Nuclear Protein Synthesis Research Articles

Effects of Irradiation on Nuclear Protein Synthesis in G 2 Phase of the Cell Cycle

A method was developed to determine the synthesis of nuclear proteins throughout the cell cycle which was resolved into six compartments on the basis of DNA and nuclear protein content (i.e., early and late G1, early and late S, etc). Using this technique cell-cycle-specific synthesis of certain nuclear proteins was observed. Of particular interest was a 170-kDa protein(s) whose synthesis was initiated in early S phase and reached a maximum rate in late G2. Following irradiation with 6.8 Gy of 137Cs gamma rays the synthesis of the 170-kDa protein(s) declined in the G2 population with near total inhibition seen by 24 h. Synthesis of the 170-kDa protein(s) appeared to be slightly enhanced, and the postirradiation inhibition of its synthesis was reversed, in the presence of 3 mM caffeine. Also, the synthesis of 55-kDa nuclear protein(s) was stimulated throughout the cell cycle in the presence of 3 mM caffeine. These observations suggest new possibilities regarding the mechanism of the X-ray-induced G2 block and its reversal by caffeine. However, the exact role of these nuclear proteins in cellular events remains to be ascertained.

Timing of Nuclear Progression and Protein Synthesis Necessary for Meiotic Maturation of Bovne Oocytes1

In cows, protein synthesis is required for germinal vesicle breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus were washed and incubated in groups of 10 for up to 24 h in 50-microliters drops of TCM-199 supplemented with follicle-stimulating hormone (NIAMADD, 0.5 micrograms/ml), luteinizing hormone (LH) NIAMADD, 5 micrograms/ml), estradiol-17 beta (1 microgram/ml), pyruvate (20 microM), and 10% heat-treated fetal calf serum. Medium was overlaid with paraffin oil. Oocytes (n = 891) were fixed at the end of each 3-h interval from 0 to 24 h of culture, or at 24 h after addition of cycloheximide (10 micrograms/ml at 10 different times during maturation (0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h; n = 175). At each time point, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spent on each step was determined. The germinal vesicle (GV) was present from 0 to 6.6 h, GVBD at 6.6 to 8.0 h, chromatin condensation at 8.0 to 10.3 h, metaphase I at 10.3 to 15.4 h, anaphase I at 15.4 to 16.6, telophase I at 16.6 to 18.0 h, and metaphase II at 18.0 to 24 h. Cycloheximide blocked oocyte maturation at GVBD, if added from 0 to 3 h; at chromatin condensation, if present from 6 to 24 h; and at metaphase I, when present from 9 to 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear protein synthesis in animal and vegetal hemispheres of Xenopus oocytes

Experiments were conducted to determine if nuclear proteins are preferentially synthesized in the vicinity of the nucleus, a factor which could facilitate nucleocytoplasmic exchange. Using Xenopus oocytes, animal and vegetal hemispheres were separated by bisecting the cells in paraffin oil. It was initially established that protein synthesis is not affected by the bisecting procedure. To determine if nuclear protein synthesis is restricted to the animal hemisphere (which contains the nucleus), vegetal halves and enucleated animal halves were injected with [ 3H]leucine and incubated in oil for 90 min. The labeled cell halves were then fused with unlabeled, nucleated animal hemispheres that had been previously injected with puromycin in amounts sufficient to prevent further protein synthesis. Thus, labeled polypeptides which subsequently entered the nuclei were synthesized before fusion. Three hours after fusion, the nuclei were isolated, run on two-dimensional gels, and fluorographed. Approximately 200 labeled nuclear polypeptides were compared, and only 2 were synthesized in significantly different amounts in the animal and vegetal hemispheres. The results indicate that nuclear protein synthesis is not restricted to the cytoplasm adjacent to the nucleus.

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.

A cytochemical study of the transcriptional and translational regulation of nuclear transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids.

Immunocytochemical localization and in situ hybridization techniques were used to investigate the presence of spermatid nuclear transition protein 1 (TP1) and its mRNA during the various stages of spermatogenesis in the rat. A specific antiserum to TP1 was raised in a rabbit and used to show that TP1 is immunologically crossreactive among many mammals including humans. During spermatogenesis the protein appears in spermatids as they progress from step 12 to step 13, a period in which nuclear condensation is underway. The protein is lost during step 15. An asymmetric RNA probe generated from a TP1 cDNA clone identified TP1 mRNA in late round spermatids beginning in step 7. The message could no longer be detected in spermatids of step 15 or beyond. Thus, TP1 mRNA first appears well after meiosis in haploid cells but is not translated effectively for the several days required for these cells to progress to the stage of chromatin condensation. Message and then protein disappear as the spermatids enter step 15. In agreement with a companion biochemical study (Heidaran, M.A., and W.S. Kistler. J. Biol. Chem. 1987. 262:13309-13315), these results establish that translational control is involved in synthesis of this major spermatid nuclear protein. In addition, they suggest that TP1 plays a role in the completion but not the initiation of chromatin condensation in elongated spermatids.

Regulation of expression driven by human immunodeficiency virus type 1 and human T-cell leukemia virus type I long terminal repeats in pluripotential human embryonic cells.

Human pluripotential embryonic teratocarcinoma cells differentially expressed gene activity controlled by the human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) long terminal repeats (LTRs) when differentiation was induced by the morphogen all-trans retinoic acid. The alterations occurred after commitment and before the appearance of the multiple cell types characteristic of these pluripotential cells. After commitment, gene activity controlled by the HIV-1 LTR markedly increased, whereas that controlled by the HTLV-I LTR decreased. Steady-state mRNA levels and nuclear run-on transcription indicated that the increased HIV-1-directed activity during differentiation occurred posttranscriptionally, whereas the decreased HTLV-I activity was at the transcriptional level. Phorbol esters did not cause commitment but strongly enhanced expression by both viral LTRs at the transcriptional level. A specific inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, indicated that the enhanced activity involved the activation of protein kinase(s) C; altered cyclic nucleotide metabolism was apparently not involved. Differentiating cells gradually lost the ability to respond to phorbol ester stimulation. Experiments with a deletion mutant of the HIV-1 LTR suggested that this was due to imposition of negative regulation during differentiation that was not reversed by phorbol ester induction. Cycloheximide, with or without phorbol ester, slightly stimulated HIV-1-directed activity at the transcriptional level and massively increased the amounts of steady-state mRNA by posttranscriptional superinduction. It appeared, however, that new nuclear protein synthesis was required for maximal transcriptional stimulation by phorbol esters. Thus, changing cellular regulatory mechanisms influenced human retrovirus expression during human embryonic cell differentiation.

Nuclear protein synthesis in thymocytes of X-irradiated rats.

The biosynthesis and phosphorylation of nuclear proteins of thymocytes were investigated in rats after 4.0 Gy whole-body X-irradiation during the period which precedes DNA degradation in lymphoid cells. Proteins were separated by two-dimensional gel-electrophoresis, detected by Coomassie blue staining. Incorporation of [35S]methionine into proteins and phosphorylation of proteins with [32P]inorganic phosphate were determined by scanning densitometry of autofluorograms and autoradiograms, respectively. No change in the quantity of proteins was observed 1 h after irradiation. Decrease in specific activity of [35S]methionine-labelled proteins was seen in most protein fractions. Significant enhancement of phosphorylation of three proteins was established, characterized by molecular weight and pH: MW 20 kD, pH 6.8; MW 35 kD, pH 5.8 and MW 48 kD, pH 5.8. These results suggest that immediately after X-irradiation a short-term increase of chromatin-bound non-histone protein phosphorylation occurs. This finding, along with the previously described enhancement of RNA polymerase II in thymocytes (Zhivotovsky et al. 1982) suggests a temporary gene activation shortly after X-irradiation of the rat.

Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (friend) cells

An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed.

Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes.

Stimulation of quiescent fibroblasts to growth by polypeptide growth factors is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. In contrast to fibroblasts, A431 cells respond to epidermal growth factor (EGF) with a decreased growth rate. Here we report that, in spite of its growth inhibitory effect, EGF rapidly induces transient expression of c-fos mRNA, followed by the synthesis of nuclear c-fos protein. In addition, EGF treatment resulted in elevated levels of c-myc expression. Practically identical results were obtained with variant A431 clones that are resistant to the inhibitory effect of EGF on cell proliferation. These observations suggest that in A431 cells c-fos and c-myc induction is a primary consequence of growth factor-receptor interaction. Indeed, efficient induction of both genes was also observed with cyanide bromide-cleaved EGF, which has previously been shown to be non-mitogenic but able to trigger early events induced by EGF. We observed strong induction of c-fos and to a lesser extent of c-myc also by TPA, and by the calcium ionophore A23187, indicating an important role for kinase C in proto-oncogene activation by growth factors.

On the effect of glucagon on mitochondrial calcium retention in isolated hepatocytes.

Exposure of isolated hepatocytes to glucagon for 45 min caused a 2.5-fold increase in the time (Ca2+ retention time) for which mitochondria subsequently isolated from the cells retained a load of exogenous Ca2+ before its spontaneous release. Half maximal effect of glucagon was observed at a concentration of 0.6 nM. An increase in the Ca2+ retention time was observed after 30 but not 15 min exposure of cells to the hormone. Incubation of hepatocytes with dexamethasone, epinephrine, vasopressin, dibutyryl cyclic AMP or 8-bromo cyclic GMP also induced an increase in mitochondrial Ca2+ retention time. The effect of glucagon was associated with an increase in cellular cyclic AMP and was inhibited by puromycin, cycloheximide and cordycepin, but not by actinomycin D or chloramphenicol. Puromycin caused only a small inhibition of the stimulation by glucagon of mitochondrial pyruvate carboxylation. It is concluded that the effects of glucagon on mitochondrial Ca2+ retention require nuclear DNA-directed protein synthesis and differ, in this respect, from the rapid-onset effects of the hormone on other mitochondrial properties, including pyruvate carboxylation.

Developmental changes in the synthesis of nonhistone nuclear proteins relative to the appearance of a short nucleosomal DNA repeat length in cerebral hemisphere neurons.

Fluctuations in the pattern of synthesis of nonhistone nuclear proteins were noted in cerebral hemisphere neurons during early postnatal development of the rat. Noteworthy changes included the synthesis of an acidic nuclear protein of relative molecular weight 41,000 (41K), two chromatin-associated basic proteins (37K and 38K) and several high molecular weight chromatin acidic proteins. These changes in the synthesis of nonhistone nuclear proteins occur at a developmental stage when a short nucleosomal DNA repeat length has appeared in cerebral hemisphere neurons.

Effects of FSH on acidic nuclear protein synthesis in cultured pig Sertoli cells

We studied the effect of FHS on nuclear protein synthesis by a primary culture of immature porcine Sertoli cells. The cells were cultured in a chemically defined medium and treated with FSH (50 ng/ml) for various periods (4, 8, 25 and 64 h). About 65 spots (pH i < 8) were identified by two-dimensional polyacrylamide gel electrophoresis of the radiolabelled nuclear proteins. After 25 h of FHS treatment, we observed an increase of 5 proteins, a slight decrease of two and a disappearance of one. For the short periods of FSH treatment (4 and 8 h), no effect of FHS on nuclear protein synthesis was observed. After 64 h of FSH treatment, the synthesis of all nuclear proteins appears to be decreased.

Events associated with restoration by zinc of meiosis in apomictic Saccharomyces cerevisiae.

The effects of nutritional alterations (carbon source and zinc) on nuclear division and protein synthesis during apomictic and meiotic development in Saccharomyces cerevisiae 19e1 were investigated. Unlike cells cultivated under meiosis-promoting conditions, cells cultured under apomixis-promoting conditions exhibited extensive protein synthesis during the first 3 h of incubation in sporulation medium, and nuclear divisions were evident during this time. Cycloheximide treatment of the latter cells induced meiosis, and maximum yields of meiotic asci resulted when this treatment was given for the first 3 h in sporulation medium. The results indicate that the decision concerning which developmental route cells will follow is made shortly after transfer to sporulation medium. Electrophoretic analysis of labeled proteins synthesized during sporulation revealed bands unique to both developmental routes.

Genome-independent effects of 1,25-dihydroxy vitamin D-3 on membrane potential

Cell membrane potential, V m , was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10 −10M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of V m (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of V m in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on V m , in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function.

Time-dependent effects of α-amanitin on nuclear maturation and protein synthesis in mammalian oocytes

ABSTRACT The addition of α-amanitin to extrafollicular, cumulus-enclosed ovine oocytes at explantation inhibits meiotic maturation and prevents many of the changes in protein synthesis that normally accompany maturation. By contrast, these inhibitory effects are considerably reduced by either delaying the addition of the drug for 1-4 h or by denuding the oocytes of all associated cumulus cells at the onset of culture. The observations that the inhibitory effect of cordycepin on nuclear maturation is also time-dependent and cumulus-cell-dependent and that the oocyte is susceptible to cordycepin for longer than its sensitivity to α-amanitin are consistent with the differential effects of these drugs on RNA synthesis. It is concluded that a transcriptional event at the onset of maturation is essential for the initiation of those changes in protein synthesis required for the regulation of nuclear and cytoplasmic maturation. It is uncertain, however, whether this transcriptional event occurs within the cumulus cells or within the oocyte.

The behaviour of nuclear proteins during nitrosamine-induced carcinogenesis.

Dimethylnitrosamine given after partial hepatectomy reduces the synthesis of DNA, histone, and, to a lesser extent, of nonhistone proteins, the inhibition being general rather than for specific nuclear proteins. The extent of inhibition of macromolecular synthesis is greatest when the carcinogen is given a few hours after the operation. As a higher incidence of hepatocellular carcinoma is induced when dimethylnitrosamine is injected later, during the wave of DNA replication, it appears that inhibition of nuclear protein synthesis is not a relevant factor in carcinogenesis, unless it relates to loss of a specific non-histone protein present in small amounts. Analysis of sequentially extracted groups of non-histone proteins by 2D electrophoresis did not reveal any changes produced by treatment with dimethylnitrosamine. Animals fed a diet containing diethylnitrosamine showed an increased incorporation of amino acid into histone. Electrophoretic analysis of non-histone proteins revealed two reproducible effects of feeding the carcinogenic diet: relative to the bulk of nuclear non-histone protein, there was a reduction in the amount of a slightly basic 65000 mol. wt. polypeptide, and an increase in the level of a high molecular weight protein that was almost undetectable in material from normal rats. As these changes were not induced by partial hepatectomy of normal animals, it is possible that they are related to malignancy rather than to the associated increase in cell replication.

Correlation of patterns of nuclear protein synthesis with differentiation of mitogen-stimulated B-lymphocytes.

Nuclear proteins synthesised by mouse lymphocytes stimulated by the B-lymphocyte mitogen lipopolysaccharide have been analysed by 2-dimensional polyacrylamide gel electrophoresis. It has been shown that the rate of synthesis of both nucleoplasmic proteins and nonhistone chromatin proteins is stimulated dramatically by LPS and many nuclear proteins are synthesised that were undetectable in resting lymphocytes. The majority of these proteins are synthesised by lymphoblasts and plasma cells, the lymphocytes that remain small after LPS stimulation synthesizing relatively few proteins. Two chromatin proteins that have previously been shown to appear in the nucleus within 4h of mitogen stimulation exhibit a continuous increase in their rate of synthesis during lymphocyte differentiation. The possible roles of these proteins in differentiating lymphocytes are discussed.

Volumen und Proteinsynthese der Kerne des Stratum Basale in der Stumpfepidermis während des Wundverschlusses nach Amputation der Vorderextremität bei Triturus vulgaris: (limb regeneration/wound closure/autoradiography/protein synthesis/automatic image analysis).

After injection of tritiated phenylalanine, microautoradiographs of longitudinal sections of the stumps of the forelegs of Triturus vulgairs 1 hour, 2, 3 and 9 hours after amputation and also of the opposite limb (reference) were prepared for quantitative measurements. The epidermal tissue (stratum basale) was examined with regard to proximo-distal changes of nuclear volume and protein synthesis. In the stratum basale of the stump the values for the parameters examined were similar to those of the reference limbs 1h and 2h after amputation, but 3h and 9h after amputation areas of increased nuclear volume were observed as well as an increased rate of protein synthesis per unit of nuclear volume. Protein synthesis in the epidermis of the stump was extremely low 1h after amputation. Protein synthesis in the reference epidermis was also reduced at this time, but then increased although the nuclear volume remained constant.

Isoproterenol modulation of nuclear protein synthesis during rat liver regenereration: An electrophoretic and capillary isotachophoretic study

The administration of isoproterenol in partially hepatectomized rats increased synthesis of the Saline-Tris-EDTA extractable proteins from the 2C liver nuclei when compared to nontreated hepatectomized animals. Nuclei were collected from various regions of the cell cycle by electrostatic sorting. Protein synthesis was examined by two-dimensional gel electrophoresis and capillary isotachophoresis, and the correlation of protein migration observed by the two techniques was made.