Polyribosomal structures inactive for protein synthesis present in erythroid cells during terminal stages of differentiation.

During the terminal stages of differentiation nucleated erythroid cells from the fetal mouse synthesize hemoglobin at a lower rate because after the last cycle of cell division about half of their polyribosomal structures are rendered inactive for protien synthesis though they maintain their aggregated shape. Partially inactive polyribosomes are tested in comparison with normal polyribosomes for the capacity to support polypeptide chain synthesis in cell-free conditions. The following observations are made: a) no difference is found for the profile on sucrose density gradients; b) partially inactive polyribosomes carry growing polypeptide chains in reduced amounts in comparison with active polyribosomes; c) partially inactive polyribosomes are not capable to release "run off" 80 S ribosomal monomers and to dissociate to active ribosomal subunits. These data are interpreted as the evidence for a block of chain termination producing inactivation of polyribosomes during the late maturation of nucleated erythroid cells.     

Effects of protein synthesis inhibition during plant mitosis

The role of protein synthesis in onion root tips during mitosis has been studied, by using synchronous cell populations. Incubation in cycloheximide (CHM) or anisomycin during early or middle prophase induces the return of these cells to interphase. Therefore, it is suggested that essential proteins are synthesized, which determine the continuation of the cells in mitosis. In late prophase these treatments caused a certain delay in the entry into further stages, suggesting that a protein synthesis probably occurs which determines the duration of the transition from metaphase to anaphase. Mitotic processes which develop after metaphase do not seem morphologically dependent on protein synthesis, in spite of the fact that one of them, the nucleolar reconstruction, is markedly dependent on RNA synthesis. Unexpectedly this reorganization increases its rate in the absence of protein synthesis.     

Increased histone mRNA levels during inhibition of protein synthesis

Inhibition of protein synthesis by cycloheximide or puromycin specifically increases the amount of translatable histone mRNA in exponentially growing and in synchronous G₁ HeLa cells by 5-fold in 3 hours. In this case histone gene expression is uncoupled from DNA replication. We conclude that the level of histone mRNA is regulated by a labile protein and is only indirectly dependent on DNA synthesis.     

Mechanism of protein synthesis inhibition in CNS during postischaemic reperfusion.

We have used brain (dog, rat) and spinal cord (dog, rabbit) cell-free systems to study early postischaemic inhibition of protein synthesis. Ischaemia alone produced a relatively small decrease in activity of all subcellular systems used. When 15 min of normoxic reperfusion was used, more than 30% decrease (p less than 0.01) in [14C]-leucine incorporation was detected. A translational inhibitor that appeared in the postribosomal supernatant fraction at the early stage of reperfusion reduced translational capacity of an initiating cell-free system. It also phosphorylated the small (38 kDa) subunit of eukaryotic initiation factor 2 (eIF-2) in vitro. Effect of the inhibitor can be reversed by addition of partially purified intact eIF-2 and/or high concentration (2 mmol/l) of GTP. A prevention of postischaemic free oxygen radical formation by the reoxygenation with hypoxaemic blood, containing 37.5 mm Hg O2 at 0-5 min and 56 mm Hg O2 at 6-10 min of recirculation, that was followed by 5 min of normoxic reperfusion, resulted in a significant increase (p less than 0.02) of polypeptide chain synthesis in vitro when compared with normoxic reperfusion.     

Increased carbohydrate substitution of lipoteichoic acid during inhibition of protein synthesis.

Decreases in electrophoretic mobilities of intracellular lipoteichoic acid, intracellular deacylated lipoteichoic acid, and extracellular deacylated lipoteichoic acid were observed during inhibition of protein synthesis in Streptococcus faecium after exposure to chloramphenicol or valine deprivation. Increased carbohydrate content, and thus an increased mass-to-charge ratio, rather than changes in ester alanine content or novel fatty acid substitutions, appeared to account for the decreased electrophoretic mobilities. The increase in carbohydrate content, as judged from mobility measurements, was progressive over time and appeared to occur on biosynthetically new lipoteichoic acid as well as on lipoteichoic acid made before inhibition of protein synthesis.     

Impairment of liver regeneration during inhibition of mitochondrial protein synthesis by oxytetracycline

Under standard conditions, liver regeneration is not impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 μg/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 μg/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.     

Quantitative changes in protein synthesis during oogenesis in Xenopus laevis

Protein synthesis rates in Xenopus laevis oocytes from stage 1 through stage 6 were measured. In addition, the translational efficiencies, total RNA contents, and percentages of ribosomes in polysomes in growing oocytes at several stages were determined. Stage 1 oocytes synthesize protein at a mean rate of 0.18 ng hr⁻¹ while stage 6 oocytes make protein at a rate of 22.8 ng hr⁻¹. Polysomes from growing and full-grown oocytes sedimented in a sucrose gradient with a peak value of 300 S, corresponding to a weight-average packing density of 10 ribosomes per mRNA. Ribosome transit times of endogenous mRNAs were essentially unchanged at all stages examined. While the oocyte's total ribosomal RNA content was observed to increase about 115-fold during oogenesis, the percentage of ribosomes in polysomes remained constant at approximately 2%. Taken together, the data suggest that the 127-fold increase in protein synthesis which occurs during Xenopus oogenesis involves the progressive recruitment onto polysomes of mRNA from the maternal stockpile.     

Mechanism of protein synthesis inhibition during stationary phase in Bacillus stearothermophil US

Summary The appearance of a protein (association factor I) in ribosomes from Bacillus stearothermophilus at stationary phase of growth is described. Association factor I is present on 30S subunits and 30S–50S ribosomal couples, but not on 50S subunits. This protein is responsible for the low levels of polyphenylalanine synthesis shown by stationary phase ribosomes. Association factor I is able to bind to free 30S–50S ribosomal couples but not to polysomes, and exerts its effect by inhibiting the initiation step of protein synthesis. Ribosomes preincubated with association factor I have a decreased ability for polypeptide snythesis directed phage mRNA or poly(U).     

Reversible inhibition of protein synthesis in HeLa

Protein synthesis in suspended HeLa S₃ cells is inhibited by more than 50% immediately after addition of 100 μg pronase/ml or 500 μg trypsin/ml. Polyribosome profiles are not altered by exposure of cells to 1 or 2 mg trypsin/ml suggesting that the inhibition affects peptide chain elongation. Protein synthesis resumes after removal of proteases by sedimentation and resuspension of the cells.     

Brugia pahangi: inhibition of protein synthesis

The effects of inhibitors of protein synthesis and electron transport on the incorporation of [14C]leucine and [35S]methionine into protein by the filarial worm Brugia pahangi have been investigated. Cycloheximide inhibits the accumulation of both [14C]leucine and [35S]methionine by the worms and their incorporation into protein. In addition, inhibitors of electron transport and some anti-parasitic compounds also significantly inhibit filarial protein synthesis. Antimycin A and cyanide inhibit [14C]leucine incorporation into protein 63 and 72%, respectively, without affecting either motility or lactate production. Interestingly, the anti-malarial compounds chloroquine and quinacrine also significantly inhibit both accumulation and incorporation of [14C]leucine by B. pahangi. In addition, fluorographs of sodium dodecyl sulfate-polyacrylamide gels of homogenates from filariids incubated in [35S]methionine and cycloheximide with and without chloramphenicol indicate that there is a discrete population of proteins, possibly mitochondrial in origin, that are synthesized in the presence of cycloheximide and are not inhibited by chloramphenicol.