Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes.

The release of arachidonic acid is the rate limiting step in eicosanoid synthesis. In mammalian cells, the release of arachidonic acid is catalyzed by several enzymes. The 85 kDa cytosolic phospholipase A2 (cPLA2) is the key enzyme for the release reaction because of its specific acyl selectivity in phospholipid substrates. We have previously reported that vitamin E enrichment potentiates the arachidonic acid release as well as the spontaneous prostacyclin release in human endothelial cells. In contrast, similar enrichment of diets caused a dose-dependent suppression of platelet thromboxane synthesis. Therefore, the present study was undertaken to determine the effect of vitamin E on arachidonate release and phospholipaseA2 activity in a platelet precursor cell, the MEG-01 megakaryocyte cell line. When these cells were incubated with different concentrations of vitamin E, cellular incorporation was linear with the dosages of this vitamin. Determination of arachidonate release after labeling cells with [3H]-arachidonate showed that vitamin E enrichment caused a dose-dependent increase in ionophore A23187-induced [3H]-arachidonic acid release. Analysis of PLA2 activity showed that activity was detected in the cytosol and this activity was completely abolished by the addition of anti-cPLA2, antibody. Determination of cPLA2 activity demonstrated that vitamin E enrichment caused an increase in enzyme activity. Analysis of cPLA2 protein by Western blot revealed that vitamin E caused an increase in enzyme protein. These data showed that the potentiation of arachidonic acid release and cPLA2, activity by vitamin E was mediated by the enhanced expression of cPLA2 protein.

Phosphorylation of the precursor sequence of rat B-type natriuretic peptide by p34cdc2 and MAP kinase.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), two distinct members of the natriuretic peptide family, share many features in common. However, differences in expression indicate that the processing mechanisms must be different. The leader sequence of rat BNP contains three potential phosphorylation sites for proline-directed kinases that are not present in the leader sequence of ANP. This study has examined how these sites are used by two somewhat different proline-directed kinases. A peptide containing these sites was phosphorylated in vitro by HeLa p34cdc2 kinase and by sea star p44mpk kinase at rates that were comparable to the rates with peptide substrates that are used to assay these enzymes. Sequence analysis of the phosphopeptide shows that both kinases phosphorylate only the two potential phosphorylation sites surrounding the cleavage site of the BNP precursor. The enzymatic potential for such a phosphorylation of BNP in cardiac tissue is demonstrated by immunoblots and kinase assays, showing that in fetal and in adult rat heart both the atria and the ventricles contain a mitogen-activated protein kinase homologue that can phosphorylate this preproBNP sequence.

Aortic stenosis produces hypertrophy of the rat heart without causing an accumulation of N1-acetylspermidine.

Isoprenaline treatment causes cardiac hypertrophy and an accumulation of N1-acetylspermidine in the rat heart. To determine whether the cardiac hypertrophy is the cause of the increase in N1-acetylspermidine, we produced cardiac hypertrophy by constriction of the aorta and analyzed polyamines in the hearts of these rats 1, 3, and 10 days after the aortic constriction. Our results show that compared to sham-operated animals, this treatment caused a 60% increase in putrescine and a 30% increase in spermidine by day 10, but not the expected increase in N1-acetylspermidine. We conclude that N1-acetylspermidine is not induced by a cardiac overload.

Cardiac mRNA levels during the development and growth of the rat.

Developmental variations of RNA content and of total mRNA activity were examined in fetal, neonatal and adult rats. The amount of cardiac RNA extractable with the LiCl urea extraction procedure decreased from 3 mg/g tissue in the 18-day-old fetal heart to 1 mg/g in the 30-day-old heart and then to 0.5 mg/g in 100-day-old animals. Translations in the reticulocyte lysate translation system showed a similar pattern for total mRNA activity. It is shown that over this entire period both RNA content and mRNA activity vary as an exponential function of the growth rate of the heart and an explanation for this relationship is proposed. An examination of the translation products of the abundant mRNAs shows that the development of the heart is characterized by a general reduction in all mRNAs.

Decreased ratio of alpha- to beta-globin mRNA in reticulocyte membrane RNA.

The amount of RNA obtained from rabbit reticulocyte membrane-bound ribosomes by direct phenol extraction of washed membranes was inversely related to the hematocrit of the animals. Translation of the RNA in the reticulocyte translation system showed that the Mr = 30,000 protein reported to be a marker of membrane polysomes was also made by an endogenous mRNA in this translation system. Analyses of the translation products made in the wheat germ system on Triton X-100 acid urea gels show that membrane RNAs display a characteristic alpha- to beta-globin ratio of 0.77 which differentiates them from RNAs prepared from cytoplasmic polysomes and from the postpolysomal supernatant. These results show that free and membrane-bound ribosomes can be distinguished by the main protein that they produce.

Heating RNA before cell-free translation gives no selective increases among the translation products of RNA from rat heart and mammary gland, rabbit reticulocyte membranes, or trout liver.

A recent study has reported that the heating of a population of guanidinium-extracted mRNAs prior to translation causes a selective increase in the translation of certain mRNAs. To determine if this phenomenon is a general property of mRNAs, we carried out a comparison of the translation products obtained when phenol-extracted rat heart and mammary gland RNAs, rabbit reticulocyte membrane RNAs, and trout liver RNAs were translated in the reticulocyte translation system, with and without a prior heat treatment. Our results show that no selective increase in the translation of mRNAs was observed for any of these samples. Among the 14 RNA preparations examined, one total mammary RNA preparation did display a twofold increase in the translation of all mRNAs after heat treatment. It is shown that the heat enhancement of translational activity observed for this sample was due to the reversible formation of intermolecular aggregates with a contaminant that can be removed by chromatography on oligo(dT)-cellulose. Since heat treatment did not selectively enhance the activity of any mRNA in these samples, our results show that the current practice of translating phenol-extracted RNAs without a prior heat treatment should be satisfactory for the translation of most mRNA populations.

Concentrations of endogenous free amino acids in rabbit reticulocyte lysates and the effect of incubation.

Amino acid analyses show that while the free amino acids found in the rabbit reticulocyte translation system do not increase during nuclease treatment or during prolonged storage, the endogenous levels of many amino acids are so high that the choice of a radioactive precursor for a translation should be based not only on the abundance of the amino acid in the translation product but also on its concentrations in the lysate preparation. It is shown that the variation of amino acid concentrations reported in this study to calculate the amount of radioactive amino acid necessary for satisfactory incorporation.

N1-monoacetylation abolishes the inhibitory effect of spermine and spermidine in the reticulocyte lysate translation system.

At optimum magnesium, the translation of rat heart mRNA in the nuclease treated rabbit reticulocyte lysate system was inhibited by low concentrations of spermidine or spermine but not of putrescine. Spermidine and spermine cause a general reduction in the translation of all the heart mRNAs since no differential effects were observed when the translation products were examined by gel electrophoresis. Spermine was a five times more potent inhibitor than spermidine but no inhibition was obtained with N1-acetylspermidine or N1-acetylspermine. Since analyses of endogenous polyamines demonstrate that the inhibitory concentrations of spermine could be obtained by converting a small fraction of the endogenous spermidine to spermine, these results indicate that interconversions of the polyamines might be a sensitive regulatory mechanism for protein synthesis.

Variation in the lack of polyadenylation of the rat milk protein mRNAs during the lactation cycle.

Translationally active milk protein mRNAs were found as nonpolyadenylated mRNAs in the rat mammary gland during pregnancy, lactation and involution. Analyses of whey protein mRNA and casein mRNA with the corresponding cDNAs showed that the lack of polyadenylation of these mRNAs at different time points of the lactation cycle is not consistent with the hypothesis that polyadenylation may be incomplete in the mammary gland when large amounts of mRNA are synthesized. The fraction of whey protein mRNA and casein mRNA that lacked polyadenylation was inversely proportional to the concentration of each sequence in the tissue during pregnancy, lactation and involution. A model is proposed to explain the finding that in each animal the ratio of casein mRNA to whey protein mRNA was similar in polyadenylated RNA and in nonpolyadenylated RNA at all stages of the lactation cycle.

Variation of the poly(A) size classes in the rat mammary gland during the lactation cycle.

The sizes of the poly(A) tracts associated with rat mammary RNA were determined at several time points in the lactation cycle. The poly(A) tracts in the lactating gland displayed two predominant size class peaks at 80-85 and 45-47 residues. The 9S whey protein mRNA and the 15S casein mRNA purified from the 12 day lactating mammary gland both contained poly(A) tracts displaying a similar size distribution. The 45 residue tracts were a characteristic of lactation; they were not found at 8 days of pregnancy and only small amounts of these shorter poly(A) tracts were found in the 16 day pregnant gland. The poly(A) tracts of the involuted gland displayed the same size characteristics as those of late pregnancy. At all the developmental stages that were examined, the fraction of 45 residue poly(A) tracts was always proportional to the total poly(A) content of the mammary cells.

Methylglyoxal inhibits the translation of natural and chemically decapped mRNAs.

Methylglyoxal was a weak inhibitor of translation in the reticulocyte-lysate cell-free system and it did not display cap-dependent inhibition. A similar inhibition was obtained in a wheat-germ cell-free system that displayed extensive cap-dependent inhibition with the cap analogue 7-methylguanosine phosphate. These results show that the chemical reaction of methylglyoxal with 7-methylguanosine is not the mechanism for the inhibition of protein synthesis by methylglyoxal and that methylglyoxal is a weak general inhibitor of translation.

Reproducible preparation of a wheat germ cell-free system.

This study has examined whether the conditions used for the precipitation of plant mRNPs are suitable for the reduction of endogenous template activity in the wheat germ cell-free system. These results have led to a modification of the procedure for the preparation of the wheat germ system that allows the consistent preparation of an active in vitro translation system with low endogenous activity.

Effect of 7-methylguanosine-5'-phosphate on the translation of rat milk-protein mRNAs in the wheat-germ cell-free system.

The translation of polyadenylated and of non-polyadenylated RNA obtained from lactating rat mammary gland was almost totally inhibited by 0.5 mM 7-methylguanosine-5'-phosphate in the wheat-germ cell-free system. This inhibition was maintained during the preparation of the 9S whey-protein mRNA and of the 12S and 15S casein mRNAs. Chemical decapping of these mRNAs caused a similar reduction of their activity. Although a large fraction of milk-protein mRNAs have been reported to lack 3'-polyadenylation, these results show that the mRNAs in the mammary gland do contain a 5'-terminal 7-methylguanosine cap.

Milk-protein mRNAs and poly(A) in the involuting rat mammary gland return to the levels found during late pregnancy.

Analyses of the rat mammary gland show that the increase in the milk-protein mRNAs during the development of lactation and the rapid disappearance of these sequences during involution are not accompanied by similar changes in the poly(A) content. During the development of lactation the casein mRNA is initially in great excess to the whey-protein mRNA and this differential expression of the genes for the two types of milk proteins is again observed during early involution. Since the amounts of poly(A) and of both milk-protein mRNAs are also similar to the amounts found in the gland during late pregnancy, these results indicate that during early involution the mammary gland has reverted to the pattern of mRNA metabolism that occurs during late pregnancy.

Distribution of rat alpha-lactalbumin and casein mRNA between polyadenylated and nonpolyadenylated RNA.

mRNAs for the two types of milk protein were determined in the polyadenylated (poly(A)+) and nonpolyadenylated (poly(A)-) RNA fractions prepared from lactating rat mammary gland, to assess if a high fraction of poly(A)- mRNA is a general characteristic of milk protein mRNAs. Use of the [3H]poly(U) assay to evaluate the effectiveness of oligo(dT)-cellulose and poly(U)-Sepharose for the preparation of these samples showed that similar, essentially poly(A)-free, RNA samples could be prepared with either resin when the appropriate sample to resin ratio was used. The poly(A) tracts in rat mammary poly(A)+ RNA displayed bimodal distribution with peaks at 40 and 80 nucleotides. Translation of these samples confirmed previous observations that mammary poly(A)-RNA contains high amounts of translational activity; however, it is shown that translation gives an overestimate of the mRNA activity in this fraction. Analyses with complementary DNAs indicated that only 24-35% of the casein mRNA and 14-33% of the alpha-lactalbumin mRNA are present in poly(A)-RNA. Milk protein mRNAs are, therefore, comparable with other abundant mRNAs. Evidence is presented indicating that partial degradation can greatly reduce the extent of polyadenylation with little effect on the amount and on the translational activity of the mRNA.

Optimization of the ethanol precipitation of RNA from formamide containing solutions.

This study shows that high variability and low recoveries are obtained when conventional ethanol precipitation is used to recover RNA from the 70%-90% formamide containing solutions that are used to elute poly(U) Sepharose columns. Precipitations of RNA at different formamide concentrations show that the variability of the recovery increases with increasing formamide concentration and the recovery decreases by 7% for each 10% increase in the formamide concentration. The formamide concentration curve shows that these factors can be optimized by diluting the formamide containing solutions at least to 30% formamide prior to the precipitation.

Analysis of the ratio of alpha- to beta-globin and globin messenger ribonucleic acid content of fractionated rabbit erythroid bone-marrow cells.

The ratio of alpha- to beta-globin mRNA was measured by hybridization of a constant amount of highly purified alpha- or beta-globin cDNA (complementary DNA) with increasing amounts of RNA in the range up to 20% cDNA hybridization, where an essentially linear reaction is obtained. Statistical analysis indicates that the ratio of alpha- to beta-globin can be measured within a maximal error of +/- 0.3 and in most cases is better than +/- 0.15. Under these conditions there is no significant deviation from the ratio of 1.3 in the alpha- to beta-globin mRNA ratio of RNA isolated from erythroid cells rich in pronormoblasts through to reticulocytes. If the ratio of alpha- to beta-globin mRNA exceeded 1.7 or was less than 0.9 in pronormoblasts, it would be detected in these experiments. The overall globin mRNA content increases to a maximal value in the fractions rich in basophilic normoblasts of 30,000--50,000 molecules/cell. However, the accuracy of these determinations is not as great as for the ratio determinations, and no significant deviations were seen except in the cells rich in pronormoblasts, which contained less globin mRNA than the later stages.

A specific dimerization of rabbit beta-globin messenger ribonucleic acid.

Rabbit globin mRNA, when layered in low salt on 0.1 M-NaCl/sucrose gradients, separates into two peaks of material. Translation of these two RNA fractions in the wheat-germ cell-free system, hybridization against globin complementary DNA (cDNA) and cross-hybridization against cDNA species prepared from each fraction show that the first peak sedimenting at 10S is a alpha-globin mRNA and the second peak, sedimenting at approx. 15S, is beta-globin mRNA. The sedimentation rate of the beta-globin mRNA is concentration-dependent. By changing concentration and pH, it is indicated that in low-salt beta-globin mRNA adopts a conformation that leads to specific, but weak, self-dimerization during centrifugation in 0.1M-NaCl. This property permits rapid preparation of intact and relatively pure alpha- and beta-globin mRNA species.