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.     

Can membrane-bound carotenoid pigment zeaxanthin carry out a transmembrane proton transfer?

Polar carotenoid pigment zeaxanthin (β,β-carotene-3,3′-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13 × 10⁷ Ω cm² (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H⁺-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV-Vis linear dichroism analysis of multibilayer formed with the same lipid-carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21° with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of β-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane.     

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.     

Correlation between chloroplast motility and elastic properties of tobacco mesophyll protoplasts

Translocations of chloroplasts induced by blue light were investigated in both leaves and protoplasts isolated from leaf mesophyll of Nicotiana tabacum. In the leaf tissue, the responses of chloroplasts were similar to those observed in other, higher and lower plant species. Weak and strong light induced movements of chloroplasts towards cell walls perpendicular and parallel to the light direction, respectively. Treatment with cytochalasin D, an actin-disturbing agent, blocked the movements. This shows that actin is involved in the motile system of chloroplast translocation in tobacco. By monitoring the response of chloroplasts to light in isolated protoplasts, we addressed the question whether the presence of the cell wall is necessary for the translocations of chloroplasts to occur. In control protoplasts (isolated at room temperature from unstressed leaves), no clear light intensity-dependent changes were observed in chloroplast distribution pattern. In contrast, in protoplasts obtained from plants treated with 4 °C for 8 h the chloroplasts maintained their responsiveness to light. Atomic Force Microscopy was used to measure elastic properties of the protoplasts. Young’s modulus, which reflects rigidity of the material, was 10 times higher for protoplasts of the coldstressed plants as compared to those isolated from the control plants. The rigidity of protoplasts isolated from the plants treated with low temperature was reduced four-fold by exposure to cytochalasin D. It appears that the status of protoplast actin is a factor responsible for elasticity of protoplasts. We speculate that unknown, cold stress-induced factors, maintain the orientational movements due to anchorage of the actin cytoskeleton in the plasma membrane despite the cell wall removal.     

Alternations in phenolic acids content in developing rye grains in normal environment and during enforced dehydration

A series of high pressure liquid chroamtography analyses revealed the presence of five phenolic acids in rye caryopses (vanillic, caffeic, p-coumaric, ferulic and sinapic), three of which (p-coumaric, ferulic and sinapic) were found in the free phenolic fraction. Ferulic acid was predominant, both among free acids and total phenolic acids (i.e. free, liberated from soluble esters and glycosides). The highest content of the free phenolic acids in rye caryopses was observed at the beginning of development, when on 22 DAF it was estimated at 11.55 μg·g⁻¹ DW. During dehydratation the total level of free phenolic acids in rye caryopses decreased in all investigated samples. Although total phenolic acids contents in all samples of unripe rye caryopses always decreased after dehydration, in rye sample collected in full ripeness (57 DAF), the amount of these compounds increased after the enforced dehydration. It should be added that in ester-bound-soluble phenolic acids fraction (the largest part in the total phenolic acids fraction), irrespective of the total amount decrease, much increase of sinapic acid content in this fraction was observed after dehydratation treatment in all investigated samples of caryopses of various ripeness. During the development and ripening of rye caryopses, a gradual increase in the precocious germination ability of the grain was observed. The enforced dehydration stimulated the process of precocious germination of developing and ripening rye caryopses. A possible role of phenolics in preventing precocious germination and acclimation to dehydration of developing and ripening rye grains is discussed.     

Analysis of serum copper and zinc concentrations in cancer patients

Several studies have shown that plasma copper concentrations are increased in various carcinomas. Zinc acts as a cellular growth protector, including growth of neoplastic cells, and its deficiency was demonstrated to be involved in several stages of malignant transformation. However, the usefulness of the serum zinc and copper determinations in cancer prevention, detection, monitoring treatment, and prognosis requires further investigations. The aim of the present study was to compare the serum copper and zinc levels in patients with cancer of the lung (PC), breast (BC), gastrointestinal tract (GIC), and gynecological (GYNC) malignancy with progress of the disease. The results of the study have shown a significant increase in the mean total serum Cu levels and the serum Cu/Zn ratio in all patient groups with cancer compared to a control group. Increased mean serum concentrations and Cu/Zn ratios were found in the whole group (ALLC), and for the GIC and GYNC groups with local as well as metastasized (Meta) disease in comparison with the control group. The mean serum concentrations of Zn were decreased only in metastasized ALLC and GYNC groups.     

Diversity of rotifers from northeastern U.S.A. bogs with new species records for North America and New England

In order to assess the effects of pollution on recruitment of brown trout (Salmo trutta f. fario L.) exposure experiments with brown trout early life stages were performed in two differently contaminated small streams near Stuttgart, southwest Germany, and in a control situation in the laboratory. Pollution in the more polluted stream is mainly due to effluents of municipal sewage treatment plants and to the influx of surface waters from areas of intense agriculture activity. Additionally, the water carries high particle loads, especially after rainfall events. Water quality of the less polluted stream is occasionally influenced by agriculture activity in the vicinity. For the exposure of trout early life stages incubators were used, which allowed frequent examinations without serious disturbance of the exposed organisms and thus the development could be monitored over time. Compared to the less polluted stream and the control, in the more polluted stream high mortality rates, the lowest hatching success, and lowest values for growth occurred. In the less polluted stream, mortality rates were comparably low in prehatching stages, but high in hatchlings and juveniles due to heavy infestations with protozoic ectoparasites Chilodonella cyprini (Moroff, 1902) and Ichthyobodo necator (Henneguy, 1883). However, pollution-related effects were not detected in this stream. Differences in developmental rates between the different treatments were correlated with different water temperatures in the two streams and the control. For the more polluted stream, previous studies have indicated embryotoxic potentials mediated by pollutants. The present study indicates that in this steam, additionally, fine solids which infiltrate stream gravels may seriously affect development of brown trout early life stages.