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.     

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.     

Occurrence of the methylisobutylxanthine-stimulated cyclic GMP binding protein in various rat tissues

A new type of cGMP binding protein, the activity of which is characteristically stimulated by methylisobutylxanthine, has been previously discovered in rat lung and platelets (Hamet, P. and Coquil, J.F. (1978) J. Cyclic Nucleotide Res. 4, 281-290). In the present study, we demonstrate the occurrence of this protein in soluble extracts of a variety of rat tissues fractionated by a DEAE-Sepharose chromatography. In several tissues (spleen, lung and brain) the binding activity of this protein was of the same order of magnitude as that of the cGMP-dependent protein kinase.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.     

Binding of HMG14 non-histone protein to histones H2A, H2B, H1 and DNA in reconstituted chromatin

The interaction between calf thymus HMG14 and rat liver chromatin components has been studied via reconstitution and chemical cross-linking. Selective labeling of HMG14 with photoactivable reversible heterobifunctional reagents has allowed a clear identification of the histones interacting with it (histones H2A, H2B and H1). These results are not dependent on whether the chromatin samples used were bulk chromatin, mononucleosomes, or core particles (for H2A and H2B). In addition to histone proteins, DNA also seems to be involved in HMG14 attachment to nucleosome.     

Copper(II)- and iron(II)-complexes of methyl 2-(2-aminoethyl)-aminomethyl-pyridine-6-carboxyl-histidinate (AMPHIS), a peptide mimicking the metal-chelating moiety of bleomycin. An ESR investigation

Methyl 2-(2-aminoethyl)-aminomethyl-pyridine-6-carboxyl-histidinate (AMPHIS), a synthetic analogue of the chelating part of bleomycin (BLM), has been studied for its metal binding properties. Electron spin resonance parameters of AMPHIS-Cu(II) and BLM-Cu(II) have been found to be closely similar likewise spectra of oxygen radicals spin-adducts induced by AMPHIS-Fe(II)-O2 and BLM-Fe(II)-O2 systems. Thus, AMPHIS could constitute a very useful tool for the study of BLM mode of action.