cAMP induces co-translational modification of proteins in IPC-81 cells.

An elevated cAMP concentration results in growth arrest and protein synthesis-dependent apoptosis in the promyelocytic leukaemia cell line IPC-81. A comparison of two-dimensional gels of extracts from these cells labelled with [(35)S]methionine revealed that five distinct protein spots were induced by cAMP in a protein-synthesis-dependent manner. The spots seemed to result from the acidic shift of a precursor protein.

cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells.

Treatment of IPC-81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin-independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP-dependent protein kinase (cAPK) in vitro induces such activator-independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis.

Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis.

Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis.

On the mechanism of stimulation of peroxisomal beta-oxidation in rat heart by partially hydrogenated fish oil.

By feeding rats a diet containing 20% (w/w) partially hydrogenated fish oil (PHFO), an apparent 6.3-fold increase in the cyanide insensitive palmitoyl-CoA-dependent NAD+ reduction was observed for the heart peroxisomal fractions. This finding was confirmed by a 7.

The peroxisomal beta-oxidation enzyme system of rat heart. Basal level and effect of the peroxisome proliferator clofibrate.

Peroxisomes, isolated from homogenates of rat hearts (myocard), contain a beta-oxidation enzyme system which is indistinguishable from that found in liver, but the total capacity of beta-oxidation is only 0.8% of the liver value (expressed per g of tissue). Fatty acyl-CoA oxidase was assayed by an H2O2 based fluorescent assay avoiding important interfering side reactions.

The expression of cAMP-dependent protein kinase subunits is differentially regulated during liver regeneration.

The levels of the regulatory (RI and RII) and catalytic (C) components of cAMP-dependent protein kinase and of their messages were studied during the first 36 h of liver regeneration after 70% hepatectomy. Both RI alpha mRNA and RII alpha mRNA started to increase 4 h after the resection, reaching peak levels after 9 h. RI mRNA decreased abruptly 9-12 h after resection, whereas RII mRNA stayed elevated.

On the mechanism of induction of the enzyme systems for peroxisomal beta-oxidation of fatty acids in rat liver by diets rich in partially hydrogenated fish oil.

In this paper, we describe the early biochemical changes in liver cells that occur in rats fed a semisynthetic diet containing 20% (w/w) partially hydrogenated fish oil. Within hours the level of ornithine decarboxylase (ODC) increased, peaked at about 24 h (11-fold increase) and returned to subnormal levels within 48 h. The diet evoked a similar rapid increase in the cellular level of mRNA for the bifunctional enzyme of peroxisomal beta-oxidation (enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase (HD)) (12-fold), followed by increases in the specific content of HD protein (3-fold) and the capacity for beta-oxidation in peroxisomes (5.

Relation between induction of ornithine decarboxylase and specific gene expression in rat liver in response to the tumor promoter agent clofibrate.

The time course of changes in a number of biochemical parameters in rat liver was studied during 10 days of clofibrate administration. Ornithine decarboxylase (ODC) and putrescine levels began to increase within hours of the first dose and reached maxima at about 36 h (40 and 10 times control levels, respectively) and then returned to normal levels by 48 h. This ODC induction by clofibrate is different from that seen in compensatory liver hyperplasia or diethylnitrosamine administration in that it was not accompanied by elevations in cAMP or increased activation of cytoplasmic cAMP-dependent protein kinases, type I or II.

Properties of an intracisternal A-particle-associated endonuclease activity which is stimulated by ATP.

An endonuclease activity associated with purified proteinase K-treated intracisternal A-particles was identified and characterized. The activity required divalent cations, preferring Mn2+ to Mg2+. Salt concentrations above 50 mM inhibited the activity.

Cell-free translation of messenger RNA for a myeloma light chain prepared from synchronised plasmacytoma cells.

Crude preparations of biologically active mRNA, which code for a myeloma (MPC-11) light chain, were isolated by two successive sucrose gradient centrifugations of RNA extracted from membrane-bound ribosomes, mRNA thus obtained was separated into a poly(A)-rich and a poly(A)-poor fraction by oligo(dT)-cellulose chromatography. Both these fractions were able to direct the synthesis of light chains in reconstituted cell-free systems derived from heterologous cells (ascites tumor lysates) and homologous cells (MPC-11 cells grown in suspension culture). The identity of the products in vitro was confirmed by comparing their migration with that of light chains produced in vivo upon electrophoresis in sodium dodecylsulphate/polyacrylamide gels, and from the profiles of tryptic peptides obtained by chromatography on Aminex A-5 ion-exchange columns.

Determination of polyadenylate-rich ribonucleic acid in the nucleus and in the cytoplasm of plasmacytoma cells.

A number of parameters affecting the adsorption of rRNA and poly(A)-containing RNA to Millipore filters were investigated separately. Binding of both types of RNA to the filter was dependent on the concentration of RNA, pH and Mg2+ concentration of the reaction mixture. Both types of RNA bound to the filter optimally at slightly acid pH values.