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H B Osborne

Publications and source records attributed to H B Osborne.

At least 37 records · Page 2Linked to original sources
PubMed1991

Expression and post-transcriptional regulation of ornithine decarboxylase during early Xenopus development.

In this paper we show that large changes in ornithine decarboxylase (ODC) activity occurred during early Xenopus development. Following fertilization, this enzyme activity rises with a quantitatively correlated accumulation of putrescine and spermidine. This increase in ODC activity was associated with an increased translation of the maternal ODC mRNA, which was stable in the embryo and whose polyadenylation increased slightly between fertilization and the mid-blastula transition (MBT). ODC activity was stable in cycloheximide-treated embryos, indicating that before the MBT this enzyme was not degraded. After the MBT, ODC activity fell, but no decrease in this mRNA was observed. In gastrulae, ODC mRNA was both increased in amount and polyadenylated. The reduced ODC activity at this stage of development was not associated with a fall in ribosome loading of the mRNA. Treatment of post-MBT embryos with cycloheximide lead to an accentuation of the normally observed decrease in ODC activity. Expression of Xenopus ODC in mutant ODC-deficient Chinese hamster ovary cells (C 55.7 cells) showed that the Xenopus enzyme was rapidly degraded and can be regulated post-translationally by polyamines, indicating that the post-MBT fall in ODC activity could be caused by a change in protein turnover or by polyamine-mediated regulation.

Animals↗
PubMed1991

Degradation of a developmentally regulated mRNA in Xenopus embryos is controlled by the 3' region and requires the translation of another maternal mRNA.

By injecting the appropriately constructed plasmids into one-cell Xenopus embryos, we determined that the 3' region of the maternal Xenopus Eg2 mRNA confers instability on the chimeric mRNA transcribed from these plasmids. This instability, like that of the maternal Eg2 transcript, was abolished by treatment of the embryos with cycloheximide. Analysis of the polysome distribution of the maternal Eg2 mRNA in cycloheximide-treated and untreated embryos showed that Eg2 mRNA was released from polysomes after fertilization and that the stabilization caused by cycloheximide treatment was not due to a reloading of ribosomes onto the mRNA. Insertion of a stable hairpin loop (delta G = -50 kcal/mol) 5' to the reporter gene in the injected plasmid caused a 10- to 20-fold decrease in translation from the transcribed mRNAs. This decrease in translation did not abolish the instability conferred by the 3' Eg2 region. Therefore, the degradation of these chimeric mRNAs in Xenopus embryos requires the translation of another maternal mRNA coding for a trans-acting factor involved in mRNA degradation. Further restriction of the 3' Eg2 region, placed 3' to the reporter gene, showed that a cis-acting instability-conferring sequence is contained in a 497-nucleotide fragment.

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PubMed1990

Stability of maternal mRNA in Xenopus embryos: role of transcription and translation.

The first 12 cell divisions of Xenopus laevis embryos do not require gene transcription. This means that the regulation of gene expression during this period is controlled at post transcriptional levels and makes Xenopus early development a potentially interesting biological system with which to study the mechanisms involved. We describe here the stability characteristics of several maternal Xenopus mRNAs which are deadenylated soon after fertilisation (J. Paris and M. Philippe, Dev. Biol., in press). We show that these mRNAs were only degraded in the embryo after the midblastula transition (MBT), when gene transcription was initiated. The kinetics with which the deadenylated maternal mRNAs decreased in the post-MBT embryos showed sequence specificity. The degradation of these mRNAs after the MBT was inhibited by cycloheximide but was not affected by dactinomycin. Therefore, the destabilization of these mRNAs does not appear to be initiated by new embryonic gene transcripts. Sequence comparisons of the 3' untranslated region of these mRNAs identified several motifs which may be involved in the posttranscriptional control of these gene products.

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PubMed1990

Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

Animals↗
PubMed1989

Polyamine levels during Xenopus laevis oogenesis: a role in oocyte competence to meiotic resumption.

The results presented here show that a decrease in the concentration of total polyamines, due to a decrease in putrescine and spermine, occurs during oogenesis in Xenopus laevis. The microinjection of spermine or spermidine decreases the hormonal responsiveness (maturation) of the fully-grown oocytes. This effect is synergistic with that already described for the microinjection of casein kinase II (Mulner-Lorillon, O. et al. (1987) Eur. J. Biochem. 171, 107-117), a polyamine dependent enzyme. Therefore a decrease in polyamine concentration, via its effect on endogeneous casein kinase II, could constitute one of the molecular changes required for the acquisition of competence to mature.

Animals↗
PubMed1989

Regulation of ornithine decarboxylase in murine erythroleukemic cells by N,N'-diacetyl-1,6-hexanediamine and N-acetyl-1,6-hexanediamine.

The levels at which the ornithine decarboxylase gene is regulated in murine erythroleukemic cells treated with N,N'-hexamethylene bisacetamide (N,N' diacetyl-1,6-hexanediamine) and with N-acetyl-1,6-hexanediamine, the monoacetylated catabolite of this inducer of erythrodifferentiation, have been investigated. Although these two molecules are structurally related and both cause a decrease in ornithine decarboxylase activity, they did not similarly affect ornithine decarboxylase mRNA. In the presence of 4 mM N,N'-hexamethylenebisacetamide, at the concentration generally used to induce differentiation in these cells, a decreased steady-state level of ornithine decarboxylase mRNA, due to decreased gene transcription, was observed. In the presence of N-acetyl-1,6-hexanediamine the decrease in enzyme activity was shown to be associated with a decrease in the half-life of the enzyme in the absence of a change in gene transcription. These results show that in proliferating cultured cells changes in ornithine-decarboxylase-gene transcription can be uncoupled from changes in cell growth and that N-acetyl-1,6-hexanediamine only regulates ornithine decarboxylase expression post-transcriptionally.

Acetamides↗
PubMed1988

Changes in the polyadenylation of specific stable RNA during the early development of Xenopus laevis.

The distribution of four specific RNA species between the poly(A)+ and poly(A)- fractions has been studied during the first hours of Xenopus laevis development, before the mid-blastula transition (MBT). Two of these specific RNA species correspond to clones selected by differential hybridization from a Xenopus egg cDNA library. Another corresponds to Xenopus c-raf mRNA and the last one to RNA revealed by a mouse ornithine decarboxylase probe. We show that two of these RNAs are adenylated after fertilization and remain in the poly(A)+ population. During the same period, the other two RNAs are deadenylated and these new poly(A)- RNAs remain stable at least until the MBT. These results show (i) that polyadenylation of specific RNA species occurs after fertilization in Xenopus and (ii) that, in the absence of transcription, adenylation and deadenylation can occur simultaneously in the fertilized egg.

Animals↗
PubMed1987

Effect of alpha-difluoromethylornithine on DNA methylation in murine erythroleukaemic cells. Relationship to stimulation of induced differentiation.

Murine erythroleukaemic (MEL) cells cultured with alpha-difluoromethylornithine (DFMO) accumulated decarboxylated S-adenosylmethionine(decarboxylated AdoMet). In the absence of the inducer hexamethylenebisacetamide (HMBA), this accumulation of decarboxylated AdoMet was associated with a concomitant and proportional increase in DNA hypomethylation. In the presence of HMBA, DFMO, which stimulates the erythrodifferentiation of MEL cells, enhanced the differentiation-associated DNA hypomethylation. However, this differentiation-associated DNA hypomethylation was neither temporally nor quantitatively correlated with the accumulation of decarboxylated AdoMet in these cells. Therefore DFMO probably stimulates the HMBA-induced differentiation of MEL cells and the associated DNA hypomethylation via the effect of this drug on polyamine biosynthesis.

Acetamides↗
PubMed1987

Modulation of murine erythroleukemic cell differentiation by inhibitors of polyamine biosynthesis.

The differentiation of murine erythroleukemic cells induced by hexamethylene bisacetamide is shown to be differently affected by two inhibitors of polyamine biosynthesis. Methyl glyoxal bis(guanyl hydrazone) (inhibitor or S-adenosyl methionine decarboxylase) inhibited this differentiation process. By using a novel experiment protocol the inhibitory effect of this drug on the induced differentiation was dissociated from pleiotropic effects on cell growth. Methyl glyoxal bis(guanyl hydrazone) only inhibited the induced differentiation if present during the first 6 h of culture of the cells with the inducer. No effect on the induced differentiation was observed if the drug was added to the culture medium 6 h after the inducer. alpha-Difluoro methylornithine (inhibitor of ornithine decarboxylase) stimulated the differentiation of these cells. Polyamine analysis demonstrated that alpha-difluoro methylornithine increased the rapidity and the amplitude of the changes in intracellular polyamines associated with this induced differentiation. The presence of methyl glyoxal bis(guanyl hydrazone) during the first 3 h with the inducer was sufficient to produce opposing changes in the intracellular polyamines. These results suggest that changes in either intracellular polyamines or the activities of polyamine biosynthetic enzymes play a regulatory role in the differentiation process induced in murine erythroleukemic cells by hexamethylene bisacetamide.

Acetamides↗
PubMed1986

Catabolites produced by the deacetylation of hexamethylenebisacetamide play a key role in murine erythroleukaemic-cell differentiation.

N-Acetyl-1,6-diaminohexane and 1,6-diaminohexane, formed by deacetylation of the inducer hexamethylenebisacetamide (HMBA), are shown to accumulate rapidly inside murine erythroleukaemic cells. The appearance of these molecules preceded the differentiation-associated changes in intracellular polyamines. A quantitative relationship was observed between the accumulation of these molecules and the changes in intracellular polyamines. In the absence of HMBA, exogenous N-acetyl-1,6-diaminohexane was able not only to cause changes in polyamine biosynthesis, but also to induce the complete differentiation process. These results imply that these catabolites of HMBA are directly responsible for the changes in polyamine biosynthesis and probably also for initiating other events regulatory for the differentiation of these cells.

Acetamides↗
PubMed1986

Relationship between ornithine decarboxylase activity and hexamethylene bisacetamide-induced differentiation of murine erythroleukemia cells.

A transitory increase in ornithine decarboxylase (ODC) activity is shown not to be a prerequisite for the differentiation induced by hexamethylene bisacetamide (HMBA) in murine erythroleukemic (MEL) cells. On the contrary, conditions are described, where inhibition of the ODC activity with alpha-difluoromethyl ornithine (DFMO) stimulated the induced differentiation. Polyamine analysis demonstrated that a reduction in intracellular putrescine and spermidine occurred in MEL cells before commitment to erythrodifferentiation. The presence of DFMO increased the rapidity and the amplitude of these changes. No effect of dexamethasone on these changes in ODC activity or intracellular polyamines was observed.

Acetamides↗
PubMed1984

Kinetics of histone H10 accumulation and commitment to differentiation in murine erythroleukemia cells.

The accumulation of histone H10 (also denoted IP 25) in murine erythroleukemia cells, induced to differentiate with hexamethylene bis-acetamide, was shown to precede by 15-20 h the appearance in the culture of cells irreversibly committed to differentiate. In addition the rates of accumulation of H10 and of committed cells vary in a similar manner with the HMBA concentration. Flow microfluorimetric analysis demonstrated that the accumulation of H10 did not occur simultaneously in all the cells. This accumulation of histone H10 was initiated first in cell in the G2 phase of the cell cycle and subsequently in the cells situated in all the phases of the cell cycle.

Acetamides↗
PubMed1983

Effect of pyridoxine on the inhibition by dexamethasone of murine erythroleukemia cell differentiation.

Glucocorticoids are known to inhibit the induced differentiation of murine erythroleukemia cells. I show here that the binding of glucocorticoid receptors to the intact nuclei of these cells is reduced by exogenous extracellular pyridoxine (1-4 mM). This reduction of glucocorticoid-receptor binding did not abrogate the inhibition by dexamethasone of the induced differentiation.

Animals↗
PubMed1982

Effect of dexamethasone on hexamethylene bisacetamide-induced Friend cell erythrodifferentiation.

Friend erythroleukemic cells can be induced to differentiate by chemicals such as dimethyl sulfoxide and hexamethylene bisacetamide (HMBA) in a dose-dependent manner. Others have shown that dexamethasone and other steroid hormones can inhibit the differentiation induced by dimethyl sulfoxide. We show here that dexamethasone has a dual mode of action on the HMBA-induced differentiation of a Friend cell line, DS19. At concentrations above 10(-10) M, dexamethasone inhibits the HMBA-induced differentiation in DS19 cells. We further found that as the concentration of HMBA is reduced, the amount of dexamethasone required to inhibit differentiation increases. Such inhibition seems to act primarily by decreasing the probability that a cell will become committed to differentiate. Besides, dexamethasone does not inhibit hemoglobin synthesis of cells which are committed in its absence. In addition, we show that sensitivity to the inhibition by dexamethasone is inversely related to the inducibility of cell populations. The second action of dexamethasone, observed at concentrations between 10(-10) and 10(-13) M, is to increase the proportion of hemoglobin-containing cells relative to that for cells cultured in its absence. The degree of this synergistic effect for induced differentiation is inversely related to the level of induction in the absence of dexamethasone and thus is best observed in the cells cultured at low levels of HMBA. We present evidence that this synergistic effect may be due to an increased viability of the induced cells and possibly an increase in the proliferative capacity of these cells.

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