Analysis of brain chromatin subunit composition using different endonucleases]

A comparison of the processes of chromatin digestion in brain and liver nuclei by Ca, Mg-dependent and staphylococcal endonucleases demonstrates a similarity of the subunit composition of chromatin from both tissues and reveals the same type of linked DNA regions. However, a formation of low molecular weight DNP fragments during hydrolysis and the DNA spectra of soluble and insoluble DNP fragments suggest that brain chromatin contains these fragments alongside with the regions, which are specific for this particular tissue, predominate in it and are resistant to staphylococcal and, particularly, to Ca, Mg-dependent endonucleases. This is paralleled with a non-histone protein enrichment of different brain chromatin fractions and an expansion of the electrophoretic monomer band towards the fragment with a greater molecular weight. It may be assumed that brain nucleosomes are characterized by a higher size heterogeneity of linked DNA, part of which are mostly covered by non-histone proteins, and/or are characterized by a greater set variety.     

Studies of deoxyribonucleoproteins progressively depleted of proteins in solutions of variousionic strengths

The conformation of deoxyribonucleoprotein (DNP) from calf thymus at different stages of deproteinization was studied. The dissociation of the first portion of histone produces no effect on the hydrodynamical and optical behavior of DNP particles. The conformational transition of a macromolecule was observed as soon as the ratio of protein to DNA ? 0.9. The effect of ionic strength on the conformation of DNP particles with high protein content was more strongly pronounced than that for DNA. On the contrary, DNP particles depleted of proteins (protein/DNA < 0.9) were found to be less sensitive than DNA to the variation of ionic strength. These data imply that the DNP molecules rich in proteins possess a superstructure that is destroyed as the protein/DNA ratio becomes 0.9. The data were analyzed in view of current theories on various model concepts. The most probable model to describe the DNP molecule was chosen by comparing the calculated and experimentally obtained parameters. We believe that DNP is best described as a “compressed coil,” possibly including superhelical regions.     

Fractionation of chromosomal deoxyribonucleoproteins by partition in two-phase aqueous polymer systems

Deoxyribonucleoprotein (DNP)¹ prepared by shearing chromatin of mouse cells may be fractionated in 2-phase aqueous Dextran-polyethyleneglycol mixtures. A partial separation of DNPs with different non-histone protein/DNA ratios may be obtained in a single-step partition. Separation of a spectrum of fractions of DNP has been obtained by countercurrent distribution using the same 2-phase polymer system. DNP fractions which bear nascent RNA (representing approximately $\text{1}\text{3}$ of the total DNA) may be separated from the major fraction of DNP; they are found in the same region of the distribution pattern as DNP fractions with the highest non-histone protein/DNA ratio.     

The effect of gamma irradiation on the content in the thymus and liver chromatin of rats of DNA-bound proteins resistant to dissociation by phenol and sodium dodecylsulfate]

Two groups of proteins of 50-68 kD (A) and 12-14 kD (B) are the components of DNP preparations from rat thymus and liver obtained by washing with 0.075 M NaCl-0.024 M EDTA solution and deproteinization with phenol and dodecylsulfate (SDS). Immediately after irradiation with a dose of 10 Gy, there observed an approximately 1.5-fold increase in the content of only B proteins in the rat thymus fraction precipitated upon treatment with SDS-NaCl. The acidic amino acid content of this fraction and DNP preparation obtained without treatment with SDS amounts to 25 mol%; the ratio to basic amino acids was 1.3-1.4. The comparison of the amino acid content in the above DNP preparation and the "supramolecular DNA" preparation, described in the literature, that was obtained by the same phenol deproteinization and contained about 50 mol% of acidic amino acids, indicates the presence in the "supramolecular DNA" preparation of a component that increases upon irradiation: the component consists almost completely of acidic amino acids and is eliminated completely from the DNP preparation by washing with 0.075 M NaCl-0.024 M EDTA prior to deproteinization. The amino acid composition of the protein fraction A is presented.     

Effects of herbicide 2,4-dinitrophenol on mitosis,DNA, RNA and protein synthesis inNigella sativa L.

Effect of 2,4-dinitrophenol (DNP) was studied onNigella sativa to note the changes in mitosis, DNA, RNA and protein synthesis. The chemical affected division frequency considerably and chromosomal abnormalities like sticky bridge, fragmentation, micronucleietc. were recorded. By using precursors of nucleic acid and protein synthesis, it was found that DNP also inhibited DNA, RNA and protein synthesis. The decrease in division frequency can be correlated with the DNA synthesis.     

Electron microscopic mapping of proteins bound to herpes simplex virus DNA.

Exonuclease digestion experiments have suggested that there is a protein(s) bound close to one or both ends of herpes simplex virus-1 (HSV) DNA. The existence of such bound proteins has been positively demonstrated and their positions on the HSV genome determined by application of a newly developed method for electron microscopic mapping of proteins bound to nucleic acids. Purified HSV DNA was treated with dinitrofluorobenzene under conditions that covalently attach the dinitrophenyl (DNP) group to the proteins in a protein-nucleic acid complex. The HSV DNA-protein-(DNP)n complex was treated with rabbit anti-DNP IgG, and, in some cases, additionally treated with monovalent Fab fragments of goat anti-rabbit IgG, and mounted for examination in the electron microscope. Electron opaque dots representing the protein-(DNP)n-(IgG)m complex were seen on the HSV DNA. Direct measurements of the positions of the protein, as well as partial denaturation mapping, indicate that there are four positions for protein bound to HSV DNA: two near but not at the two ends and two at sites corresponding to the internal inverted repeats of the ends. These results suggest that there is a specific protein binding sequence within the direct terminal repeat of HSV DNA. The previous observation that HSV DNA is more sensitive to digestion by a 3' than by a 5' exonuclease then indicates that the bound protein(s) is more intimately associated with one strand of the specific sequence than with the complementary strand.     

In vivo and in vitro phosphorylation of DNA-binding proteins from Escherichia coli.

A deoxyribonucleoprotein (DNP) complex has been isolated from Escherichia coli cells by chromatography on Sephadex G-200. The DNP complex contains phosphoproteins and the content of phosphorus bound to the DNP protein is 3 times higher than in cytoplasmic proteins not bound to DNA. These results have been confirmed by in vivo (32-P-KH2PO4) and in vitro (32-P-ATP) phosphorylation of E. coli DNA-binding proteins isolated by chromatography on DNA--cellulose.     

Dinitrophenol Inhibits the Rejoining of Radiation-Induced DNA Breaks by L-Cells

The production and rejoining of X-ray-induced single-stranded DNA breaks was studied using the alkaline sucrose density gradient technique and by measuring the disappearance of both 5' termini and 3'-OH termini using polynucleotide kinase and DNA polymerase, respectively. All studies were conducted using L-cell suspensions irradiated both in the presence and absence of 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation. Results show that the induction of single-stranded DNA breaks probably includes a nucleolytic component in addition to indirect free radical effects. A greater number of breaks were produced in the absence of DNP, suggesting that depressed adenosine triphosphate (ATP) levels reduce endogenous nucleolytic activity. The rejoining mechanism is enzymatic and requires an available ATP supply for operation. In the presence of DNP no DNA rejoining was observed following 30 min incubation after 10,000 rad. These results suggest that DNA breaks produced may be characterized by 5'-PO(4)-3'-OH termini and are rejoined by DNA ligase.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Exploring the binding mode of donepezil with calf thymus DNA using spectroscopic and molecular docking methods

Donepezil (DNP) is one of approved drugs to treat Alzheimer's disease (AD). However, the potential effect of DNP on DNA is still unclear. Therefore, the interaction of DNP with calf thymus DNA (DNA) was studied in vitro using spectroscopic and molecular docking methods. Steady‐state and transient fluorescence experiments showed that there was a clear binding interaction between DNP and DNA, resulting from DNP fluorescence being quenched using DNA. DNP and DNA have one binding site between them, and the binding constant (K_b) was 0.78 × 10⁴ L·mol^?1 at 298 K. In this binding process, hydrophobic force was the main interaction force, because enthalpy change (ΔH) and entropy change (ΔS) of DNP–DNA were 67.92 kJ·mol^?1 and 302.96 J·mol^?1·K^?1, respectively. DNP bound to DNA in a groove‐binding mode, which was verified using a competition displacement study and other typical spectroscopic methods. Fourier transform infrared (FTIR) spectrum results showed that DNP interacted with guanine (G) and cytosine (C) bases of DNA. The molecular docking results further supported the results of spectroscopic experiments, and suggested that both Pi‐Sigma force and Pi‐Alkyl force were the major hydrophobic force functioning between DNP and DNA.     

Dendroaspis natriuretic peptide protects the post-ischemic myocardial injury

The present study used isolated rat hearts to investigate whether (1) Dendroaspis natriuretic peptide (DNP) is protective against post-ischemic myocardial dysfunction, and (2) whether the cardioprotective effects of DNP is related to alteration of Bcl-2 family protein levels. The excised hearts of Sprague-Dawley rats were perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. Left ventricular end-diastolic pressure (LVEDP, mmHg), left ventricular developed pressure (LVDP, mmHg) and coronary flow (CF, ml/min) were continuously monitored. In the presence of 50 nM DNP, all hearts were perfused for a total of 100 min consisting of a 20 min pre-ischemic period followed by a 30 min global ischemia and 50 min reperfusion. Lactate dehydrogenase (LDH) activity in the effluent was measured during reperfusion. Treatment with DNP alone improved the pre-ischemic LVEDP and post-ischemic LVEDP significantly comparing with the untreated control hearts during reperfusion. However, DNP did not affect the LVDP, heart rate (HR, beats/min), and CF. Bcl-2, an anti-apoptotic protein expressed in ischemic myocardium of DNP+ischemia/reperfusion (I/R) group, was higher than that in I/R alone group. Bax, a pro-apoptotic protein expressed in ischemic myocardium of DNP+I/R group, has no significant difference compared with I/R alone group. These results suggest that the protective effects of DNP against I/R injury would be mediated, at least in part, through the increased ratio of Bcl-2 to Bax protein after ischemia-reperfusion.     

Inhibition of nucleic acids synthesis in Chlorella by 2,4-dinitrophenol a unique concentration effect on DNA synthesis

Nucleic acids and protein synthesis in synchronously growing Chlorella cells were inhibited by 2,4-dinitrophenol. RNA and protein synthesis decreased gradually from about 100% at 0.1 mM to almost 0% at 10 mM dinitrophenol. DNA synthesis was strongly inhibited at 0.5 mM but less at 1 mM concentration of the inhibitor. Beyond 1 mM the inhibitory effect increased again. A transient exposure to 0.5 and 10 mM dinitrophenol was fully reversible and cell division after the inhibition proceeded normally except for a slight delay.Abbreviation DNP 2,4-dinitrophenol     

2,4‐DINITROPHENOL PARTIALLY ALLEVIATES FERROCYANIDE‐INDUCED TOXICITY IN Drosophila melanogaster

The toxicity of potassium ferrocyanide (PFC) and protective effects of 2,4‐dinitrophenol (DNP) under PFC treatment were tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with PFC at concentrations of 1.0 mM and mixtures with DNP in concentrations of 0.50 and 1.25 mM, either alone or in combination with 1.0 mM PFC. Food supplementation with PFC decreased larvae viability or pupation height, whereas when larvae were fed by PFC and DNP combination the decrease was less pronounced. Larval exposure to PFC and mixtures of DNP and PFC lowered activities of aconitase. Larval treatment with PFC resulted in higher carbonyl protein, uric acid, and low molecular mass thiols content and higher activity of thioredoxin reductase in adult flies, while DNP in mixtures with PFC relieved these effects. Furthermore, treatment with PFC/DNP mixtures resulted in higher activities of superoxide dismutase and glutathione‐S‐transferase. It is proposed that PFC toxicity is mainly related to the cyanide and iron ions, released during its decomposition. The potential mechanisms of protective DNP effects against PFC toxicity are discussed.     

Effects of 2,4-dinitrophenol on membrane lipids of roots

Previous work has shown that the undissociated form of 2,4-dinitrophenol (DNP) increases the permeability of barley (Hordeum vulgare var. trebi) roots to ions. The present studies were undertaken to determine whether the effects of undissociated DNP were directly on membrane lipids. Relative amounts of the principal fatty acids from the lipids of barley root membranes were assayed as a function of DNP concentration, pH, and time of treatment under conditions similar to the previous studies of DNP effects on permeability. Undissociated DNP increases the proportions of palmitic and oleic acids and decreases linoleic and linolenic acids with no changes in the amounts of total fatty acids. The effects are immediate, as are the effects on permeability. Only the undissociated DNP is effective. Anionic DNP has no effect, although it is the major species taken up by the roots both at pH 5 and pH 7. DNP has no effect on respiration at either pH, indicating that undissociated DNP effects are on the membranes and not a general metabolic effect. The close parallelism between the effects of DNP on the composition of membrane lipids and on permeability suggests that the increase in permeability produced by undissociated DNP is due to a direct effect on the root membranes.     

The effect of polyene antibiotics on fractions of DNP both bound and not bound to dog kidney nuclear membranes]

Effects of amphotericine B and nistatine on nuclear membrane-bound DNP (DNPm) and free DNP (DNPf) from dog kidney are studied. Intravenous injection of amphotericine B resulted in the increase of binding of DNP particles with nuclear membrane: the content of DNA in DNPm fraction was 50-fold increased. The injection of nistatine did not affect DNP binding with nuclear membrane. Amphotericine B alone increased the protein-DNA ratio and decreased the RNA/DNA ratio in DNPf fraction. Both amphotericine B and nistatine sharply increased the protein/DNA ratio and practically did not change the RNA/DNA ratio in DNPm fraction. Amphotericine B produced considerable changes in temperature denaturation of DNA in DNPo, while nistatine produced no effect. Both antibiotics considerably changed the composition of acid soluble proteins in DNPm and DNPf, non-histone proteins in DNPf, and also they caused the changed and quantitative redistribution of separate lipid components in DNPm lipids. Polyene antibiotics are suggested to effect on animal cell nuclear structures.     

Application of x-ray analysis for the study of the orientation in DNP films]

A planar texture in dried film samples of DNP was found by X-ray study. Axes of DNA macromolecules are located in the film plane and the 77 A reflection is directed along this axis. Periodic structures corresponding to 55 and 34 A are packed parallel to the film plane. The results obtained do not contradict with a supercoil DNP model, however they all allow other possible packing models of DNP molecules, in particular, histone layers alternating with DNA layers in oriented films.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

The effects of linoleate hydroperoxide on respiration and oxidative phosphorylation of rat liver mitochondria.

Linoleate hydropepoxide, purified by silica gel chromatography and at concentrations 70-100 nmol/mg mitochondrial protein, activated state 4 respiration and Mg-ATPase activity of mitochondria to levels of 80% and 25%, respectively, of those induced by 300 microM DNP, and completely inhibited oxidative phosphorylation. These effects are the same as those caused by linoleate, but the hydroperoxide caused more rapid degeneration of the activated respiration of mitochondria than linoleate. Further addition of the hydroperoxide induced oligomycin-insensitive Mg-ATPase to a level 3 times that obtained with DNP, accompanied by clearing of the mitochondrial suspension and release of malate dehydrogenase from the matrix. The extent of the effects caused by the methyl ester of linoleate hydroperoxide was much less than by the free acid.