Thermodynamic Study of Interactions of Distamycin A with Chromatin in Rat Liver Nuclei in the Presence of Polyamines

We studied the thermodynamics of melting of isolated rat liver nuclei with different degrees of chromatin condensation determined by the concentration of polyamines (PA) and the solution ionic strength, as well as the effect of the antibiotic distamycin A (DM) on melting. Differential scanning calorimetry (DSC) profiles of nuclear preparations contained three peaks that reflected melting of three main chromatin domains. The number of peaks did not depend on the degree of condensation; however, nuclei with more condensed chromatin had a higher total enthalpy. DM stabilized peaks II and III corresponding to the melting of relaxed and topologically strained DNA, respectively, but destabilized peak I corresponding to the melting of nucleosome core histones. At the saturating concentration (DM/DNA molar ratio = 0.1), DM increased T_m of peaks II and III by ~5°C and decreased T_m of peak I by ~2.5°C. Based on the dependence of ΔH on DM concentration, we established that at low DM/DNA ratio (?0.03), when DM interacted predominantly with AT-rich DNA regions, the enthalpy of peak II decreased in parallel with the increase in the enthalpy of peak III, which indicated that DM induces structural transitions in the nuclear chromatin associated with the increase in torsional stress in DNA. An increase in free energy under saturation conditions was equal to the change in the free energy of DM interaction with DNA. However, the increase in the enthalpy of melting of the nuclei in the presence of DM was much greater than the enthalpy of titration of nuclei with DM. This indicates a significant increase in the strength of interaction between the two DNA strands apparently due, among other things, to changes in the torsional stress of DNA in the nuclei. Titration of the nuclei with increasing PA concentrations resulted in the decrease in the number of DM-binding sites and the non-monotonous dependence of the enthalpy and entropy contribution to the binding free energy on the PA content. We suggested that the observed differences in the thermodynamic parameters were due to the different width of the minor groove in the nuclear chromatin DNA, which depends on PA concentration.     

Influence of Distamycin,Chromomycin, and UV-irradiation on Extraction of Histone H1 from Rat Liver Nuclei by Polyglutamic Acid

Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A3 (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this dif- ference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and s~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (s~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV- stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (s~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.     

Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides

Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2в, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind pre-dominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.     

Trophic level modulates carabid beetle responses to habitat and landscape structure: a pan‐European study

1. Anthropogenic pressures have produced heterogeneous landscapes expected to influence diversity differently across trophic levels and spatial scales. 2. We tested how activity density and species richness of carabid trophic groups responded to local habitat and landscape structure (forest percentage cover and habitat richness) in 48 landscape parcels (1 km²) across eight European countries. 3. Local habitat affected activity density, but not species richness, of both trophic groups. Activity densities were greater in rotational cropping compared with other habitats; phytophage densities were also greater in grassland than forest habitats. 4. Controlling for country and habitat effects, we found general trophic group responses to landscape structure. Activity densities of phytophages were positively correlated, and zoophages uncorrelated, with increasing habitat richness. This differential functional group response to landscape structure was consistent across Europe, indicated by a lack of a country × habitat richness interaction. Species richness was unaffected by landscape structure. 5. Phytophage sensitivity to landscape structure may arise from relative dependency on seed from ruderal plants. This trophic adaptation, rare in Carabidae, leads to lower phytophage numbers, increasing vulnerability to demographic and stochastic processes that the greater abundance, species richness, and broader diet of the zoophage group may insure against.     

Ecological and environmental footprint of 50 years of agricultural expansion in Argentina

Agriculture expanded during the last 50 years from the Pampas to NW Argentina at the expense of natural forests and rangelands. In parallel, productivity was boosted through the increasing application of external inputs, modern technology and management practices. This study evaluated the impact of agricultural expansion between 1960 and 2005 by assessing the implications of land use, technology and management changes on (i) carbon (C), nitrogen (N) and phosphorous (P) stocks in soil and biomass, (ii) energy, C, N, P and water fluxes and (iii) water pollution, soil erosion, habitat intervention and greenhouse gas (GHG) emissions (impacts). Based on different data sources, these issues were assessed over～1.5 million km² (63% of Argentina), involving 399 political districts during three representative periods: 1956–1960, 1986–1990 and 2001–2005. The ecological and environmental performance of 1197 farming system types was evaluated through the AgroEcoIndex model, which quantified the stocks, fluxes and impacts mentioned above. Cultivation of natural ecosystems and farming intensification caused a noticeable increase of productivity, a strengthening of energy flux, an opening of matter cycles (C, N, P) and a negative impact on habitats and GHGs emission. However, due to the improved tillage practices and the application of less aggressive pesticides, erosion and pollution risk are today lower than those of the mid‐20th century. The consistency of some assumptions and results were checked through uncertainty analysis. Comparing our results with international figures, some impacts (e.g. soil erosion, nutrient balance, energy use) were less significant than those recorded in intensive‐farming countries like China, Japan, New Zealand, USA, or those of Western Europe, showing that farmers in Argentina developed the capacity to produce under relatively low‐input/low‐impact schemes during the last decades. [Correction added after online publication 4 October 2010: In the first sentence of the Abstract, NE was corrected to NW.]     

DNA-protein cross-links in different organs of mice induced by the combined action of zinc and gamma-irradiation

Fractional whole-body gamma-irradiation of mice at total doses of 0. 5-1.5 Gy induces increased DNA-protein cross-links (DPCs) in thymus, spleen, and brain, whereas in liver no DPCs are detected. Chronic administration of zinc ions in drinking water at concentration 10 mg/liter for 20-30 days increased DPCs in thymus, spleen, brain, and liver of mice. The combined action of zinc ions and gamma-radiation produced a significantly lower amount of DPCs than was induced by the separate action of these agents.     

DNA-protein cross-links in cells of internal organs of rats after fractional irradiation at various doses

The influence of daily fractional irradiation of male Wistar rats for 30 days on DNA-protein cross-links (DPC) in spleen, thymus, and liver cells was studied. The level of DPC depended strongly on the daily dose of irradiation and the studied organ. After irradiation at dose 0.5 Gy per day increased DPC level was detected in all organs. The highest level was in the lymphoid organs and the lowest in the liver. After irradiation at dose 0.3 Gy per day DPC formation was detected only in the thymus. The data suggest the existence of a dose threshold for DPC formation during fractional irradiation.     

Tonoplast intrinsic protein isoforms as markers for vacuolar functions

Plant cell vacuoles may have storage or lytic functions, but biochemical markers specific for the tonoplasts of functionally distinct vacuoles are poorly defined. Here, we use antipeptide antibodies specific for the tonoplast intrinsic proteins alpha-TIP, gamma-TIP, and delta-TIP in confocal immunofluorescence experiments to test the hypothesis that different TIP isoforms may define different vacuole functions. Organelles labeled with these antibodies were also labeled with antipyrophosphatase antibodies, demonstrating that regardless of their size, they had the expected characteristics of vacuoles. Our results demonstrate that the storage vacuole tonoplast contains delta-TIP, protein storage vacuoles containing seed-type storage proteins are marked by alpha- and delta- or alpha- and delta- plus gamma-TIP, whereas vacuoles storing vegetative storage proteins and pigments are marked by delta-TIP alone or delta- plus gamma-TIP. In contrast, those marked by gamma-TIP alone have characteristics of lytic vacuoles, and results from other researchers indicate that alpha-TIP alone is a marker for autophagic vacuoles. In root tips, relatively undifferentiated cells that contain vacuoles labeled separately for each of the three TIPs have been identified. These results argue that plant cells have the ability to generate and maintain three separate vacuole organelles, with each being marked by a different TIP, and that the functional diversity of the vacuolar system may be generated from different combinations of the three basic types.     

Microspectrophotometry of mitochondrial cytochrome P-450 in single adrenal cells

Synopsis The cytochrome P-450 is known to be a key enzymic component in steroid hydroxylation. Biochemically it is localized in adrenal cells, both in the mitochondrial and in the microsomal fractions. Owing to its characteristic light absorptivity, attempts were made at its localization and measurement in a microspectrophotometric system. Specific difference spectra were obtained in the cytoplasm of some cells from an adrenal cell suspension before and after saturation with carbon monoxide. Scanning at 450 nm showed slender absorption maxima after CO saturation randomly distributed in the cytoplasm. These may be attributed to mitochondria or larger cytoplasmic functional units including mitochondria.