Prepartation of soluble chromatin and specific chromatin fractions with restriction nucleases.

By digestion of rat liver nuclei with EndoR HaeIII, EndoR EcoRI, and EndoR Bam and subsequent lysis of the nuclei approx. 90%, 40%, and 45%, respectively, of the chromatin were solubilized. The plateau values of solubilization are in agreement with a model in which the chromatin strands are crosslinked and/or attached to a supporting structure. The distribution of DNA lengths in the soluble and insoluble chromatin fractions were determined. According to digestion experiments with restriction nucleases rat liver DNA contains highly repetitive sequences, some of which are arranged in tandem repeats of 95 and 380 nucleotide pairs, respectively. With EndoR EcoRI chromatin containing the repetitive RNA was preferentially solubilized and, by subsequent sucrose gradient centrifugation, purified to about 90%. The useful properties of chromatin prepared by the specific action of restriction nucleases are discussed.     

Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI

BACKGROUND: This study investigated the relationship between chromatin compactness, which is directly related to chromatin condensation, and DAPI uptake. Materials and Methods For the structural characterization of in situ chromatin, we used fluorescence microscopy and differential scanning calorimetry on calf thymocytes. The compactness of nuclear chromatin was altered by permeabilizing native cells with NP40 detergent. A time-dependent analysis of detergent effects was performed by acquiring nuclear images at different time intervals after permeabilization. In order to compare nuclei of different sizes, we implemented a geometrical correction in the calculation of the integrated fluorescence intensity. For a quantitative evaluation of chromatin condensation we introduced two new parameters, "average chromatin packing ratio" and "average dye spatial density." RESULTS: This approach allowed us to estimate the effects of NP40 detergent at the level of in situ chromatin. Detergent effects could be modulated by changing the ionic composition of buffer. Moreover, changes of chromatin condensation induced by detergent were inversely related to modifications of nuclear volume. CONCLUSIONS: The combination of complementary information obtained by fluorescence microscopy, supported by a proper geometrical correction, and differential calorimetry allowed us to interpret the patterns of fluorescence intensities inside the nucleus in terms of chromatin structure.     

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin

To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.     

Associations of benzo[a]pyrene with rat-liver chromatin and the chromatin protein

Rat-liver nuclei were prepared in the course of time after the i.p. injection of [G-3H]benzo[a]pyrene ([3H]BP). The nuclei were lysed in the hypotonic buffer and centrifuged at 4000 X g. The recovery of the radioactivity of resulting supernatant (chromatin) was thus 91% at 24 h, 68% at 48 h and 74% at 168 h after the i.p. injection. The incorporation into nucleosome-oligomer fraction was always much more than into those of monomer and DNA-rich fractions. The preferential incorporation was found in the fraction which was enriched in non-histone chromatin proteins (NHCPs) of 49 000-55 000 daltons. This fraction steadily raised the incorporation level until at 168 h after the i.p. injection. In contrast, the levels of histone and DNA fractions were always very low. The significant incorporation was observed in the fraction which was composed of five classes of histones and low molecular-weight NHCPs (less than 30 000 daltons), despite the very low incorporation into the histone fraction. The fluorographic analysis revealed the predominant incorporations at the positions of molecular weight of 65 000, 52 000 and 44 000 daltons. In addition, the incorporations were clearly observed at the positions of 59 000, 49 000, 45 000, H1 histone, A24 protein and another one. On the other hand, these fractions were, at the final preparation steps, subjected to either dialysis of SDS-phenol treatment and/or acetone precipitation. The total recovery of radioactivity was thus 21% at 24 h, 32% at 48 h and 52% at 168 h after the i.p. injection. These results suggest that the chromatin contains considerable amounts of water-soluble, phenol and/or acetone-soluble BP-conjugates in the early period after the i.p. injection.     

Interaction of nogalamycin with chromatin

Number of available nogalamycin binding sites in Sarcoma-180 chromatin is less than that present in Sarcoma-180 DNA. Gradual removal of proteins from chromatin by salt leads to increase in available drug binding sites, without appreciable alteration in binding affinity. Histones restrict the accessibility of nogalamycin to chromosomal DNA, whereas high mobility group (HMG) proteins have no effect. Association of histone H1 with chromosomal DNA has a more marked inhibitory effect on nogalamycin binding than other types of histones. Chromosomal protein induced conformational change in DNA appears to be the main factor in determining the availability of strong binding sites.     

Interaction of mithramycin with chromatin

Mithramycin (MTR) is an anti-cancer antibiotic that blocks the macromolecular biosynthesis via reversible interaction with DNA template in the presence of bivalent metal ion such as Mg2+. In absence of DNA, mithramycin forms two types of complexes with Mg2+, complex I (with 1:1 stoichiometry in terms of MTR: Mg2+) and complex II (with 1:2 stoichiometry in terms of MTR: Mg2+). In an eukaryotic system, the drug would interact with chromatin, a protein-DNA complex. We have employed the spectroscopic techniques such as absorption and fluorescence to study the interaction of MTR: Mg2+ complexes with rat liver chromatin. In this report, we have shown that the two types of ligands have different binding potentials with the same chromatin. This supports our proposition that complexes I and II, are different molecular species. We have also shown that the histone protein(s) reduce the binding potential and the number of available sites for both ligands.     

Ultrastructure of buffalo,Syncerus caffer,platelets: Comparison with bovine and human platelets

Platelets are found in the blood of all mammals and serve the same basic hemostatic functions in all. Species differences in the morphology and activities of platelets in human and domesticated animals have been observed, but there is little published information regarding the blood pictures of wild animals. In this study, the ultrastructure of buffalo platelets was compared with that of bovine and human platelets. Buffalo platelets were found to be smaller than human platelets and intracellularly had larger α-granules, possessed distinctive dense granules and a more distinct microtubuli system, but they lacked the open canalicular system observed in the human type. This morphology is similar to that of cattle platelets. © 1996 Wiley-Liss, Inc.     

The phospholipid and fatty acid compositions of seal platelets: a comparison with human platelets

1. Platelet phospholipid compositions were studied in four species of phocid seals consuming herring or herring and shrimp and in human subjects consuming a normal mixed diet. 2. There were no major differences in platelet phospholipid, cholesterol and protein levels between different species of seal nor between seals and human subjects, nor in the relative abundance of the individual types of phospholipid. 3. The seal platelet phospholipids (phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were greatly enriched in the omega 3 fatty acid, eicosapentaenoic acid (EPA) and depressed in arachidonic acid (AA) relative to the corresponding human platelet phospholipids. 4. Much less accumulation of EPA in phosphatidylserine (PS) and phosphatidylinositol (PI) was found. 5. The EPA contents of the individual seal platelet phospholipids exhibited considerable differences (including EPA discrimination from PI) but gave patterns which were generally similar to those reported for human volunteers consuming fish/fish oils enriched in EPA. 6. These results suggest that the seal platelet may be a useful model for studying the metabolism and function of the omega 3 fatty acids, such as EPA, in relation to platelet reactivity, phospholipid turnover and the formation of AA- and EPA-derived eicosanoids.     

Intrauterine transfusion with red cells and platelets.

Having direct access to the fetoplacental circulation by ultrasound-directed needle puncture has led to therapeutic interventions for fetal anemia and thrombocytopenia. Most cases of red cell alloimmunization associated with fetal anemia are caused by the antibody to the D red cell antigen. The intravascular transfusion of red cells to a hydropic fetus in such cases has notably improved survival. Nonimmune hydrops fetalis due to maternal parvovirus infection has also been treated successfully with the intravascular transfusion of red cells, whereas fetomaternal hemorrhage has not proved amenable to such therapy. Sensitization to the PLA-1 platelet antigen is the most common cause of fetal thrombocytopenia in maternal platelet alloimmunization. Fetal platelet transfusions have not proved to be a practical therapeutic modality for this disorder owing to the short half-life of the platelets. Platelets transfusions to the fetus just before delivery may avert the need for cesarean section in cases of severe thrombocytopenia.