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We have previously described ECIF-1, a DNA-binding factor present in nuclear extracts of murine embryonal carcinoma cells which specifically recognizes a region within the human beta-interferon promoter. We show that the promoter region located between -112 and -93 is sufficient for this binding activity, which is not due to binding of interferon-regulatory factor 1 or 2. By mutational analysis of the ECIF-1 site, it was determined that the central nucleotides which are critical for binding contain an octameric motif: ATTTACAT. The binding activity of ECIF-1 with its cognate site within the beta-interferon promoter decreases upon differentiation concurrently with the onset of interferon inducibility. Furthermore, by using an in vitro transcription assay with deleted promoter elements of the beta-interferon gene, we show that undifferentiated P19 nuclear extracts contain a repressing activity which depends on the presence of the ECIF-1 site. This repression is not observed using nuclear extracts from differentiated P19 cells. Comparison of the binding activity of this octamer site with others previously shown to be active in embryonal carcinoma cells reveals similarities and differences in the spectrum of proteins binding there.

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The structure of ApoD and its sites of synthesis have been discovered. These characteristics differ from those of the other apolipoproteins. The role of ApoD in the plasma lipoprotein system remains to be discovered, but the recent, rapid increase in our knowledge of this protein suggests that it plays an important role in the homeostasis or housekeeping of probably all organs. One of its functions is likely to be the transport of a hydrophobic ligand (a lipid) in a one-to-one molar ratio with itself. This transport is likely to occur unidirectionally between neighboring cells in an organ, and between perivascular cells and the blood circulation. The chemical structure of the natural ligand, or ligands, of ApoD in normal cells in vivo or in culture is not known, but ApoD has been shown to bind some steroids and bilirubin. Remarkable upregulation of synthesis of ApoD has been observed during regeneration of injured peripheral nerves. Perhaps the physiologic role of ApoD will prove to be more interesting and of equal importance in biology to the roles of the other apolipoproteins in cardiovascular disease.

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Based on our previous observation that monoclonal antibody anti-apoD-4E11 reacted with several HDL proteins we studied them further with three questions in mind: i) is there common protein polymorphism in healthy individuals? ii) how many proteins are present and what are their characteristics? iii) are they all apolipoproteins and do they have the same lipoprotein distribution as apoD? Isolated, delipidated apoD was used as a standard for radioimmunometric assay of plasma with antibody 4E11. The antigen varied from 3 to 11 mumol-equivalents of apoD per liter of plasma (equivalent to 5-20 mg apoD/dl plasma) with means of 6.1 and 6.8 mumol/l in men and women, respectively. Two-dimensional electrophoresis of plasma found up to eight 4E11-antigenic-proteins of different Mr, each heterogeneous in pI. All plasmas tested contained apoD and an Mr 38,000 antigen, the latter being the most immunoreactive. Six proteins of Mr 70,000-94,000 were found, but the number varied between subjects. Eighty nine percent of the plasma antigen was associated with lipoproteins: 83% with HDL and VHDL, 5% with LDL and VLDL. Lipoproteins of all sizes, separated by polyacrylamide gradient gel electrophoresis, contained the antigen. ApoD was almost the only 4E11-antigen in LDL, and was in two states: the one free, the other an apoD-apoB mixed disulfide complex. The apparent proportions of higher Mr antigens increased with increasing lipoprotein density, and the proportion of apoD decreased reciprocally. None of these 4E11-antigenic-proteins cross-reacted with antiserum to retinol-binding protein.

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We have produced five hybridomas which secreted monoclonal antibodies that reacted with human plasma apolipoprotein D. On analysis by polyacrylamide gel electrophoresis (PAGE) high density lipoproteins and lecithin:cholesterol acyltransferase (EC 2.3.1.43)-enriched fractions of plasma contained many protein bands that reacted with the antibodies. Purified apolipoprotein D had the lowest Mr (29,000), the lowest pI (4.8-5.2), and the greatest migration on alkaline urea-PAGE of all the immunoreactive bands. These characteristics agreed with those described for apolipoprotein D in the literature. The other immunoreactive proteins had apparent Mr from about 39,000 to 98,000, they migrated more slowly than apolipoprotein D on alkaline urea-PAGE, and there were 10 polymorphs on isoelectric focusing. These cross-reacting proteins were present in the high density lipoproteins of each of four individuals sampled on several occasions and in pooled plasma. All of the monoclonal antibodies reacted both with apo-D and the higher Mr cross-reacting proteins. Each of our five monoclonal antibodies bound to one of two distinct antigenic sites on apo-D, determined by antibody competition immunoassays. Neither of these two sites was composed of carbohydrate, but expression of both sites seemed to be influenced by thiol-reducing agents: site 5G10 gained but 4E11 either lost immunoreactivity or was unchanged by reduction according to the conditions. We conclude that apolipoprotein D is only one of several plasma proteins, which contain two homologous polypeptide antigenic sites, recognized by monoclonal antibodies and also by a specific goat antiserum. Apolipoprotein D had the least Mr of these proteins.

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Protein methylesterase, an enzyme that hydrolyzes protein methyl esters, has been purified. The purification procedure includes ammonium sulfate and acid precipitations, and chromatographies on Sephadex G-100, Polybuffer exchanger 94, and matrex gel Green A. With this procedure, protein methylesterase was purified 1190-fold with a 28% recovery in activity. Its molecular weight has been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 31,000. Protein methylesterase has an optimum pH of 4.0 and an isoelectric point of 4.45. The enzyme is stable over a wide range of pH (2-10). The Km for ovalbumin methyl esters was estimated at 5.9 microM. Monovalent ions had little effect on activity while divalent ions at concentrations above 50 mM inhibited protein methylesterase activity in a concentration-dependent manner.

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In Drosophila Kc cultured cell lines, heat shock induces an increased synthesis of one of the core histones, H2B. This is accompanied by a reduction in the rate of synthesis of H1 and the other core histones, suggesting a noncoordinated expression of histones during heat shock. Arsenite which has a heat-shock mimicking effect does not induce an increase in the synthesis of H2B. The increased expression of H2B during heat shock shows a temperature dependency similar to that of the low molecular weight heat-shock proteins being observed at temperatures higher than 33 degrees C. A full heat-shock response is observed after a short 15-min shock at 37 degrees C, suggesting a rapid transcriptional response of the H2B gene and possibly a decreased transcription of the other histones and (or) an accelerated decay of their corresponding mRNAs. This increased synthesis of H2B seems under transcriptional control since it can be inhibited, like the other major heat-shock proteins, by the addition of actinomycin D.

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The post-translational methylation of histones in the presence of inhibitors of protein synthesis, was studied during a heat-shock to Drosophila cells in culture. In control cells (23 degrees C), both histones H3 and H4 are methylated. After heat-shock (37 degrees C), there is a dramatic reduction in the methylation of H3 and an increase in the methylation of another core histone identified as H2B. These changes in the pattern of methylation vary with the temperature of the heat-shock. The increased methylation of H2B is also observed in arsenite-treated cells but the methylation of H3 is unchanged, being similar to that observed in control cells. Inhibition of synthesis of heat-shock proteins has no effect on the methylation changes, suggesting that heat-shock proteins are not directly involved in the methylation reaction. These changes could be involved in the extensive transcriptional regulation occurring in these cells during heat-shock.

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