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H R Arnstein

Publications and source records attributed to H R Arnstein.

At least 37 records · Page 2Linked to original sources
PubMed1986

The anti-oestrogen drug tamoxifen is an elongation inhibitor of eukaryotic protein biosynthesis.

The drug tamoxifen is widely used in the chemotherapy of breast cancer but its action is not explained completely by its anti-oestrogen properties. We now present evidence indicating that it is also a potent inhibitor of eukaryotic protein synthesis as demonstrated in Xenopus oocytes, intact reticulocytes and reticulocyte lysates. The inhibition affects general protein synthesis, is transient in oocytes and not reversed by oestrogen. The drug appears to act by inhibiting polypeptide chain elongation. This action of tamoxifen is independent of oestrogen receptors and may explain its therapeutic effectiveness in oestrogen-independent tumours.

Animals↗
PubMed1986

The 66 kDa component of eukaryotic initiation factor 3 interacts with globin mRNA and 18 S rRNA in preinitiation complexes.

The 66 kDa protein present in a complex with globin mRNA and 18 S rRNA [(1984) Eur. J. Biochem. 143, 27-33] has been reincorporated into functional eukaryotic initiation factor 3 (eIF-3) under conditions of protein synthesis. Additionally, two-dimensional polyacrylamide gel electrophoresis has been used to demonstrate the identity of the 66 kDa protein with the 66 kDa subunit of eIF-3.

Animals↗
PubMed1986

Stimulation of the adenylate cyclase activity of rabbit bone marrow immature erythroblasts by erythropoietin and haemin.

The effect of two agents of erythroid cell differentiation on the adenylate cyclase activity of fractionated rabbit bone marrow erythroblasts has been investigated. Addition of 0.2U/ml erythropoietin to cell cultures causes a transient increase in the activity of plasma membrane adenylate cyclase, which is maximal by 20 min and disappears within 4 h. The magnitude of the response to hormonal stimulation depends on the stage of erythroid cell development and is greater in the more immature cells. Addition of 50 microM haemin to cultures of erythroblasts also causes an increase in the activity of adenylate cyclase, which differs from the effect of erythropoietin in kinetics and specificity of target cells. With immature cells the haemin-induced stimulation starts after the first hour and continues to increase up to 20 h of culture. Erythropoietin but not haemin can stimulate the basal activity of adenylate cyclase in an in vitro assay containing plasma membranes of immature erythroid cells. The degree of activation depends on the concentration of erythropoietin and is maximal with 0.2-0.5 U/ml hormone (5-12 nM). In the presence of guanine nucleotides the activation of adenylate cyclase by erythropoietin is increased further but the effect is not additive. With respect to the basal and the guanine-nucleotide-stimulated activities of adenylate cyclase erythropoietin acts differently from the beta-agonist l-isoprenaline. The in vitro effect of erythropoietin is abolished by the beta-thio analogue of GDP, GDP[beta S], and extensive washing of membranes makes hormone action GTP-dependent. The stimulation of adenylate cyclase by the addition of erythropoietin to the reaction mixture is inversely related to the extent of previous hormonal stimulation of the cells from which the membranes were prepared. This loss of hormonal responsiveness is due to desensitization or receptor down-regulation and persists for up to 20 h. We conclude that in immature erythroblasts erythropoietin acts via a receptor and a guanine nucleotide-binding protein with high affinity for GTP (EC50 less than 10 nM), whereas haemin appears to activate adenylate cyclase indirectly, as a consequence of progressive perturbations of the plasma membrane.

Adenylyl Cyclases↗
PubMed1986

Control of protein synthesis by amino acid supply. The effect of asparagine deprivation on the translation of messenger RNA in reticulocyte lysates.

The enzyme asparaginase, which hydrolyses asparagine to aspartic acid, inhibited cell-free protein synthesis by reticulocyte lysates. The inhibition was rapid and complete when sufficient enzyme was added but could be prevented or reversed by the addition of asparagine. The initial effect of asparaginase appears to be a block in polypeptide chain elongation due to asparagine deprivation, but there are some indications that prolonged incubation under these conditions may give rise to a secondary decrease in initiation of protein synthesis.

Amino Acids↗
PubMed1985

Changes in histone acetylation during the development of rabbit bone marrow erythroid cells.

The acetylation of histones in rabbit bone marrow erythroid cells was investigated by measuring the incorporation of labelled acetate into erythroblasts which were separated by velocity sedimentation at unit gravity into five fractions corresponding to different stages of development. Histone acetylation decreased during erythroid development in concert with a decline in DNA and histone synthesis. Some acetylation persisted after condensation of the nucleus and cessation of DNA synthesis in late orthochromatic cells. This residual acetylation may be related to the low level of transcription which is still present at this stage. Sodium butyrate increased the acetylation of histones 2- to 7-fold, with the greatest stimulation occurring in the most immature cells. The general decline in acetylation of histones during erythroid cell development was similar in the presence and absence of butyrate.

Acetates↗
PubMed1985

Messenger RNA turnover during bone marrow erythroid cell differentiation.

Incubation of bone marrow cells from anaemic rabbits in the presence of actinomycin D led to a decrease in total protein synthesis and an increase in the relative synthesis of globin. This increase in the proportion of globin was observed with in vivo labelling of cellular proteins and in vitro translation of isolated RNA, which indicates that the messenger RNA for globin is much more stable than the other bone marrow cell messages. This was further shown by pulse-labelling the RNA and characterization of the different species by separation on a cDNA-oligo(dT)-cellulose column. Within 12 h after pulse-labelling the relative levels of globin mRNA had risen 10-fold, while a rapid decrease in the level of the poly(A)-rich RNA fraction was observed. Investigations into the mechanisms of this differential stability indicate that the more metabolically active cells from the early stages of erythropoietic development are more susceptible to inhibitors of RNA synthesis such as actinomycin D and alpha-amanitin. A preliminary study using a lysosomal inhibitor, chloroquine, indicates that there appear to be at least two degradative mechanisms, involving a lysosomal and a non-lysosomal pathway, with selective specificity for different messages.

Amanitins↗
PubMed1984

The effect of haemin on RNA synthesis and stability in differentiating rabbit erythroblasts.

Haemin accelerates the maturation of erythroid cells but whether this is the result of increased globin gene transcription or processing and stabilization of globin mRNA is not clear. The effect of haemin on the synthesis and stability of non-globin messengers is also unknown. We examined the changes that occur in RNA metabolism when anaemic rabbit bone marrow erythroblasts, fractionated into immature and mature fractions, are cultured with 20 microM or 50 microM haemin for brief periods (5-8 h). With both cell types haemin increases the incorporation of [3H]uridine into newly synthesized RNA, particularly into the poly(A)-rich fraction which can increase threefold. Haemin also increases the synthesis of globin mRNA (up to 500% absolutely and 50% relative to the synthesis of total RNA) in the immature, but not in the mature, cells. These results suggest that haemin increases the transcription of both globin and non-globin mRNAs and that the relative increase of each depends on the stage of erythroid cell development. When the [3H]RNA from prelabelled cells was chased in the presence of haemin (with or without actinomycin D) the proportion of 3H remaining in globin mRNA increased in the immature, but not in the mature, cells. These changes in the relative concentration of globin mRNA were also shown by the translation of extracted bone marrow RNAs in a nuclease-treated reticulocyte lysate. We conclude that a secondary effect of haemin is on RNA stability and that it enhances the accumulation of globin mRNA by both molecular and cellular mechanisms.

Animals↗
PubMed1984

Formation of a 22S mRNA X rRNA X protein complex during translation of globin messenger RNA.

Poly(A)-rich RNA was isolated from rabbit reticulocyte polyribosomes by affinity chromatography on oligo(dT)-cellulose and fractionated in sucrose gradients under non-denaturing conditions. Most of the translatable RNA sedimented in sucrose gradients both as free 9S mRNA and as a 22S complex containing 18S ribosomal RNA and a protein of Mr 66 000. The complex was characterized by identification of the translation products. Experiments with both labelled globin mRNA and Mr-66 000 protein indicate that the complex is not an artefact, but rather that it is formed during the initiation of protein synthesis. The Mr-66 000 protein appears to be a component of the 48S pre-initiation complex and recycles before 80S complex formation.

Animals↗
PubMed1984

The nonhistone proteins of developing mammalian erythroid cells.

Anaemic rabbit bone marrow cells, labelled with [35S]methionine, were separated at unit gravity. Chromatin was isolated from these cells and proteins were separated from DNA, using urea/salt extraction. Two-dimensional gel electrophoresis of the nonhistone proteins showed that these proteins appeared to change quantitatively but not qualitatively, with one important exception, as cell development proceeded. The one protein that did change had a molecular weight of approximately 20,000 and was very basic. This protein was synthesised at low levels in the early cells, but its synthesis was seen to increase at the polychromatic stage of cell development, just prior to nuclear condensation. Treatment of bone marrow cells with sodium butyrate was shown to increase the synthesis of this protein in the early cells.

Animals↗
PubMed1983

Messenger RNA-ribonucleoprotein interaction during the initiation of protein synthesis.

The interaction of globin mRNA with proteins during translation has been investigated in order to establish whether and to what extent messenger-ribonucleoprotein complexes are involved in protein synthesis. We present evidence for the functional importance of two minor messenger RNA-associated proteins (55 kDa and 60 kDa) during the initiation of globin mRNA translation in reticulocyte lysates. The formation of an mRNA complex containing the major 78 kDa and 52 kDa messenger-ribonucleoproteins was not detected.

Animals↗
PubMed1983

Efficient translation and polyribosome binding of 125I-labelled rabbit globin messenger ribonucleoprotein.

Rabbit polyribosomal globin messenger ribonucleoprotein (mRNP) was labelled under mild conditions, using 125I and Iodogen, in the protein moiety so that the fate of mRNA-associated proteins could be followed during translation. 125I-mRNP was shown to retain functional activity in the nuclease-treated reticulocyte lysate translation system under optimal labelling conditions. Polyribsome binding of 125I-mRNP and its sensitivity to cycloheximide indicated a functional- and translation-dependent binding of mRNP proteins. The results constitute a successful and direct approach to the study of mRNA-associated proteins in translational control.

Animals↗
PubMed1983

Characteristics of the beta-adrenergic adenylate cyclase system of developing rabbit bone-marrow erythroblasts.

After fractionation of rabbit bone marrow into dividing (early) and non-dividing (late) erythroid cells, the adenylate cyclase activity of membrane ghosts was assayed in the presence of guanine nucleotides ((GTP and its analogue p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate))), the beta-adrenergic agonist L-isoprenaline (L-isoproterenol) and the antagonist L-propranolol. Both GTP and p[NH]ppG increased the adenylate cyclase activity of early and late erythroblasts, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing bone-marrow cells. The effect of L-isoprenaline was completely inhibited by the antagonist L-propranolol, confirming that the response was due to stimulation of beta-adrenergic receptors on the plasma membrane. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind the selective ligand [125I]iodohydroxybenzylpindolol with almost equal affinities, the apparent dissociation constants, Kd, being 0.91 X 10(-8)M and 1.0 X 10(-8) M respectively. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p[NH]ppG during erythroblast development was observed: the dissociation constants of both guanine nucleotides were almost identical with early and late erythroblast membrane preparations [2-3 (X 10(-7) M]. With dividing cells, however, in the presence of L-isoprenaline the dissociation constants of GTP and p[NH]ppG were lower (6 X 10(-8) M). The dose-response curves for isoprenaline competition in binding of [125I]iodohydroxybenzylpindolol by dividing cells showed that the EC50 (effective concentration for half maximum activity) value for isoprenaline was higher in the presence of p[NH]ppG. With non-dividing cells the EC50 value for isoprenaline was equal in the presence and in the absence of p[NH]ppG and similar to that observed with dividing-cell membranes in the presence of the nucleotide. Thus differentiation of rabbit bone-marrow erythroid cells seems to be accompanied by uncoupling of the beta-adrenergic receptors from the adenylate cyclase catalytic protein as well as by a decrease in the number of receptors per cell, but not by changes in the catecholamine and guanine-nucleotide-binding affinities.

Adenylyl Cyclases↗
PubMed1983

Polyribosome binding of rabbit globin messenger RNA and messenger ribonucleoprotein labelled with bacteriophage-T4 RNA ligase and 5'-[32P] phosphocytidine 3'-phosphate.

Rabbit polyribosomal globin messenger RNA (mRNA) and messenger ribonucleoprotein (mRNP) were labelled at the 3' poly(A) tail to high specific activity with T4 RNA ligase and [5'-(32)P]pCp without consequent loss of functional activity. Labelled message was translated in both micrococcal nuclease treated and untreated rabbit reticulocyte lysates, as shown by the formation of labelled polyribosomes. The utilisation of labelled messenger was abolished by T2 toxin or sodium fluoride which are known to inhibit protein synthesis.Images

Animals↗
PubMed1983

Stimulation of adenylate cyclase activity by catecholamines and prostaglandins E during differentiation of rabbit bone marrow erythroid cells.

After fractionation of rabbit bone marrow into erythroid cells at different developmental stages adenylate cyclase activity of membrane ghosts was assayed in the presence of sodium fluoride, catecholamines or prostaglandins E. Both basal and fluoride-stimulated adenylate cyclase decreased continuously during differentiation. Only catecholamines having beta 2-adrenergic activity stimulated adenylate cyclase and their effect was restricted to the most immature cells, the proerythroblasts and, to a lesser extent, the basophilic erythroblasts. Thus, uncoupling of beta-adrenergic receptors occurs early in erythroblast development and hormone responsiveness is lost before the final cell division. Prostaglandin E receptors and adenylate cyclase remain coupled throughout erythroid cell development.

Adenylyl Cyclases↗
PubMed1983

Characteristics of the adenylyl cyclase system of differentiating rabbit bone marrow erythroblasts.

Changes in the cellular content of cyclic AMP and in the activities of adenylyl cyclase, cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinases during differentiation of rabbit bone marrow erythroid cells were investigated. The cells were separated by velocity sedimentation at unit gravity into six fractions corresponding to different stages of development: proerythroblasts, basophilic erythroblasts, polychromatic cells, early orthochromatic and late orthochromatic cells and reticulocytes. Adenylyl cyclase activity was found to decrease continuously as the cells developed, from approx. 180 pmoles cyclic AMP formed/mg of protein/20 min in proerythroblasts to 10 pmoles in circulating reticulocytes. The proerythroblasts were the richest cells in cyclic AMP which is present at a cellular concentration of approx. 1.4 microM. In basophilic cells the cyclic AMP content was about 80% lower than in proerythroblasts. No further changes in cyclic AMP levels were observed after the final cell division. Cyclic AMP phosphodiesterase was found to be very active in the most immature cells, the proerythroblasts. After differentiation into basophilic erythroblasts, a 4-fold decrease in cyclic AMP phosphodiesterase activity occurred. In polychromatic cells there was a further drop in phosphodiesterase activity and after the last cell division the enzyme activity was constant and very low. Both cytosolic cyclic AMP-binding capacity and cytosolic cyclic AMP-dependent protein kinase activity decreased in dividing rabbit bone marrow erythroblasts when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative but only quantitative changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent Kd for the interaction of binding proteins with cyclic AMP was 4 . 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and their affinity for cyclic AMP. The phosphorylation of rabbit erythroblast plasma membrane proteins by membrane-associated protein kinase(s) was found to be cyclic AMP-dependent in dividing cells during the early stages of differentiation. When the erythroid cells reach the non-dividing stage in their development, autophosphorylation of membrane ghosts was no longer stimulated by cyclic AMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗
PubMed1983

Characteristics of the adenylate cyclase system of differentiating rabbit bone marrow erythroblasts.

Adenylate cyclase activity of rabbit bone marrow erythroblasts decreased continuously as the cells developed. The proerythroblasts were the richest cells in cAMP. No changes in cAMP levels were observed after the final cell division. cAMP-phosphodiesterase activity declined rapidly during the early period of erythroid cell development and remained constant but extremely low after condensation of the nucleus. Both cytosolic cAMP-binding capacity and cAMP-dependent protein kinase activity decreased in dividing erythroblasts when calculated in terms of cell number but remained constant per cell volume. Membrane-associated protein kinase was found to be cAMP-dependent only in the dividing cells. The adenylate cyclase activity of both early and late erythroblasts was stimulated by GTP and p (NH)ppG, whereas the stimulating effect of the beta-adrenergic drug L-isoprenaline was limited to the immature dividing cells. The lack of response of non-dividing erythroblasts to beta-adrenergic stimuli is not due to loss of beta-receptors, since both dividing and non-dividing cells bind 125I-iodohydroxybenzylpindolol with equal affinities. The number of beta-adrenergic receptors per cell was 2-fold higher in the dividing cells. No significant change in binding affinity for GTP and p (NH) ppG during erythroblast development was observed.

3',5'-Cyclic-AMP Phosphodiesterases↗