Bisphosphonate chelating agents. Coordination ability of 1-phenyl-1-hydroxymethylene bisphosphonate towards Cu(2+) ions

Potentiometric and EPR data allow for evaluation of the coordination equilibria in the Cu(2+)-bisphosphonate system. The bisphosphonic ligand was found very efficient in Cu(2+) chelation with formation of monomeric and dimeric species. Two phosphonate groups are basic binding sites for metal ion. The involvement of hydroxyl in metal ion coordination is also likely, especially when one phosphonate is protected by dimethyl ester. As the metal bound phosphonate groups are relatively bulky (six oxygens) and their negative charge above pH 4 is high (four per ligand) the equimolar species is a dominant complex at physiological pH.     

Dual effect of 2-methoxyestradiol on cell cycle events in human osteosarcoma 143B cells

We have demonstrated for the first time that the steroid metabolite, 2-methoxyestradiol (2-ME) is a powerful growth inhibitor of human osteosarcoma 143 B cell line by pleiotropic mechanisms involving cell cycle arrest at two different points and apoptosis. The ability of 2-ME to inhibit cell cycle at the respective points has been found concentration dependent. 1 microM 2-ME inhibited cell cycle at G1 phase while 10 microM 2-ME caused G2/M cell cycle arrest. As a natural estrogen metabolite 2-ME is expected to perturb the stability of microtubules (MT) in vivo analogously to Taxol--the MT binding anticancer agent. Contrary to 2-ME, Taxol induced accumulation of osteosarcoma cells in G2/M phase of cell cycle only. The presented data strongly suggest two different mechanisms of cytotoxic action of 2-ME at the level of a single cell.     

Proinflammatory interferon-gamma--inducing monokines (interleukin-12, interleukin-18, interleukin-15)--serum profile in patients with systemic lupus erythematosus

Concentrations of three interferon-gamma - inducing monokines, IL-12, IL-18 and IL-15, were investigated in the serum of 60 patients with systemic lupus erythematosus (SLE) and 20 healthy subjects. IL-12 and IL-18 were detectable in the serum of all patients with SLE and in all healthy controls. The level of IL-12 was significantly higher in SLE patients (median 264.9 pg/ml) than in the control group (median 163.6 pg/ml, p < 0.02). Similar differences were observed in the level of IL-18 (median values 602.2 pg/ml and 252.7 pg/ml, respectively, p < 0.001). Correlations between serum levels of IL-12 and IL-18 and SLE activity were not statistically significant (p > 0.05). We found a significant, positive correlation between IL-12 and IL-18 (rh? = 0.419, p < 0.001) in SLE patients. The level of IL-18 was higher in the SLE patients with antinuclear antibodies (ANA) (median 660.0 pg/ml) than in ANA-negative patients (median 326.5 pg/ml, p < 0.03), as well as in patients with immunoglobulin deposits at the dermal-epidermal junction (median 746.0 pg/ml and 444.0 pg/ml respectively, p < 0.04). The level of IL-12 was also higher in patients with skin immunoglobulin deposits (328.9 pg/ml) than those without this phenomenon (257.0 pg/ml, p < 0.05). The levels of both cytokines in the patients treated with immunosuppressive drugs and those patients not treated were similar. The serum levels of IL-15 were low and not significant both in SLE patients (median 2.9 pg/ml), and in healthy controls (median 1.6 pg/ml). In conclusion, serum levels of IL-12 and IL-18 are higher in SLE patients than in healthy controls which may indicate a role in the disease pathogenesis. However, they do not correlate with disease activity and seem to be unresponsive to immunosuppressive treatment.     

Heavy metals in human hair and teeth

Biological samples were collected simultaneously with environmental quality investigations. Studies of metal levels in biological (hair and teeth) and environmental (soil and air) samples were performed in Zwardoń during 1991/1992. Zwardoń is a small mountain resort village, situated on the border pass of Zwardoń, in the close proximity of the southwestern border of Poland. Heavy metal levels in soil, air, and chemical metals forms in the soil were examined. Pearson’s product correlation in soil (for total concentration of heavy metals and each chemical form) in hair and in teeth was calculated to investigate bioavailability of heavy metals in human organism. We received essential correlations simultaneously between: Pb vs Mn in exchangeable form of metal in soil, in teeth and in soil (total); Cd vs Zn and Mn vs Co in organically bound form in soil and in teeth and soil (total); and Cu vs Zn in all investigated samples (teeth, hair, soil total, and organically bound form in soil); Mn vs Co and Cr vs Mn in residual form in soil, in teeth, and in soil (total) and between Co vs Ni for hair, soil (total), and residual form in soil.     

Structural and Kinetic Properties of Nonglycosylated Recombinant Penicillium amagasakiense Glucose Oxidase Expressed in Escherichia coli

The gene coding for Penicillium amagasakiense glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. Recombinant Escherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml⁻¹ in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10°C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg⁻¹ and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX in E. coli.Glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) is a hydrogen peroxide-generating flavoprotein catalyzing the oxidation of β-d-glucose to d-glucono-1,5-lactone. GOX is used in the food industry for the removal of glucose from powdered eggs, as a source of hydrogen peroxide in food preservation, for gluconic acid production, and in the production of beer and soft drinks, in which its reaction serves an antioxidant function (10, 39, 42). GOX is also used extensively for the quantitative determination of d-glucose in samples such as blood, food, and fermentation products (10, 39, 49). The enzyme has been purified from both Aspergillus niger (45) and Penicillium spp. (33), with A. niger NRRL3 being the most widely used strain for industrial-scale production (11). A problem with utilizing GOX from its native source is the presence of impurities such as catalase, cellulase, and amylase, which may impair some of its applications. To overcome these difficulties and to simplify the stringent purification procedures, which are relatively expensive, A. niger GOX has been cloned and expressed in Saccharomyces cerevisiae as a highly glycosylated form (17).The most frequent employment of GOX has been in biosensors, in which the biochemical event of glucose oxidation is detected by electrochemical, thermometric, or optical techniques. The most interesting possibilities appear to lie in electron transfer reactions, with artificial electron acceptors or mediators being used to transfer information from the enzyme to the electrode (49). The electrical communication between GOX and the electrode and thereby its biosensor performance are hampered by the protein-bound carbohydrate moiety of the enzyme (1, 15), which most probably impedes electron tunneling through the enzyme (32). Almost complete (24, 27) or partial (15, 32) deglycosylation of GOX is possible, but the procedure is expensive and complicated. A more efficient and effective way of obtaining nonglycosylated GOX would be to express the enzyme in a prokaryotic host. This would also enable the properties and efficiency of GOX to be improved for its use in biosensors by protein engineering techniques (49). As a first step towards this objective, GOX from Penicillium amagasakiense was cloned and expressed in Escherichia coli. GOX from P. amagasakiense was selected since the enzyme has a higher turnover rate and a better affinity for β-d-glucose than its A. niger counterpart (30, 33).In this study, we describe the cloning and expression of the gene encoding P. amagasakiense GOX and the refolding, purification, and characterization of the nonglycosylated recombinant enzyme. The activity of the recombinant GOX, expressed in the form of insoluble inclusion bodies, was reconstituted, and the active enzyme was shown to possess properties and secondary structure composition similar to those of native P. amagasakiense GOX. This is the first reported expression of a fully active nonglycosylated form of a eukaryotic glycosylated GOX in a prokaryote, which enabled us to demonstrate that in contrast to previous assumptions (4, 9, 47) the protein-bound carbohydrate moiety is not essential for the correct folding of GOX.     

A long-term study (1985–1995) of fish populations in the impounded Warta River,Poland

Three sites, two in the tailwater and one upstream of the backwater of a large man-made reservoir in the middle course of the 808 km long lowland Warta River (Oder catchment), were studied. The pre-impoundment study lasted one (1985), the post-impoundment one 10 yr (1986–1995). The dam contributed to the extinction of the anadromous Vimba vimba, and of the rheophilous Chondrostoma nasus, both cyprinids. A number of directly or indirectly negative changes related to the construction of the reservoir were recorded in the tailwater. These changes affected the species number, spawning groups, diversity, density, standing crop, mean body weight, ABC index, and proportional stock density – PSD. Numerous impacts related to engineering, water chemistry and climate were more negative for fish at the tailwater than at the backwater sites. The hydrology of the site upstream of the backwater continued to be dependent on natural factors. Fish populations there suffered only from accidental impacts of the construction of the dam, such as bank revetment or the clearance of trees and shrubs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Compositional dynamics of natural forests in the Bialowieza National Park,northeastern Poland

Abstract. This paper presents results of a long-term study on natural forest dynamics in the Bia?owieza National Park (BNP), northeastern Poland. Five permanent sample areas were used, each consisting of a transect of varying width (40 - 60 m) and length (200 - 1380 m). The total sample area is 14.9 ha. The study covers the period 1936–1992. During this period measurements were made on five occasions at approximately 10-yr intervals. On each measurement date all trees with DBH > 5 cm were identified and their spatial location, diameter, crown condition and position in the canopy determined. During the study period the stands underwent noticeable changes, mainly in terms of tree species composition. The major change was a quantitative increase of the late-successional species: Tilia cordata and Carpinus betulus, also to a lesser degree Fraxinus excelsior and, in the last period, of the early successional Alnus glutinosa. Declining species included both early- and late-succession species. Among the latter group, Picea abies ranked first. This species lost much of its importance during the last few decades. P. abies was followed by Pinus sylvestris which is an important component of the climax vegetation under the conditions prevailing in Bialowieza, at least on more oligotrophic sites. Still, this species has not been able to regenerate during the whole study period. Some other late-succession species, Acer platanoides and Quercus robur, were also amongst the declining species. Although the basal area of Q. robur increased, its population was getting older and the process of natural regeneration was markedly impeded. All typical pioneer, short-lived species: Betula pendula and B. pubescens, Salix caprea and Populus tremula also decreased, which was probably caused by a lack of major disturbances during the study period. In general, the results obtained for the semi-natural conditions of Bialowieza during the 56-yr observation period suggest a rather high compositional instability of the forest stands there. A more precise identification of the role of particular factors in the observed stand dynamics is difficult because of the paucity of appropriate historical and environmental data which refer directly to the study plots; moreover, the data are generally incompatible and of different resolution.     

Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells

The tomato yellow leaf curl virus (TYLCV) found in Israel is a whitefly-transmitted monopartite geminivirus. Although geminiviruses have been found in the nuclei of phloem-associated cells, the mechanism of viral invasion is poorly understood. The possible role of the TYLCV capsid protein (CP), the only known component of the viral coat, in virus transport into the host cell nucleus was investigated by monitoring its specific nuclear accumulation in plant and insect cells. CP was fused to the β-glucuronidase (GUS) reporter enzyme to assay nuclear import in petunia protoplasts, and micro-injection of purified fluorescently labeled CP was used to examine its nuclear uptake in Drosophila embryos. Both assays demonstrated that TYLCV CP is transported into plant-and insect-cell nuclei by an active process of nuclear import via a nuclear localization signal (NLS)-specific pathway. Using the GUS assay and deletion analysis, the TYLCV CP NLS sequence was identified in the amino-terminus of the protein.     

Inefficient processing of human protein C in the mouse mammary gland

Vitamin K-dependent plasma protein, human Protein C (HPC) has been expressed in transgenic mice, using a 4.2kb mouse whey acidic protein (WAP) promoter, 9.0 kb HPC gene and 0.4 kb 3flanking sequences. Expression was mammary gland-specific and the recombinant human Protein C (rHPC) was detected in milk at concentrations of 0.1 to 0.7mg ml^–1. SDS-PAGE revealed that the single, heavy and light chains of rHPC migrated with increased electrophoretic mobility, as compared to HPC. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation. The substantial increase observed in the amount of single chain protein, as well as the presence of the propeptide attached to 20–30% of rHPC, suggest that mouse mammary epithelial cells are not capable of efficient proteolytic processing of rHPC. TheK _m of purified rHPC for the S-2366 synthetic substrate was similar to that of plasma-derived HPC, while the specific activity was about 42–77%. Amino acid sequence analyses and low anticoagulant activity of purified rHPC suggest that -carboxylation of rHPC is insufficient. These results show that proteolytic processing and -carboxylation can be limiting events in the overexpression of fully biologically active rHPC in the mouse mammary gland.