In Vitro Characterization of the Pharmacological Properties of the Anti-Cancer Chelator,Bp4eT,and Its Phase I Metabolites

Cancer cells have a high iron requirement and many experimental studies, as well as clinical trials, have demonstrated that iron chelators are potential anti-cancer agents. The ligand, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), demonstrates both potent anti-neoplastic and anti-retroviral properties. In this study, Bp4eT and its recently identified amidrazone and semicarbazone metabolites were examined and compared with respect to their anti-proliferative activity towards cancer cells (HL-60 human promyelocytic leukemia, MCF-7 human breast adenocarcinoma, HCT116 human colon carcinoma and A549 human lung adenocarcinoma), non-cancerous cells (H9c2 neonatal rat-derived cardiomyoblasts and 3T3 mouse embryo fibroblasts) and their interaction with intracellular iron pools. Bp4eT was demonstrated to be a highly potent and selective anti-neoplastic agent that induces S phase cell cycle arrest, mitochondrial depolarization and apoptosis in MCF-7 cells. Both semicarbazone and amidrazone metabolites showed at least a 300-fold decrease in cytotoxic activity than Bp4eT towards both cancer and normal cell lines. The metabolites also lost the ability to: (1) promote the redox cycling of iron; (2) bind and mobilize iron from labile intracellular pools; and (3) prevent ⁵⁹Fe uptake from ⁵⁹Fe-labeled transferrin by MCF-7 cells. Hence, this study demonstrates that the highly active ligand, Bp4eT, is metabolized to non-toxic and pharmacologically inactive analogs, which most likely contribute to its favorable pharmacological profile. These findings are important for the further development of this drug candidate and contribute to the understanding of the structure-activity relationships of these agents.     

Backbone resonance assignments of the outer membrane lipoprotein FrpD from Neisseria meningitidis

The iron-regulated FrpD protein is a unique lipoprotein embedded into the outer membrane of the Gram-negative bacterium Neisseria meningitidis. The biological function of FrpD remains unknown but might consist in anchoring to the bacterial cell surface the Type I-secreted FrpC protein, which belongs to a Repeat in ToXins (RTX) protein family and binds FrpD with very high affinity (K _d  = 0.2 nM). Here, we report the backbone ¹H, ¹³C, and ¹⁵N chemical shift assignments for the FrpD_43–271 protein that allow us to characterize the intimate interaction between FrpD and the N-terminal domain of FrpC.     

The Use of Anchored Agonists of Phagocytic Receptors for Cancer Immunotherapy: B16-F10 Murine Melanoma Model

The application of the phagocytic receptor agonists in cancer immunotherapy was studied. Agonists (laminarin, molecules with terminal mannose, N-Formyl-methioninyl-leucyl-phenylalanine) were firmly anchored to the tumor cell surface. When particular agonists of phagocytic receptors were used together with LPS (Toll-like receptor agonist), high synergy causing tumour shrinkage and a temporary or permanent disappearance was observed. Methods of anchoring phagocytic receptor agonists (charge interactions, anchoring based on hydrophobic chains, covalent bonds) and various regimes of phagocytic agonist/LPS mixture applications were tested to achieve maximum therapeutic effect. Combinations of mannan/LPS and f-MLF/LPS (hydrophobic anchors) in appropriate (pulse) regimes resulted in an 80% and 60% recovery for mice, respectively. We propose that substantial synergy between agonists of phagocytic and Toll-like receptors (TLR) is based on two events. The TLR ligand induces early and massive inflammatory infiltration of tumors. The effect of this cell infiltrate is directed towards tumor cells, bearing agonists of phagocytic receptors on their surface. The result of these processes was effective killing of tumor cells. This novel approach represents exploitation of innate immunity mechanisms for treating cancer.     

The phylogenetic structure of microbial biofilms and free-living bacteria in a small stream

The phylogenetic composition, bacterial biomass, and biovolume of both planktonic and biofilm communities were studied in a low-order Byst?ice stream near Olomouc City, in the Czech Republic. The aim of the study was to compare the microbial communities colonizing different biofilm substrata (stream aggregates, stream sediment, underwater tree roots, stream stones, and aquatic macrophytes) to those of free-living bacteria. The phylogenetic composition was analyzed using fluorescence in situ hybridization for main phylogenetic groups. All phylogenetic groups studied were detected in all sample types. The stream stone was the substratum where nearly all phylogenetic groups were the most abundant, while the lowest proportion to the DAPI-stained cells was found for free-living bacteria. The probe specific for the domain Bacteria detected 20.6 to 45.8 % of DAPI-stained cells while the probe specific for the domain Archaea detected 4.3 to 17.9 %. The most abundant group of Proteobacteria was Alphaproteobacteria with a mean of 14.2 %, and the least abundant was Betaproteobacteria with a mean of 11.4 %. The average value of the Cytophaga–Flavobacteria group was 10.5 %. Total cell numbers and bacterial biomass were highest in sediment and root biofilm. The value of cell biovolume was highest in stone biofilm and lowest in sediment. Overall, this study revealed relevant differences in phylogenetic composition, bacterial biomass, and biovolume between different stream biofilms and free-living bacteria.     

Zinc induces DNA damage in tobacco roots

We applied the alkaline version of the single-cell gel electrophoresis (comet assay) to seedlings of heterozygous tobacco (Nicotiana tabacum L. var. xanthi) treated with zinc acetate dihydrate (20 to 80 mM Zn²⁺ for 2 h or 2 to 12 mM Zn²⁺ for 24 h). A dose dependent increase in DNA damage expressed by the tail moment values were observed in nuclei isolated from the roots after 2 and 24 h Zn²⁺ treatments. In contrast, Zn²⁺ did not induce significant DNA damage to leaf nuclei, with the exception of 10 or 12 mM Zn²⁺ for 24 h. Somatic mutations, identified as dark green, yellow, and dark green/yellow double sectors on the pale green tobacco leaves were not detected after any Zn²⁺ treatments. The accumulation of Zn in roots and shoots was determined by inductively coupled plasma optical emission spectrometry and the Zn content in roots was about three times higher than in shoots.     

Production,purification and oxidative folding of the mouse recombinant prior protein

The method leading to overexpression of the full-length mouse recombinant prion protein (mrPrP 23-231) in the cytoplasm of E. coli as a his-PrP fusion protein and its effective purification using affinity chromatography is described. A typical yield of the method was 8-10 mg his-mrPrP per L of the bacterial culture. The purity of purified protein was > 95 %. The purified his-mrPrP was converted to a soluble form and its folding to alpha-helical and beta-sheet conformations was studied. The properties of differently folded mrPrP were determined by measuring their circular dichroism spectra, partial resistance to cleavage by proteinase K and by centrifugation in sucrose gradient.     

Ion selectivity of a poly(3-pentylmethoxythiophene) LB-layer modified carbon-fiber microelectrode as a consequence of the second order filtering in voltcoulometry

Ion selective properties of poly(3-pentylmethoxythiophene) Langmuir-Blodgett film modified carbon-fiber microelectrode are described. The study of the electrode behavior indicates that important features occur if two electrochemical methods, one of them being kinetics sensitive, are used. While in case of the typical steady-state voltammetry the electrode remains sensitive to both the cations and anions, the kinetics-sensitive properties of voltcoulometry based on the second-order filtering scheme disable the observation of anions. As a model system a mixture of copper and dopamine ions is used.     

Impact of spruce forest and grass vegetation cover on soil micromorphology and hydraulic properties of organic matter horizon

Two organic matter horizons developed under a spruce forest and grass vegetation were chosen to demonstrate the impact of a different vegetation cover on the micromorphology, porous system and hydraulic properties of surface soils. Micromorphological studies showed that the decomposed organic material in the organic matter horizon under the grass vegetation was more compact compared to the decomposed organic material in the organic matter horizon under the spruce forest. The detected soil porous system in the organic matter horizon under the spruce forest consisted of two clusters of pores with different diameters that were highly connected within and between both clusters. The soil porous system in the organic matter horizon under the grass vegetation consisted of one cluster of pores with the larger diameters and isolated pores with the smaller diameter. The retention ability of the organic matter horizon under the grass vegetation was higher than the retention ability of the organic matter horizon under the spruce forest. Presented at the International Conference on Bioclimatology and Natural Hazards, Poľana nad Detvou, Slovakia, 17–20 September 2007.     

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.