Proteomic analysis of complexes formed by human topoisomerase I

Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.     

Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids

Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon. The aim of this work was the expression of potato leafroll virus coat protein-based proteins that would be able to assemble into virus-like particles in insect cells. These modified particles were tested for their ability to encapsidate nucleic acids. Two types of N-terminally His-tagged coat protein constructs were used for the expression in insect cells: one, encoding a 23 kDa protein with the C-terminal amino-acid sequence corresponding to the wild type coat protein and the second with additional clathrin binding domain at the C-terminus. The expression of these two proteins by a recombinant baculovirus was characterized by Western immunoblotting with antibodies directed against potato leafroll virus. The protection or putative encapsidation of nucleic acids by these two coat protein derivatives was shown by DNase I and RNase A protection assays.     

Contrasting effects of low-dose IL-2 on vaccine-boosted simian immunodeficiency virus (SIV)-specific CD4+ and CD8+ T cells in macaques chronically infected with SIVmac251

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.     

Folding thermodynamics and kinetics of YNMG RNA hairpins: specific incorporation of 8-bromoguanosine leads to stabilization by enhancement of the folding rate

Modified nucleotides allow fundamental energetic and kinetic properties of nucleic acids to be probed. Here, we demonstrate that an RNA hairpin containing the nucleotide analogue 8-bromoguanosine (8BrG or G), gcUUCGgc, has enhanced stability relative to the unmodified hairpin, with DeltaDeltaG(37)(degrees)= -0.69 +/- 0.15 kcal mol(-1) and DeltaT(M) = +6.8 +/- 1.4 degrees C. NMR spectroscopic data suggest that the enhanced stability of gcUUCGgc does not arise from the native state; laser temperature-jump experiments support this notion, as gcUUCGgc and gcUUCGgc have similar unfolding rate constants, but the folding rate constant of gcUUCGgc is 4.1-fold faster at 37.5 degrees C and 2.8-fold faster under isoenergetic conditions. On the basis of these findings, we propose that 8BrG reduces the conformational entropy of the denatured state, resulting in an accelerated conformational search for the native state and enhanced stability.     

Wheat (Triticum vulgare) chloroplast nuclease ChSI exhibits 5' flap structure-specific endonuclease activity

The structure-specific ChSI nuclease from wheat (Triticum vulgare) chloroplast stroma has been previously purified and characterized in our laboratory. It is a single-strand-specific DNA and RNA endonuclease. Although the enzyme has been initially characterized and used as a structural probe, its biological function is still unknown. Localization of the ChSI enzyme inside chloroplasts, possessing their own DNA that is generally highly exposed to UV light and often affected by numerous redox reactions and electron transfer processes, might suggest, however, that this enzyme could be involved in DNA repair. The repair of some types of DNA damage has been shown to proceed through branched DNA intermediates which are substrates for the structure-specific DNA endonucleases. Thus we tested the substrate specificity of ChSI endonuclease toward various branched DNAs containing 5' flap, 5' pseudoflap, 3' pseudoflap, or single-stranded bulged structural motifs. It appears that ChSI has a high 5' flap structure-specific endonucleolytic activity. The catalytic efficiency (k(cat)/K(M)) of the enzyme is significantly higher for the 5' flap substrate than for single-stranded DNA. The ChSI 5' flap activity was inhibited by high concentrations of Mg(2+), Mn(2+), Zn(2+), or Ca(2+). However, low concentrations of divalent cations could restore the loss of ChSI activity as a consequence of EDTA pretreatment. In contrast to other known 5' flap nucleases, the chloroplast enzyme ChSI does not possess any 5'-->3' exonuclease activity on double-stranded DNA. Therefore, we conclude that ChSI is a 5' flap structure-specific endonuclease with nucleolytic activity toward single-stranded substrates.     

Sulforaphane-mediated induction of a phase 2 detoxifying enzyme NAD(P)H:quinone reductase and apoptosis in human lymphoblastoid cells

The effect of sulforaphane on human lymphoblastoid cells originating from a patient of a high cancer risk was studied. Sulforaphane (SFN) is a naturally occurring substance of chemopreventive activity. In our study, changes in cell growth, induction of apoptosis and phase 2 enzymes as well as glutathione level were examined. Apoptosis was tested by confocal microscopy at three stages: change in mitochondrial membrane potential, caspase activation and phosphatidylserine externalization. We show that SFN increases the activity of the detoxification system: it increases quinone reductase activity at low concentration (0.5-1 microM) and raises glutathione level in a dose-dependent manner. At higher doses (2.5-10 microM) sulforaphane is a cell growth modulator, as it caused cell growth cessation (IC50 = 3.875 microM), and apoptosis inducer. The results obtained suggest that sulforaphane acts as a chemopreventive agent in human lymphoblastoid cells.     

The effect of Botrytis cinerea infection on the antioxidant profile of mitochondria from tomato leaves

Infection of tomato leaves with the necrotrophic fungus Botrytis cinerea resulted in substantial changes in enzymatic and non-enzymatic components of the ascorbate-glutathione cycle as well as in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione transferase (GST), and l-galactono-gamma-lactone dehydrogenase (GLDH) activities. In the initial phase of the 5 d experiment CuZn SOD was the most rapidly induced isoform (up to 209% of control), whereas later on its activity increase was not concomitant with the constant total SOD enhancement. Starting from the second day B. cinerea infection diminished the mitochondrial antioxidant capacity by decreasing activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) as well as declining ascorbate and glutathione contents. This was accompanied by dehydroascorbate (DHA) and oxidized glutathione (GSSG) accumulation that resulted in ascorbate and glutathione redox ratios decreases. The strongest redox ratio decline of 29% for ascorbate and of 34% for glutathione was found on the 3rd and 2nd days, respectively. Glutathione reductase (GR) induction (185% of control 2 d after inoculation) was insufficient to overcome the decreased antioxidant potential of glutathione. Changes in the ascorbate pool size were closely related to the activity of l-galactono-gamma-lactone dehydrogenase (GLDH). The activities of two glutathione-dependent enzymes: GSH-Px and GST were increased from day 1 to day 4. These results demonstrated that in B. cinerea-tomato interaction mitochondria could be one of the main targets for infection-induced oxidative stress.     

Involvement of glial cells in rhythmic size changes in neurons of the housefly's visual system

In the housefly's first optic neuropile, or lamina, the axons of two classes of monopolar cell interneurons, L1 and L2, exhibit a daily rhythm of size changes: swelling during the day, and shrinking by night. At least for the L2 cells this rhythm is circadian. Moreover, epithelial glial cells that enwrap each lamina cartridge, its monopolar cell axons, and their surrounding crown of input photoreceptor terminals also change size, but in the opposite direction to the changes in L1 and L2-swelling by night and shrinking by day. The rhythmic changes in glia indicate the possible involvement of these cells in the lamina's circadian system. To examine their role in regulating the rhythmic changes of L1 and L2's axon sizes we have injected three chemicals into the haemolymph of the fly's head: fluorocitrate (FL) and iodoacetate (IAA), which affect the metabolism of glial cells, and octanol (OC), which closes gap junction channels. All chemicals exerted an effect on L1 and L2, which depended on the time of injection, the drug concentration, and the postinjection times at which we examined the fly's brains. Moreover, day/night changes in the axon sizes of L1 and L2 were increased in FL- and IAA-treated flies, indicating that glial cells may normally inhibit these changes by regulating the sizes of L1 and L2's axons during the day and night. In turn, lack of a day/night rhythm in L1 and L2 after OC injections shows that the rhythm's persistence depends on communication between the lamina cells through gap junction channels.     

Disappearance of differences in antibiotic resistance of Staphylococcus aureus strains isolated in hospitals and in general practice

There is general opinion that Staphylococcus aureus strains isolated in hospitals are more frequently resistant to antibiotics than community strains, however, the increasing resemblance between hospital and community strains has been recently reported. The aim of the study was to compare the antibiotic resistance and phage-type pattern of S. aureus strains isolated from patients treated either in hospitals or in general practice in northern part of Poland. The study was conducted on 771 S. aureus strains isolated from different specimens. Phage typing was performed according to the method of Blair and Williams. The drug susceptibility was determined by the disc-diffusion method. There were no significant differences in antibiotic resistance or phage-type pattern when hospital and community methicillin-sensitive S. aureus (MSSA) strains were compared. The most MSSA were resistant to penicillin (84.6% and 82.1% respectively) and doxycycline (49.3% and 50.4% respectively) whereas they were rarely resistant to other antibiotics. The predominance of phage group II was found in both hospitals (28.0%) and general practice (29.9%). Phage group III, usually associated with hospitals, occurred in small percentage (12.9% and 9.4% respectively) while to this group predominantly (76.6%) multiresistant methicillin resistant S. aureus (MRSA) isolated in hospitals belonged. These results suggest, that there is only slight difference in antibiotic resistance between hospital and community S. aureus strains. Antibiotic resistance pattern mainly results from frequency of appearance of MRSA, mostly occurring in hospitals.