Henry R.V. Arnstein

The antioxidant action of tamoxifen and its metabolites. Inhibition of lipid peroxidation.

The anti‐oestrogen drug tamoxifen is an inhibitor of lipid peroxidation in rat liver microsomes and in phospholipid liposomes. Its cis isomer and N‐desmethyl form are weaker inhibitors, but 4‐hydroxytamoxifen is much more powerful. It is possible that the antioxidant property of tamoxifen might contribute to its biological actions.

Mechanism of inhibition of lipid peroxidation by tamoxifen and 4-hydroxytamoxifen introduced into liposomes. Similarity to cholesterol and ergosterol.

The anticancer drug tamoxifen when introduced into phospholipid liposomes during their preparation inhibited Fe(III)‐ascorbate induced lipid peroxidation to a greater extent than similarly introduced cholesterol. Ergosterol was equipotent with tamoxifen, but much less effective than 4‐hydroxytamoxifen. Possible mechanisms underlying these effects are discussed in relation to structural mimicry of the sterols by these triphenylethylene drugs as membrane stabilizers against lipid peroxidation.

Presence of haemoglobin messenger RNA in the postribosomal supernatant of rabbit reticulocytes and conditions necessary for its translation

As an extension of our earlier work on the incorporation of liver ribosomal subparticles into reticulocyte polysomes [ 1] we have developed a heterologous cellfree system containing rat liver ribosomal subparticles and rabbit reticulocyte postribosomal supernatant in order to test the initiation factor requirements for the translation of exogenous haemoglobin mRNA. Contrary to our expectations we have found that this cell-free system synthesizes globin without addition of exogenous mRNA. A number of earlier observations in the literature [2,3] suggested the presence of mRNA in the postribosomal supernatant and we now present evidence that the amount of mRNA in the supernatant may be considerable. Moreover, the mRNA is not associated with small ribosomal sub particles and codes almost exclusively for the a-chains of globin. In addition we have found that the.factors necessary for the translation de nova of haemoglobin mRNA are also present in the postribosomal supernatant.

Synthesis of α- and β-globin directed by messenger ribonucleoprotein from rabbit reticulocytes

Abstract The translation of globin messenger ribonucleoprotein (mRNP) obtained from high salt-washed rabbit reticulocyte ribosomes by treatment with EDTA was investigated using a cell-free system from mouse Krebs II ascites tumour cells. The messenger activity of the mRNP and the mRNA derived from it by mild deproteinization was compared in the presence and absence of reticulocyte initiation factors. Both forms gave identical products over a wide range of messenger concentration and there was no qualitative or quantitative difference in their efficiency as messengers. It is concluded that the proteins associated with polysomal mRNA do not alter the specificity of translation of α- and β-globin messengers or the requirement for initiation factors.

The antioxidant action of ketoconazole and related azoles: Comparison with tamoxifen and cholesterol

The azole antifungal drug ketoconazole was found to inhibit Fe(III)-ascorbate dependent lipid peroxidation using either rat liver microsomes or ox-brain phospholipid liposomes as the substrate. It also inhibited microsomal peroxidation induced by the Fe(III)-ADP/NADPH system. The related azoles, miconazole and clotrimazole, were much weaker inhibitors than ketoconazole. Ketoconazole was approximately equipotent with the triphenylethylene anticancer drug tamoxifen in the microsomal system and was almost as effective as 4-hydroxytamoxifen in the liposomal system. Ketoconazole introduced into phospholipid liposomes during their preparation inhibited Fe(III)-ascorbate induced lipid peroxidation to a greater extent than similarly introduced cholesterol, ergosterol or tamoxifen. Miconazole and clotrimazole were again poor inhibitors of lipid peroxidation in this system. These antioxidant effects of ketoconazole may be due to membrane stabilization in the systems used. The implications of our findings for the clinical applications of these drugs are discussed.

Polyribosome formation from haemoglobin ribonucleoprotein in vitro.

When reticulocyte polyribosomes are treated with EDTA, an RPN particle containing 9 S RNA is released [l-4] . This species of RNA is the mRNA for globin [5]. One of the first steps in the initiation of protein synthesis involves the binding of mRNA to the smalI subparticle [6] . This binding has been demonstrated both on native [7] and derived subparticles [8]. In order to demonstrate the subsequent steps in initiation of protein synthesis it is necessary to show formation of an initiation complex from both subparticles with mRNA and subsequent build up of disomes and larger polyribosomes as a result of repeated initiation on the added mRNA. We have found that this is possible, using a preparation of ribosomes and enzymes obtained from a rabbit reticulocyte lysate by pH 5.4 precipitation, which is capable of efficient initiation [9] . Furthermore, aurlne tricarboxylic acid (ATA) and cycloheximide are found to inhibit different steps in this process.

Interaction of haemoglobin messenger RNP with the small subparticle of reticulocyte ribosomes

When reticulocyte polysomes that have been isolated from rabbits injected 18 hr previously with 32P phosphate are dissociated with EDTA, a highly labelled component is released [l-3] . This material sediments at approximately 14 S and has been shown to contain 9 S RNA, which is believed to be the messenger for haemoglobin [4-81, together with proteins [3] . There exists considerable evidence of rapidlylabelled ribonucleoprotein complexes in other cells, including liver [9-l 11, thyroid [ 121, and L cells [ 131. Whether these complexes have any physiological significance remains to be seen, but they are thought to be the form in which mRNA is transferred from the nucleus to the polysomes. These complexes bear a remarkable resemblance to informosomes, which are polysome-bound and can be translated [ 141. The binding of mRNA to the small ribosomal subparticle is a prerequisite for the initiation of protein synthesis both in E. cob [ 15,161 and in mammalian cells [ 171. Recently [ 181 it has been shown that 9 S RNA can be isolated from the small subparticle, derived by dissociation of reticulocyte polysomes with pyrophosphate. We wish to report that a messenger ribonucleoprotein complex can be released from the small ribosomal subparticle by treatment with EDTA and that under certain conditions it can bind to the 40 S ribosomal subparticle. These subparticles have been obtained by a mild procedure involving the release of nascent pro tein with puromycin followed by dissociation. The method is a modification of that used by Lawford [ 191 with rat liver subparticles. North-Holland Publishing Company Amsterdam 2. Methods

The reassociation of fractionated rabbit reticulocyte ribosomal subunits into particles active in polyphenylalanine synthesis

The biological activity of reticulocyte ribosomal subunits, derived by treating ribosomes with high salt, has been shown [l-4] . Attempts to fractionate the subunits on sucrose gradients, however, resulted in particles which failed to reassociate into active ribosomes [4], and it was not possible, therefore, to demonstrate unequivocally that the originally observed activity was entirely due to subunits. Recently, fractionation of rat muscle ribosomes on gradients containing 0.85 M KC1 0.015 M MgCl, at room temperature was reported to produce subunits which reassociated into particles active in polyphenylalanine synthesis [5]. This communication shows that this method of fractionation produces subunits which reassociate into active particles also in the reticulocyte system.

Ribonuclease activity during erythroid cell maturation.

A progressive decrease in the soluble and ribosomal RNA content due to an enzyme system that degrades RNA has been shown to occur during the late stages of erythroid cell maturation [ 1,2] . There is substantial evidence that ribonuclease activity also declines in the same period [3,4] . The information available on RNase activity in erythroid cells is, however, still fragmentary and concerned only with those cells at the final stage of erythroid development, namely reticulocytes. Taking advantage of a technique recently developed in this laboratory for the fractionation of the immature cells from rabbit bone marrow by velocity sedimentation in a Ficoll gradient [S] , we have investigated the changes of RNase* activity during erythroid cell development in the bone marrow. From the results of this study it appears that RNase activity is related to other biochemical and morphological changes occurring during cell maturation. Thus, the enzyme activity decreases markedly after cessation of RNA synthesis and even further after extrusion of the nucleus from the orthochromatic cells. In contrast, an examination of RNase activity by polyacrylamide gel electrophoresis of erythroid cell lysates demonstrated that the RNase electrophoretic pattern showed only slight changes as the cells move from the dividing into the nondividing cell compartment.

The mobilisation of iron from the cell stroma during erythroid maturation.

Abstract Adult mammalian erythroblasts from an anaemic rabbit were separated into fractions of cells at different stages of development using the velocity sedimentation technique. The iron content of the stroma and cytoplasm of the cells in each fraction was determined by atomic absorption spectroscopy. A high proportion of the iron in the dividing erythroblasts was found to be associated with the cell stroma. After the final cell division the proportion of stromal iron rapidly declines and it appears that stromal iron is mobilised at this stage and utilised by the non-dividing erythroblasts for haemoglobin synthesis.