Melatonin increases tissue accumulation and toxicity of cadmium in the bank vole (<Emphasis Type="Italic">Clethrionomys glareolus</Emphasis>)

Recent study has shown that a short photoperiod increases the accumulation and toxicity of cadmium (Cd) in the bank vole as compared to a long photoperiod. Since many of the effects of photoperiod on physiological processes in small mammals are transduced by the pineal gland and its hormone melatonin, in this study the effect of subchronic melatonin injection (7 mol/kg/day for 6 weeks) on the hepatic, renal and intestinal Cd accumulation in the bank voles raised under a long photoperiod and exposed to dietary Cd (0.9 mol/g) was examined. Simultaneously, histological examinations of the liver and kidneys, and analyses of metallothionein (MT) and lipid peroxidation were carried out. Melatonin co-treatment brought about a significant increase in the hepatic (61%), renal (79%) and intestinal (77%) Cd concentrations as compared to those in the Cd alone group. However, the concentrations of MT in the liver and kidneys of the Cd + melatonin co-treated bank voles did not differ from those in the Cd alone group. Also, histopathological changes in the liver (infiltration of leukocytes) and kidneys (glomerular swelling and a focal tubular cell degeneration) as well as an increase (2-fold) in the renal lipid peroxidation occurred only in animals from the Cd + melatonin group. These data indicate that (1) subchronic melatonin injection has similar effect on the tissue accumulation and toxicity of Cd to that produced by a short photoperiod and (2) the Cd-induced toxicity in the liver and kidneys of melatonin co-treated bank voles is probably due to increased Cd accumulation and decreased synthesis of MT.     

Comparison of different approaches for the calculation of energy expenditure using doubly labeled water in a small mammal

The doubly labeled water (DLW) method is an isotope-based technique for the estimation of the CO(2) production, and hence energy expenditure, of free-living animals and humans. Several methods are available for the calculation of CO(2) production from the isotope fluxes, depending on different assumptions about the behavior of isotopes during the elimination process. We used the DLW method to estimate the daily energy expenditures (DEE) of 55 field voles (Microtus agrestis) held in a captive facility at 8 degrees C. We calculated DEE using both plateau and intercept approaches for estimating the sizes of the isotope dilution spaces, three different assumptions about fractionation processes, and two ways of treating the different dilution spaces of the oxygen and hydrogen isotopes. We compared the resultant DEE estimates with metabolizable energy intake (MEI) measured during a 3-d feeding trial immediately before the DLW measurements, during which the animals were in energy balance. By making different assumptions about the apparent energy absorption efficiency, we generated a range of direct estimates of MEI. When we compared DEE and MEI, we found that the two-pool model formulations consistently underestimated energy demands by up to 29.8%, depending on the assumptions made in the reference calculation. However, while our data suggest that some correction for fractionation is necessary, with the present data we were unable to separate the two most common treatments of fractionation. These data strongly support the previous suggestion that for small mammals single-pool models provide more accurate estimates of energy demands than two-pool formulation of the DLW method.     

Kinetic model of oxidation catalyzed by xanthine oxidase-the final enzyme in degradation of purine nucleosides and nucleotides

A new kinetic model is presented for analysis of experimental data of oxidation process catalyzed by milk xanthine oxidase. The kinetics for two substrates, xanthine and its analog 2-chloroadenine, in a broad pH range (5.8-9.0) are best described by an equation which is a rational function of degree 2:3 and 2:2, respectively.     

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.