectly assayed with Rhodamine B by visualization of cell fluorescence in the presence of verapamil. Changes in ABC proteins were measured by flow cytometry after exposition to various perfluorinated carboxylic acids. MCF7 and HeLa cells were found to contain the highest constitutive level of both ABCB1 and ABCC1. HEK293 exhibited much less ABCB1 and no activity of pumping out Rhodamine B. The pumping activity was found to be related to the amount of the cell-type specific 170 kDa ABCB1 protein. An 8-day exposure to 10(-4) M perfluorononanoic acid resulted in about 2-2.5-fold increase of ABCB1 level. That was confirmed also for short times by flow cytometry of cells exposed to perfluorinated acids and its natural congeners. Both ABCB1- and ABCC1-related fluorescence increased along with the carbon chain in acids from C(6) up to C(9) and decreased for C(10). Measuring of multixenobiotic resistance changes in vitro induced by chemicals may be a convenient test for screening for their potential toxicity.     

Influence of phosphorohydrazone derivatives of benzopyrones on polymerization and viscosity of fibrin

The synthesis and antitumour and antibacterial activity of coumarin and chromone phosphorohydrazones have been reported. This study describes influence of phosphorohydrazones derivatives of coumarin and chromone on the polymerization and viscosity of fibrin. The fibrin polymerization assay was performed by the Shen and Lorand method and the clot viscosity was measured on the basis of Shen and Lorand and Marchi and coworkers methods. Among the eight compounds tested, one coumarin derivative and two chromone derivatives showed significant activity.     

Pioglitazone, a PPAR-gamma ligand, exerts cytostatic/cytotoxic effects against cancer cells, that do not result from inhibition of proteasome

Thiazolidinediones are oral antidiabetic agents that activate peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and exert potent antioxidant and anti-inflammatory properties. It has also been shown that PPAR-gamma agonists induce G0/G1 arrest and apoptosis of malignant cells. Some of these effects have been suggested to result from inhibition of proteasome activity in target cells. The aim of our studies was to critically evaluate the cytostatic/cytotoxic effects of one of thiazolidinediones (pioglitazone) and its influence on proteasome activity. Pioglitazone exerted dose-dependent cytostatic/cytotoxic effects in MIA PaCa-2 cells. Incubation of tumor cells with pioglitazone resulted in increased levels of p53 and p27 and decreased levels of cyclin D1. Accumulation of polyubiquitinated proteins within cells incubated with pioglitazone suggested dysfunction of proteasome activity. However, we did not observe any influence of pioglitazone on the activity of isolated proteasome and on the proteolytic activity in lysates of pioglitazone-treated MIA PaCa-2 cells. Further, treatment with pioglitazone did not cause an accumulation of fluorescent proteasome substrates in transfected HeLa cells expressing unstable GFP variants. Our results indicate that pioglitazone does not act as a direct or indirect proteasome inhibitor.     

Microhabitat selection by Eurasian lynx and its implications for species conservation

We studied microhabitat selection of the Eurasian lynxLynx lynx (Linnaeus, 1758) at 116 hunting and 88 resting sites in Białowieża Primeval Forest (Poland) to describe its characteristics and determine the importance of habitat structure for stalking prey and for security during resting. We identified lynx-used sites by radio-tracking 3 male and 3 female lynx. When hunting, the lynx did not select for any type or age class of forest. During both summer and winter, the lynx selected sites characterized by high complexity (number of structures useful for stalking: fallen logs and branches, root plates, patches of dense bushes) and low visibility. In summer, hunting sites were often located in the vicinity of small forest glades that provided good stalking opportunities for lynx and rich foraging resources for roe deer — the main prey of lynx. The habitat at kill sites was more open than at sites where the prey was cached, with higher visibility, lower density of trees and poorer undergrowth. The most important characteristic of resting sites was very low visibility that resulted mainly from using young pine or spruce thickets in the winter and dense undergrowth of oak-lime-hornbeam and ash-alder forests in the summer. The information provided by this study could have direct implications for Eurasian lynx conservation by guiding forest restructuring to better suit the species’ biological requirements.     

Design of selective substrates of proteinase 3 using combinatorial chemistry methods

In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates’ sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X₃-X₂-X₁-ANB-NH₂ yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme-substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH₂ displayed the highest value of specificity constant (k_cat/K_M = 189 × 10³ M⁻¹ s⁻¹) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G.     

Habitat conditions of the phytocoenoses dominated by Luronium natans (L.) Rafin in Poland

Phytosociological and habitat studies of the phytocoenoses of Luronium natans have been conducted. The present results were compared with data on L. dortmanna and I. lacustris. It is demonstrated that the community of L. natans differs from the other two communities with respect to habitat conditions despite the fact that they have been reported to occur jointly and alongside in Lobelia lakes. It appears that significant differences between the communities are found not only regarding their waters, but also their substrates. L. natans dominated phytocoenoses are confined to oligotrophic, extremely soft waters, markedly poor in Ca²⁺, but richer in Na⁺ and SO₄ ^2- than those of Lobelia and Isoëtes. Luronium natans develops best on acidic, highly hydrated substrates rich in organic matter, NO₃ ^– and total N. The results obtained indicate that L. natans and the phytocoenoses formed by it are characterized by their narrow ecological amplitude in Poland as opposed to those occurring in western Europe, which tolerate a relatively wide range of habitats. The present findings confirm the data from numerous works, which point to the weak competitive ability of the species compared with species typical of eutrophic waters.     

The Differential Killing of Genes by Inversions in Prokaryotic Genomes

We have elaborated a method which has allowed us to estimate the direction of translocation of orthologs which have changed, during the phylogeny, their positions on chromosome in respect to the leading or lagging role of DNA strands. We have shown that the relative number of translocations which have switched positions of genes from the leading to the lagging DNA strand is lower than the number of translocations which have transferred genes from the lagging strand to the leading strand of prokaryotic genomes. This paradox could be explained by assuming that the stronger mutation pressure and selection after inversion preferentially eliminate genes transferred from the leading to the lagging DNA strand. Received: 12 December 2000 / Accepted: 20 April 2001     

Heavy metal localisation in mycorrhizas ofEpipactis atrorubens (Hoffm.) Besser (Orchidaceae) from zinc mine tailings

Summary The metal distribution within mycorrhizal and nonmycorrhizal roots ofEpipactis atrorubens collected from zinc mine tailings and an area rich in heavy metal ores (both located in southern Poland) was investigated. The tailings, consisting of postflotation material, were characterised by high levels of toxic elements such as Zn, Pb, and Cd, while soil outside the tailings was also strongly enriched in heavy metals. Atomic absorption spectrometry and proton-induced X-ray emission analysis revealed that heavy metals were mostly accumulated within orchid roots. Elemental maps from proton-induced X-ray emission showed that plant root epidermis and fungal coils which had developed within cortical cells of roots collected from the zinc mine tailings were the main places of Zn and Pb accumulation, associated with increased concentrations of Fe, Cd, Ti, Mn, Si, Ca, and S. The mean content of Pb and Zn in the coils was 4 to 5 times higher than in the root epidermis. In mycorrhizal roots from the tailings a statistically significant decrease in Pb and Zn content towards the inside of the root was observed. The mean content of Pb in coils from roots of plants growing outside the tailings was about 1% of the concentration in root coils from the tailings. Coils selected from orchid roots originating from a site outside the tailings contained comparatively high concentrations of Zn, Cd, and Cu, which was probably due to the high content of these elements in the soil. The results presented suggest a biofiltering effect against heavy metals by orchid mycorrhizal fungi.     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.