Specific recognition by the tripeptide lysyl-tryptophyl-alpha-lysine of structural damage induced in DNA by platinum derivatives

The antitumoral derivative cisPt binds to DNA, as do its inactive analogs, trans- and dienPt. Structural damage introduced into DNA after reaction with the Pt derivatives were probed by using the peptide LysTrpLys. This peptide was used for its preferential binding to single-stranded structures (Brun, F., Toulmé, J.J. and Hélène, C. (1975) Biochemistry 14, 558-563). Phosphorescence lifetime measurements show that the Pt-induced heavy atom effects are quite similar in the three peptide-DNA-Pt complexes whatever the nature of the Pt derivative used. In contrast, fluorescence quenching strongly depends on the nature of the Pt derivatives. This quenching was therefore attributed to the stacking interactions engaged by the tryptophan residue with nucleic acid bases. A comparison of fluorescence quenching data for native and modified DNAs demonstrates that modification by dienPt has no effect on stacking interactions and that high levels of modifications by trans Pt are required to observe a change in stacking efficiency. In contrast modification by cis Pt induces the formation of strong stacking sites. The results strongly suggest the existence of locally opened regions in DNA modified by cis Pt.     

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.     

Structural transitions of chromatin at low salt concentrations: a flow linear dichroism study

We have studied the structure of nuclease-solubilized chromatin from Ehrlich ascites cells by flow linear dichroism (LD) using the anisotropic absorption of the DNA bases and of two intercalated dyes, ethidium bromide and methylene blue. It is confirmed that intercalation occurs preferentially in the linker part of the chromatin fiber, at binding ratios (dye/base) below 0.020. Using this information, we determined the orientation of the linker in relation to the average DNA organization in chromatin. The LD measurements indicate that the conformation of chromatin is considerably changed in the ionic strength interval 0.1-10 mM NaCl: with increasing salt concentration, the LD of the intrinsic DNA base absorption changes signs, from negative to positive, at approximately 2.5 mM NaCl. The LD of the intercalated dyes also changes signs, however, at a somewhat higher salt concentration. The results are analyzed in terms of possible allowed combinations of tilt angles of nucleosomes and pitch or tilt angles of linker DNA sections relative to the fiber axis, at different salt concentrations in the interval 0.1-10 mM NaCl. Two models for the salt-induced structural change of chromatin are discussed.     

Separation and kinetic properties of the molecular forms of chicken liver cytoplasmic aspartate aminotransferase

A method is proposed for the separation of the five molecular forms, alpha, beta, gamma, delta and epsilon, of chicken liver cytoplasmic aspartate aminotransferase free from lactate dehydrogenase activity. These molecular forms varied in isoelectric point, but no differences were observed either in their Michaelis constants or in the degree of their inhibition by excess of 2-oxoglutarate or L-aspartate.     

Oligonucleotides covalently linked to intercalating agents. Influence of positively charged substituents on binding to complementary sequences

A pentamethylene chain was used to covalently link the 3'-phosphate of oligothymidylates to the 9-amino group of an acridine derivative. Positively charged substituents were further attached to the 3'-phosphate group to form 3'-phosphotriesters. These molecules form specific complexes with poly(rA) which involve the formation of a number of A X T base pairs equal to that of thymines in the oligonucleotide. Absorption changes induced in the acridine absorption bands are similar to those expected upon intercalation of the acridine dye between A X T base pairs. The acridine covalently linked to the 3'-phosphate strongly stabilizes the complexes formed with poly(rA) as compared with the corresponding unsubstituted oligodeoxynucleotide. The presence of a positively charged substituent on the 3'-phosphate together with the acridine dye further enhances the interaction. The effect of salt concentration on complex stability depends on the number of negatively charged phosphate groups of the oligodeoxynucleotide and on the nature of the substituents borne by the 3'-phosphate group. When the oligothymidylate is substituted by an acridine dye, the stability of the poly(rA) complexes increases when salt concentration increases. If an additional positively charged substituent is present on the 3'-phosphate group, stability decreases when salt concentration increases for the shortest oligonucleotide (trimer) and increases with longer oligonucleotides. Thermodynamic parameters have been calculated from the concentration dependence of melting temperatures.     

Monoclonal antibodies against histone H5. Epitope mapping and binding to chromatin

Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed.     

The composition of liver chromatin proteins from embryos, chicks and adult chickens

The chemical composition of chromatin from the livers of 12-, 15- and 19-day-old embryos, of 1-day-old chicks and of adult chickens was analysed. The process of embryonic development is accompanied by an increase in non-histone chromatin proteins and chromatin RNA, as well as in the phosphorus content of chromatin phosphoproteins. The amount of these components decreases in the livers of 1-day-old chicks and adults. Two-dimensional polyacrylamide gel electrophoresis of acid-soluble chromatin proteins showed an increase in the amount of the H1 histone in 19-day-old embryos and adult chickens. Non-histone proteins of embryo liver chromatin showed a high content of the fraction of Mr of about 40 000; this was not the case for adult chickens. The non-histone protein fraction of Mr of about 120 000, characteristic of adult chicken liver proteins, was not found in the livers of 12- and 15-day-old embryos. Non-histone chromatin proteins isolated from the livers of animals of different age exhibited also quantitative differences.     

Reciprocal syndromes caused by deficiency duplication resulting from maternal t(10;18)(p12;q22) translocation

The detection of a familial translocation, t(10;18)(p12;q22), has made possible the observation in type and countertype of two related persons with opposite chromosomal imbalance: trisomy 18q22----18qter with monosomy 10p12----10pter in one of the two and monosomy 18q22----10pter in the other. In each case the abnormalities attributable to monosomy overrule those attributable to monosomy overrule those attributable to the associated trisomy.     

Change in the physical state of platelet plasma membranes upon ionophore A23187 activation. A fluorescence polarization study

Human platelets were isolated and fluorescence-labelled by 1,6-diphenylhexatriene. Diphenylhexatriene was essentially localized in the plasma membrane, as indicated by trinitrobenzenesulfonate-quenching experiments. A decrease of the fluorescence polarization of diphenylhexatriene was observed upon ionophore A23187 addition in the absence of aggregation. 0.3 microM ionophore allowed to reach the maximum rate of the decrease of fluorescence polarization; it also maximally stimulated the light transmission change, the serotonin release and the thromboxane B2 synthesis. The amplitude of the fluorescence polarization decrease was maximum at platelet concentrations between 4 X 10(7) and 7 X 10(7)/ml. The presence of Ca2+ in the medium increased the rate constant of the polarization change. Chlorpromazine (60 microM) completely inhibited this transition, but at 30 microM its inhibitory effect was reversed by Ca2+. The membrane events implied in platelet activation very likely lead to fluidization of the plasma membrane, perhaps by its fusion with the membranes of internal granules which are relatively depleted of cholesterol. Ca2+ plays a central role in the triggering of the observed effects at the membrane level.