Deoxyribonucleoprotein Research Articles

The effect of the new combined cream on the protein synthetic activity of cells on the model of the thermal skin damage in rats

Aim. To study the mechanisms of the reparative action of a new combined cream on the model of thermal burn injury in rats.Materials and methods. On the model of thermal burn in rats the reparative properties of the new combined cream under the conditional name “Dermalipoin” containing α-lipoic acid, urea, olive oil, tea tree oil, PEG-400 were studied. To determine the level of the protein synthetic activity of cells (epitheliocytes of the stratified squamous epithelium in the epidermis and cells of the fibroblast cell in the edges of healing of thermal damage) the content of ribonucleoproteins (RNP) in the cytoplasm and deoxyribonucleoproteins (DNP) in the nuclei of the cells was assessed by the cytometric method.Results and discussion. On the model of thermal burn in rats “Dermalipoin” cream showed a more active effect on reparative processes compared to the reference drugs – methyluracil ointment and “Titriol” gel. It indicates a significant increase in the protein synthetic function of epitheliocytes of the stratified squamous epithelium in the epidermis and cells of the fibroblastic series at the healing edges. The reparative activity of the cream was, first of all, provided by the presence of thiol (sulfhydryl) groups in the molecule of lipoic acid, giving it the properties of an antioxidant. The antioxidant effect of lipoic acid promotes more efficient DNA molecule reparation after damage as a result of the oxidative stress.Conclusions. “Dermalipoin” cream shows a marked reparative effect due to the increase of the protein synthetic activity and acceleration of reparation of DNA molecules of epithelial cells after damage; as a result, the process of healing burns accelerates.

Bovine sperm chromatin is not protected from the effects of ultrasmall gold nanoparticles

The response of ejaculated bovine spermatozoa to gold nanoparticles was studied by the standard method of nuclear chromatin decondensation in vitro. After the treatment of semen samples with a hydrosol containing gold nanoparticles with an average diameter of 3.0 nm and a concentration of 1 x 10(15) particles/mL, the ability of sperm nuclei to decondense in the presence of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) dramatically changed compared to the control. The frequencies of gametes with nondecondensed ("intact"), partially decondensed, and completely decondensed nuclei correlated as 40 : 32 : 28% and 0 : 36 : 64% in the experiment and the control, respectively. Moreover, the appearance of a sufficiently large number of gametes with destructed and almost completely destroyed nuclei was noticed in the spermatozoa treated with gold nanoparticles. This article suggests the putative mechanisms of action of ultrasmall gold nanoparticles on the structural and functional integrity of the deoxyribonucleoprotein (DNP) complex of mature male gametes.

Ultrastructural analysis of Balbiani ring genes of Chironomus pallidivittatus in different states of Balbiani ring activity

Patterns of gene activity on Balbiani ring (BR) transcription units (TUs) of polytene chromosome IV of Chironomus pallidivittatus larvae were ultrastructurally characterized by electron microscopy. In Miller chromatin speads, the 9 μm long TUs of Balbiani ring 1 and 2 (BR1 and BR2), respectively, were unambiguously identified on the basis of the size and structure of their stalked ribonucleoprotein (RNP) granules. Because of the mild spreading procedure the morphology of these 50 nm BR RNP particles remained quite reminiscent of their in situ configuration. BR TUs were found as clusters of sister copies, looking almost homogeneous in structure and organization, a number of them appearing as loops. TUs of normal and hyperactive BRs (the latter resulting from stimulation of the larvae with pilocarpine) were compared. The patterns of transcriptional activity of BR TUs were rather similar but not absolutely identical on all homologous sister chromatin strands. In normal as well as in hyperactive BRs, the great majority of TUs were highly transcriptionally active, i.e. densely packed with RNP fibres all along the deoxyribonucleoprotein (DNP) axis. Mean RNP fibre frequency of BR TUs of normal larvae was 30/μm while hyperactive BRs showed a fibre frequency of 31μm. The average number of RNP fibres per BR TU is in the range of 200 to 300. Assuming a transcription time of 20 min per BR mRNA molecule, the number of RNA polymerase molecules starting transcription of the 37 kb gene could be estimated to be 10 to 15 per minute. A number of BR genes were only partially active, exhibiting short, middle or long arrays of RNP fibres. Such arrays occurred in any segment of the genes showing normal RNP fibre density and a nucleosomal configuration in the inactive part of the gene. The variations in RNP fibre frequencies suggest that transcription can be modulated at the level of the individual BR gene. However, the large increase in the rate of BR mRNA synthesis upon pilocarpine stimulation may mainly be due to activation of hitherto inactive sister copies of the BR gene.

Linker histone-DNA complexes: enhanced stability in the presence of aluminum lactate and implications for Alzheimer's disease

The binding of human brain linker histone proteins to a radiolabelled human Alu repetitive element was examined by mobility shift assay.. Analysis of the complexes formed from protein extracts of whole neocortical nuclei, under physiological conditions in vitro revealed that linker histone H1° has the highest affinity for the Alu DNA sequence. The linker histone-DNA complexes assembled in the presence of aluminum lactate were more resistant to sodium chloride-induced dissociation than those formed in the presence of sodium lactate. The enhanced stability of deoxyribonucleoprotein (DNP) complexes in the presence of the aluminum cation may be of significance in neurodegenerative conditions such as Alzheimer's disease where aluminum preferentially associates with DNA containing structures of the nucleus.

Differential decondensation of mitotic chromosomes during hypotonic treatment of living cells as a possible cause of G-banding: an ultrastructural study.

The chromosomal ultrastructure of Chinese hamster cells treated with 0.075 M KCl - a solution ordinarily used for making preparations of spread chromosomes - was studied. The hypotonic treatment was shown to result in differential decondensation of chromosomes which consists in the uneven distribution of deoxyribonucleoprotein (DNP) fibrils along chromatids. Fixation of cells with methanol acetic acid causes an abrupt restructuring of chromosomes. However, the DNP preserves its uneven distribution along chromatids. As seen on ultra-thin sections of marker nucleolus organizer chromosomes, the densely packed regions may correspond to G-bands detected in the selfsame chromosomes by standard methods of differential staining. The results suggest that the capacity of chromosomes for differential staining is based on the different resistance of G- and R-bands to the decondensing action of hypotonic solutions on living cells.

In vitro effect of aflatoxin B 1 on the transcriptional activity of DNA template, chromatin and soluble DNA-dependent RNA polymerases in buffalo liver

The effect of aflatoxin B 1 on the DNA template and DNA-dependent RNA polymerases in buffalo liver was studied. Aflatoxin B 1 inhibited both Mg 2+- and Mn 2+-activated RNA polymerases in a dosedependent manner. At 10 μg the inhibition of both enzymes was almost complete. The inhibitory effect on the solubilized enzymes was higher than the chromatin-bound, suggesting a direct effect at the enzyme level. On the other hand, incubating DNA or deoxyribonucleoprotein (DNP) with 2 μg aflatoxin reduces its transcriptional capacity with a greater effect on the Mg 2+-activated RNA polymerase than the Mn 2+-activated enzyme. These results suggest that aflatoxin B 1 inhibits in vitro transcription in buffalo liver at both enzyme and template levels.

DNA methylation and RNA transcriptional activity in amphibian lampbrush chromosomes

Lampbrush chromosomes simultaneously exhibit two types of chromatin: the actively transcribed chromatin of lateral loops and the transcriptionally inactive chromatin of the chromosome axis. In situ localization of 5-methylcytosine in lampbrush chromosomes of the amphibian Pleurodeles waltlii, using specific antibodies to 5-methylcytosine, allowed us to demonstrate an inverse relationship between RNA transcriptional activity and the level of DNA methylation by light and electron microscopy using immunofluorescence and immunogold staining. The 5-methylcytosine was exclusively located in transcriptionally inactive chromatin in the chromosome axis and in the untranscribed spacers of some loops when the deoxyribonucleoprotein (DNP) axis was not wholly transcribed. In the vast majority of loops where the DNP axis was wholly transcribed, no methylated cytosine residues were ever detected. These results indicate a close relationship between the functional state of chromatin and the level of DNA methylation and suggest that DNA methylation is closely linked to gene control mechanisms in lampbrush chromosomes, i.e. at a definite time during oogenesis.

Three-dimensional electron microscopy of the internal nucleolus-associated chromatin and of the nucleolar vacuoles during early germination of Sinapis alba.

Spatial relationships between the internal nucleolus-associated chromatin (NAC) and the numerous nucleolar vacuoles that appear during early germination have been studied in nucleoli of quiescent (non-germinated) and early germinating embryos of Sinapis using serial sections. In quiescent non-vacuolated nucleoli, the transcriptionally inactive internal NAC is a short strand about 900 nm thick that in cross-section appears as heterogeneous fibrillar centres (FCs). At 4 and 6 h after germination one or several large networks of interconnected nucleolar vacuoles develop around the dispersing internal NAC. Clumps of dense chromatin are still present within the nucleolar vacuoles and are probably unfolding into deoxyribonucleoprotein (DNP) fibres (about 110 nm thick), which rapidly intrude within the nucleolar body and form thin chromatin threads. At 24 h after germination the internal NAC is more dispersed and forms, for its greatest part, a long thread (about 240 nm in diameter) wrapped up with a few dense fibrillar component, the whole forming the first outline of a nucleolonema. In cross-section most of the internal NAC appears as homogeneous FCs but short portions remain more condensed and appear as heterogeneous FCs always associated with a nucleolar vacuole. From 48 h the internal NAC is a longer thinner strand (about 160 nm in diameter), probably continuous and surrounded entirely by a homogeneous muff of dense fibrillar component, the whole forming a typical nucleolonema (about 950 nm thick) meandering throughout the nucleolus. Small amounts of the internal NAC still remain undispersed in the form of heterogeneous FCs associated with a nucleolar vacuole. The repeated association of nucleolar vacuoles and dispersing internal NAC suggests that they could play a role in chromatin dispersion and, or, activation by creating a favourable microenvironment.

Histamine receptor-mediated changes in nuclear chromatin of cultured endothelial and smooth muscle cells.

Histamine, acting via membrane receptors, modulates a number of potentially important processes within the vascular wall. Histamine receptor activation affects cellular activities such as contractility, movement, growth, and metabolism. The present study entails a characterization of endothelial (EC) and smooth muscle cell (SMC) histamine receptors with respect to cytochemical indices of DNA content and chromatin dispersion.Both EC and SMC exhibited functionally active H1 and H2 receptors. Activation of either receptor type resulted in a substantial ploidy shift in EC but not in SMC cultures. The physico-chemical state of chromatin as assessed by Feulgen reactivity was altered in both cell types. H2 receptor activation elevated Feulgen reactivity in 2C nuclei, while in 4C nuclei both H1 and H2 receptors were active in this regard. Elevation of Feulgen reactivity of EC and SMC nuclear chromatin in response to histamine receptor agonists presumably represents a “chromatin activation reaction” reflecting alterations in the conformation of the deoxyribonucleoprotein (DNP) complex and accompanying changes in template activity. Disparity between agonist effects on 2C and 4C populations may reflect the cell cycle regulation of histamine receptors.

Elementary structures in polytene chromosomes of Drosophila melanogaster

Whole-mounted polytene chromosomes were isolated from nuclei by microdissection in 60% acetic acid and analyzed by electron microscopy. Elementary chromosome fibers in the interchromomeric regions and individual chromomeres can be distinguished in polytene chromosomes at low levels of polyteny (26–27 chromatids). Elementary fibers in the interbands are oriented parallel to the axis of the polytene chromosome. Their number roughly corresponds to the expected level of polyteny. These fibers have an irregular beaded structure, 100–300 A in diameter, and there is no apparent lateral association between them in the interchromomeric regions. Most bands, in contrast, form continuous structures crossing the entire width of the chromosome. Polytene chromosomes isolated in 2% or 10% acetic acid can be reversibly dispersed in a solution for chromatin spreading. The spread chromosomes consist of long uniform deoxyribonucleoprotein (DNP) fibers with a nucleosome structure. This supports the notion that continuous DNA molecules extend through the entire length of a polytene chromosome and that the nucleosome structure exists both in bands and interbands. Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polytene chromosome at higher levels of polyteny develops.

Nuclear chromatin changes in hepatocytes of soman treated rats

Cytochemical and morphometric indices of DNA metabolism and nuclear transcriptional-translational capabilities were used to monitor liver responses in Sprague-Dawley × Wistar rats subcutaneously injected with 0.9 LD 50 soman, a highly toxic acetylcholinesterase-inhibiting organophosphate. Thirty minutes post-soman the following were evident: (1) increased polyploidization; (2) expansion of the nuclear envelope; and (3) elevated deoxyribonucleoprotein (DNP) complex lability to acid hydrolysis. Nuclear changes were accompanied by cytoplasmic correlates of impaired hepatocyte function, i.e., vacuolation and depressed basophilia. The overall data suggest that with acute soman toxication there are concomitant adaptive and maladaptive responses occurring within liver parenchymal elements. It is postulated that nuclear changes represent an early proliferative response and DNP activation to enhance detoxification capabilities, whereas cytoplasmic changes reflect the disruption of normal function and hepatotoxic injury.

Changes in chromatin structure at the replication fork. DNase I and trypsin-micrococcal nuclease effects on approximately 300- and 150-base pair nascent DNAs.

DNase I, trypsin, and micrococcal nuclease are used to further probe the structure of nascent deoxyribonucleoprotein (DNP) fractions which appear after in vivo 20-s pulse labeling of sea urchin embryos with [3H]thymidine. We present evidence that the large nascent DNP which protects the approximately 300-base pair large nascent DNA consists of at least one nucleosome core. This is based on fractionation in denaturing polyacrylamide gels of DNA extracted from large nascent DNP fractions of a micrococcal nuclease + DNase I digest of nuclei. The data also suggest the existence of a DNase I-hypersensitive site(s) within the large nascent DNP; this is consistent with the hypothesis that the latter consists of closely packed dinucleosome cores. Histone H1 and non-histone proteins do not account for the previously reported unusual hyperresistance of the large nascent DNA against micrococcal nuclease. The protection offered this approximately 300-base pair nascent DNA was not eliminated by an 0.2-microgram/ml trypsin pretreatment which removes the above proteins from the chromatin. However, 5-10 micrograms/ml of trypsin, which remove a portion of the NH2 termini of the four core histones of nucleosomes, eliminate the hyperresistance of the large nascent DNA to subsequent micrococcal nuclease digestion, while nascent and bulk monomer DNAs remain unaffected. This indicates histone-histone and/or histone-DNA interactions within the large nascent DNP which differ from those of nascent and bulk mononucleosome cores.

Proteins and functional properties of chromatin fractions from nuclei of normal liver and experimental hepatomas

In the modern view nuclear skeletal structures (the nuclear matrix) play an extremely important role in the structural organization of chromatin [12]. There is evidence that differences in the degree of compactness of active and inactive chromatin in the nucleus may be due not only to the specific nature of the protein composition, but also to local differences in interconnection between fibrils of deoxyribonucleoprotein (DNP) with protein fibrils of the matrix: The ~topography of diffuse and condensed regions of the nucleus is preserved after removal of both DNA and histone HI from it [i0]. The possibility of fractionating chromatin, based not on choice of conditions of selective degradation of the DNA of active chromatin, but on differences in solubility of DNP fragments having points of attachment to the matrix and not bound with it, is accordingly interesting. This possibility arises because of the fact that after nuclease treatment of a certain intensity (when the mean size of DNA fragments between neighboring double-stranded breaks is definitely less than the size of DNA of a single loop) under conditions of maximal solubility of DNP in the nucleus, chromatin enriched with points of attachment to the nuclear matrix remains in the nucleus [8].

Relationship between immune response to hydralazine and to deoxyribonuclease in patients receiving hydralazine.

In a prospective study of 21 hypertensive patients receiving hydralazine for 1 year, we found a close relationship between development of antibodies to deoxyribonucleoprotein (DNP) and to hydralazine but no evidence for cross-reactivity between antibodies to these two antigens. Of 8 patients who developed increased levels of antiDNP, 7 also developed antibodies to hydralazine. Inhibition of the reaction between DNP and antiDNP as measured by radioimmunoassay in 3 patients with hydralazine lupus could not be achieved with large amounts of hydralazine. However, antibodies to DNP produced in guinea pigs immunized with hydralazine conjugates could be inhibited with hydralazine in accordance with previous studies by others on rabbits. In the human, antibodies to DNP which develop during hydralazine administration are not due to cross-reactive antibodies nor do they appear as a result of immune response to an in vivo hydralazine DNP conjugate.

Prospective study of immune response to hydralazine and development of antideoxyribonucleoprotein in patients receiving hydralazine

To examine the relationship between the immune responses to hydralazine, a drug known to induce systemic lupus erythematosus, and to deoxyribonucleoprotein (DNP) we followed prospectively 21 hypertensive patients treated with hydralazine for the first time. Within one year, antibodies to hydralazine developed in 16 of these patients and anti-DNP in seven of these. In one patient whose serum had a positive antinuclear antibody test prior to treatment, a mild hydralazine systemic lupus erythematosus syndrome developed preceded by rises in the levels of both anti-hydralazine and anti-DNP. Studies by radioimmunoassay on serums of three additional patients, not followed in this study but known to have hydralazine-induced systemic lupus erythematosus, revealed no evidence for either (1) cross-reactivity between anti-DNP and anti-hydralazine or (2) antibodies specific for a hydralazine-DNP complex. In some way, perhaps related to the mechanism by which carrier molecules enhance the immunogenuity of haptens, hydralazine increases the antigenicity of DNP. This effect depends on the development of immunity to hydralazine as well.

Non-nucleolar transcription complexes of rat liver as revealed by spreading isolated nuclei.

Miller's technique was applied to isolated nuclei of rat liver. Both the usual nucleolar and non-nucleolar transcription complexes were visualized. In addition, an unusual type of putative non-ribosomal transcription unit was revealed. It was charcaterized by a high density of the lateral ribonucleoprotein (RNP) fibrils. Although these particular units exhibited a regular increase of fibril lengths, the length of the transcript-covered deoxyribonucleoprotein (DNP) fibres and the morphological aspect of the RNP fibrils distinguished them from the nucleolar 'Christmas-tree'-like figures. The linear and granular configuration of the transcripts and the absence of terminal knobs made them similar to non-nucleolar nascent RNP fibrils.

Relationship between ultrastructure and function of hepatocyte chromatin: A study with adrenalectomized rats after cortisol administration

Using either the routine staining procedure or the Feulgen-like Gautier's method the condensed pattern of hepatocyte chromatin in adrenalectomized rats appeared unchanged 1 hr after cortisol administration, whereas at 4 hr and especially at 8 hr it looked more dispersed, in parallel with an increased synthesis of RNA and nuclear nonhistone proteins (NHP). The quantity of [3H]cortisol bound to deoxyribonucleoproteins (DNP) was the same at 1 and 4 hr after hormone treatment and it was unaffected by amanitin, which induced a chromatin condensation. Eight hours after cortisol injection almost all the DNA structures, revealed by the Gautier technique, were in the unraveled form; concurrently, a large quantity of perichromatin fibrils was visualized with the Bernhard method for ribonucleoproteins (RNP). Amanitin poisoning of 8-hr stimulated rats induced, 1 hr after treatment, a light condensation of the DNA structures and a strong condensation after 2 hr; the perichromatin fibrils were lightly reduced 1 hr after toxin injection and had almost disappeared 2 hr thereafter. Amanitin reduced RNA synthesis to about 25% of control value both 1 and 2 hr after poisoning. No modification of NHP synthesis was observed after toxin treatment. Our results demonstrating a strict relationship only between the chromatin pattern and the quantity of perichromatin fibrils, morphological expression of heterogeneous nuclear (hn) RNA which has been synthesized (J. P. Bachellerie, E. Puvion, and J. P. Zalta,Eur. J. Biochem. 58, 327, 1975; S. Fakan, E. Puvion, and G. Spohr,Exp. Cell Res. 99, 155, 1976), suggest that the chromatin ultrastructural changes induced by cortisol are a consequence and not a cause of gene transcriptional activity.

Thin deoxyribonucleoprotein fibers revealed by gamma-irradiation.

Deoxyribonucleoprotein (DNP) extracted from calf thymus is gamma-irradiated at a dose of 100 krad. Freeze-replicas of the irradiated DNP are made, and examined by means of electron microscopy. The non-irradiated DNP fibers with a mean diameter of 183Å are formed by coiling of finer fibers, 23Å in mean diameter. In contrast, the irradiated DNP fibers appear to be thin fibers, being 49Å in mean diameter.

The protein synthesis dependent on the RNA directed by irradiated calf thymus DNA and deoxyribonucleoprotein.

The translational ability of RNA directed by irradiated calf thymus DNA or deoxyribonucleoprotein (DNP) in vitro was investigated. Both the template activity of calf thymus DNA for RNA synthesis and the incorporation of amino acids into acid insoluble materials in vitro markedly decreased by irradiation with 20 increasing dose. The activity of calf thymus DNP, however, increased with irradiation at a dose lower than krads. The incorporation of amino acids also increased with the same dose. It was shown that the translation dependent on the RNA directed by irradiated templates was qualitatively different from that of unirradiated templates.

On the structure of cellular and viral chromatin.

Some of the recent experimental data obtained in our laboratory are briefly reviewed. 1. A mild staphylococcal nuclease digestion of either chromatin or nuclei from mouse Ehrlich tumour cells results in chromatin subunits (mononucleosomes) of three discrete kinds. The smallest mononucleosome (MN 1 contains all histones except H1 and a DNA fragment 140 base pairs long. The intermediate mononucleosome (MN 2 ) contains all five histones and a DNA fragment 170 base pairs long. The third mononucleosome (MN 3 ) also contains all five histones, but its associated DNA is longer and somewhat heterogeneous in size (180-200 base pairs). Most of the MN 3 particles are rapidly converted to mononucleosomes MN 2 and MN 1 by nuclease digestion. However, there exists a relatively nuclease-resistant subpopulation of the MN 3 mononucleosomes. These 200 base-pair MN 3 particles contain not only histones but also non-histone proteins and are significantly more resistant to nuclease then even the smaller mononucleosomes MN 1 and MN 2 . 2. Nuclease digestion of hen erythrocyte nuclei or chromatin, in which histone H1 is partially replaced by histone H5 produces the mononucleosomes MN 1 and two electrophoretically resolvable kinds of MN 2 mononucleosomes, one containing histone H1 and the other one histone H5. A relatively nuclease-resistant subset of the mononucleosomes MN 3 is preferentially accumulated at later stages of the digestion. 3. Although pancreatic DNase (DNase I) and spleen acid DNase (DNase II) attack the DNA in chromatin in a manner different from that of staphylococcal nuclease, the deoxyribonucleoprotein (DNP) products of digestion are similar for all three enzymes under identical solvent conditions, as revealed by gel electrophoresis of the DNP at low ionic strength. 4. There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (i.e. particles smaller than the mononucleosomes). In particular, the subnucleosome SN 1 is a set of naked double-stranded DNA fragments ca. 20 base pairs long. Subnucleosome SN 2 is a complex of a specific highly basic non-histone protein and a DNA fragment ca. 27 base pairs long. Subnucleosomes SN 7 and SN 8 each contain all of the histones except H1 and DNA fragments ca. 100 and 120 base pairs long, respectively. 5. Nuclease digestion of isolated mono- and dinucleosomes does not produce all of the subnucleosomes. These and related findings indicate that the cleavages required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- or dinucleosomes. Nuclease digestion of isolated minichromosomes of Simian virus 40 (SV40) (which contain all five histones including H1) produces mononucleosomes MN 1 and MN 2 but does not produce some of the subnucleosomes or the relatively nuclease-resistant subset of the MN 3 mononucleosomes. 6. The rate of sedimentation of the SV40 minichromosomes ( ca. 60 S ) under ‘physiological’ ionic conditions ( μ ≈ 0.15) is about two times higher than that in a low ionic strength buffer ( μ ≈ 0.005). Occurrence of the compact state of the minichromosome critically depends upon the presence of histone H1 and can be irreversibly fixed by treatment with formaldehyde.