Genotoxicity of cis-PtII complex of 3-aminoflavone in comparison with cis-DDP in A549 cells evaluated by comet assay

Cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most successful antineoplastic drugs. However, besides effectiveness it gives many side effects. Therefore, current studies are concentrated on searching for new analogs equally effective in chemotherapy but less toxic. Comparison of genotoxic properties of cis-Pt(II) complex of 3-aminoflavone and cis-DDP in a comet assay with and without H2O2 application was performed in A549 cell line. The higher tail moment values were noticed for the former compound contrary to the latter one in both variants. It suggests mainly DNA breaks (besides cross-links) appearance after cis-Pt(II) complex of 3-aminoflavone application and might indicate DNA degradation in comparison with cis-DDP.     

Structural determinants of the selectivity of KTS-disintegrins for the alpha1beta1 integrin

KTS-disintegrins are a subfamily of short monomeric disintegrins that are potent and selective inhibitors of alpha1beta1 integrin. The amino acid sequence of the new KTS-disintegrin, viperistatin, differs from previously characterized obtustatin in three residues at position 24 (within the integrin binding loop), 38 (hydrophobic core) and 40 (C-terminal region). Noteworthy, viperistatin is about 25-fold more potent than obtustatin inhibiting the binding of this integrin to collagen IV. Synthetic peptides representing the full-length of integrin-binding loops of these disintegrins showed that the Leu24/Arg substitution appears to be partly responsible for the increased inhibitory activity of viperistatin over obtustatin.     

Cytogenetic studies of Glomeris (Diplopoda: Glomeridae)

Karyotypes and meiosis of Glomeris hexasticha and G. connexa (Diplopoda: Glomeridae) from Poland were described using C-heterochromatin distribution and observations of the location of NORs. These species were characterized by 2n>=16 and the XY sex determination system. Differences were found in the amount of C-heterochromatin in X and Y chromosomes between the studied species. In G. hexasticha, supernumerary B chromosomes were described.     

Metabolic and antiproliferative consequences of activated polyamine catabolism in LNCaP prostate carcinoma cells

Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor alpha-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.     

Processing of the dual targeted precursor protein of glutathione reductase in mitochondria and chloroplasts

Pea glutathione reductase (GR) is dually targeted to mitochondria and chloroplasts by means of an N-terminal signal peptide of 60 amino acid residues. After import, the signal peptide is cleaved off by the mitochondrial processing peptidase (MPP) in mitochondria and by the stromal processing peptidase (SPP) in chloroplasts. Here, we have investigated determinants for processing of the dual targeting signal peptide of GR by MPP and SPP to examine if there is separate or universal information recognised by both processing peptidases. Removal of 30 N-terminal amino acid residues of the signal peptide (GRDelta1-30) greatly stimulated processing activity by both MPP and SPP, whereas constructs with a deletion of an additional ten amino acid residues (GRDelta1-40) and deletion of 22 amino acid residues in the middle of the GR signal sequence (GRDelta30-52) could be cleaved by SPP but not by MPP. Numerous single mutations of amino acid residues in proximity of the cleavage site did not affect processing by SPP, whereas mutations within two amino acid residues on either side of the processing site had inhibitory effect on processing by MPP with a nearly complete inhibition for mutations at position -1. Mutation of positively charged residues in the C-terminal half of the GR targeting peptide inhibited processing by MPP but not by SPP. An inhibitory effect on SPP was detected only when double and triple mutations were introduced upstream of the cleavage site. These results indicate that: (i) recognition of processing site on a dual targeted GR precursor differs between MPP and SPP; (ii) the GR targeting signal has similar determinants for processing by MPP as signals targeting only to mitochondria; and (iii) processing by SPP shows a low level of sensitivity to single mutations on targeting peptide and likely involves recognition of the physiochemical properties of the sequence in the vicinity of cleavage rather than a requirement for specific amino acid residues.     

Low-temperature-induced transmembrane potential changes in mesophyll cells of Arabidopsis thaliana, Helianthus annuus and Vicia faba

Glass microelectrodes were inserted into mesophyll cells of intact leaves from higher plants: Arabidopsis thaliana, Helianthus annuus and Vicia faba var minor , and transient membrane potential changes were recorded in response to a sudden temperature drop. The cold-induced potential changes were unaffected by an anion channel inhibitor (anthracene-9-carboxylic acid) and potassium channel inhibitor (tetraethyl ammonium ion). Verapamil, a calcium channel inhibitor, caused significant suppression of the cold-induced potential changes. In the presence of calmoduline antagonists (trifluoperazine and N -6-aminohexyl-5-chloro-1-naphtalenesulphonamide), their amplitudes decreased and their durations were prolonged. Neomycin, which suppresses phospholipase C, also caused substantial inhibition of the amplitudes of the cold-induced potential changes. It is concluded that cold-evoked membrane potential changes are due to calcium influxes from both the apoplast and internal stores.     

Aggregation and G-quadruplex DNA-binding study of 6a,12a-diazadibenzo-[a,g]fluorenylium derivative

The aggregation and DNA binding behavior of a new G-quadruplex selective ligand, 6a,12a-diazadibenzo-[a,g]fluorenylium derivative, was studied by UV-vis absorption and fluorescence spectroscopy. The formation of ligand aggregates with different spectral characteristics was observed at low and high concentration of NaCl, respectively. The ligand binds to G-quadruplex with much higher affinity than to single- and double-stranded DNA.