ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

Experimental changes in the amount of maternally stored ribosomes affect the translation efficiency of ribosomal protein mRNA in Xenopus embryo

The amount of maternal free ribosomes in developing Xenopus embryos has been experimentally modified; an increase was obtained by microinjection of purified ribosomes into fertilized eggs, and a decrease was induced by treatment with a drug which reduces the amount of free ribosomes. The effect of this manipulation on the partition of the ribosomal protein mRNA (rp-mRNA) was analyzed during embryo development; it was observed that when ribosomes available for translation are in excess, polysome loading with rp-mRNA decreases. Conversely, when ribosomes are scarce, polysome loading of rp-mRNA increases. These experiments, which artificially stress events observed in the course of development, indicate that there is a relationship between the availability of ribosomes in the cells and the utilization of rp-mRNA for synthesis of ribosomal proteins, as already suggested by previous observations on r-protein synthesis during embryogenesis.     

Dissociation of polysome aggregates by protease k

Apparent large size-classes of zein-synthesizing polysomes from developing kernels of Zea mays L. were converted to smaller polysomes after treatment with Protease K. The reduction in polysome size was not a result of ribonuclease activity, inasmuch as the enzyme did not affect the free polysomes or the size of the mRNA from the membrane-bound polysomes. High concentrations of MgCl(2) in polysome buffer inhibited ribonuclease activity and appeared to cause protein interaction between nascent zein polypeptides. Although Protease K inhibited the polysome's capacity for protein synthesis, it was a useful reagent for determining if polysomes were aggregated by protein.     

Rapid Changes in Levels of Polyribosomes in Zea mays in Response to Water Stress

Sucrose gradient profiles of polyribosomes from the coleoptilar node region of seedlings of Zea mays L. were obtained without pelleting and redispersion of the particles. Water stress caused a shift of ribosomes from the polymeric to the monomeric form, starting about 30 minutes after stress initiation and when the water potential of the tissue began to decrease measurably. After about 4 hours of stress (a decrease in tissue water potential of about 5 bars), most of the higher polymers of ribosomes had shifted to monoribosomes. Release of stress caused the ribosomes to revert from monomeric to polymeric form after a lag period apparently determined by the extent of prior stress. Use of bentonite and isolation of polyribosomes from combined stressed and control tissue gave results indicating that the reduced polyribosomal level was not an artifact caused by ribonuclease during isolation.     

A ribosome-free extract from cultured cells improves recovery of polysomes from the mosquito fat body: analysis of vitellogenin and ribosomal protein rpL34 gene expression

We have examined the association of ribosomal protein rpL34 mRNA with polysomes in Aedes albopictus C7-10 cells in culture using a simple, two-step sucrose gradient. In growing cells, 40-50% of the ribosomes were engaged on polysomes. This proportion could be increased to 80% when metabolism was stimulated by refeeding the cells with fresh medium. Conversely, ribosomes shifted off polysomes when cells were starved with phosphate-buffered saline or cell lysates were treated with puromycin. When similar approaches were used with fat body from blood-fed female Aedes aegypti mosquitoes, we were unable to obtain the polysome fraction that contained vitellogenin mRNA, which is abundantly translated after a blood meal. Addition of post-mitochondrial supernatant from fat body to polysomes from cultured cells shifted the polysome profile towards smaller polysomes and monosomes, in a dose-dependent fashion. Disruption of fat body tissue in a post-ribosomal supernatant from refed cells improved the recovery of polysomes, demonstrating both the engagement of vitellogenin mRNA on polysomes and the mobilization of rpL34 from messenger-ribonuceloprotein particles onto polysomes in blood-fed mosquitoes. These observations suggested that ribonucleases remain active when polysomes are prepared from mosquito fat body, and that cell culture supernatant contains a ribonuclease inhibitor.     

Polyribosomes in protoplasts isolated from tobacco leaves

Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.Abbreviations EGTA ethylene glycol bis (2-aminoethyl ether)-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - mRNA messenger ribonucleic acid - RNase ribonuclease - Tris tris(hydroxymethyl)aminomethane - TCA trichloroacetic acid     

Translational initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells

The association of mRNA and ribosomes with the cytoskeleton of eucaryotic cells may be important for protein synthesis and its regulation. HeLa cells were gently lysed with detergent, and soluble and cytoskeletal framework subfractions were prepared by centrifugation. We analyzed these fractions for ribosomes and confirmed earlier findings that polysomes are preferentially associated with the cytoskeletal fraction. The levels of initiation factors elF-2, elF-3, elF-4A, and elF-4B were quantitated by immunoblotting; all are enriched in the cytoskeletal fraction relative to the soluble fraction. Heat shock, fluoride, pactamycin, and cytochalasin caused the release of both ribosomes and initiation factors into the soluble fraction. However, treatment of the cytoskeletal fraction with EDTA or low levels of ribonuclease resulted in polysome degradation but no release. Therefore initiation factor association with the cytoskeletal framework correlates with the presence of ribosomes, whereas ribosome association does not require intact mRNA.     

Temperature-sensitive breakdown in vivo of polysomes with mutants of Chlamydomonas reinhardii

Summary Two temperature-sensitive mutants of Chlamydomonas reinhardii Dangeard which are defective in protein synthesis were examined. Both show breakdown of their polysomes at the restrictive temperature into monosomes which do not contain fragments of mRNA. Many of the ribosomes still contain nascent peptides able to react with puromycin. The polysome breakdown involves only cytoplasmic (80S) ribosomes and is prevented or reversed when ribosome translocation is inhibited with cyloheximide.     

Batchwise Purification of Liver Ribosomes and Polysomes from Unfasted Mice on Hydroxylapatite

Batchwise purification of liver ribosomes and polysomes on hydroxyl-apatite is a rapid procedure to remove glycogen, hemoglobin, ribonuclease and other contaminants from ribosomal preparations. Ribosomes and polysomes are adsorbed to hydroxylapatite in a Büchner filter funnel and the contaminants are eluted from the hydroxylapatite with 0.15 M KH₂PO₄. The ribosomes and polysomes are then eluted with 0.3–0.4 M KH₂PO₄ and concentrated by centrifugation. The resolution of the polysome profiles was greatly improved following purification. The purified ribosomes could be dissociated into subunits at 0.3 M KCl, and showed no loss of activity in poly-U directed phenylalanine synthesis.     

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.     

Effects of actinomycin D,cycloheximide and kinetin on ribonuclease and beta-fructofuranosidase in water stressed tomato cotyledons

Three days old excised tomato cotyledons were subjected to mannitol induced water stress in the presence of actinomycin D and cycloheximide. Within a few hours, in the presence of actinomycin D but not cycloheximide, water stress induced increase in ribonuclease activities and decrease in beta-fructofuranosidase activities. The water stress action in the presence of actinomycin D was reversible by addition of kinetin. It was postulated that water stress had some immediate fundamental action on the protein synthesis sites at the ribosomes.     

Polysome stability in relaxed and stringent strain of Escherichia coli during amino acid starvation

The influence of amino acid starvation on polysome content was examined in relaxed and stringent strains of Escherichia coli which were isogenic for the RC locus. No difference was observed between the polysome profiles obtained from two different sets of stringent and relaxed strains starved for the same amino acid. In both relaxed and stringent strains, starvation for amino acids other than methionine resulted in only a slight breakdown of polysomes with a concomitant increase of 70S ribosomes. However, starvation for methionine in both RC stringent and relaxed strains of E. coli resulted in a more extensive degradation of polysomes and accumulation of 70S ribosomes. The 70S ribosomes obtained as a result of methionine starvation were more sensitive to degradation to 50 and 30S subunits in 10(-3)m Mg(2+) than 70S monomers obtained either by degradation of polysomes with ribonuclease or by starvation of cells for amino acids other than methionine. The 70S ribosomes from methionine starvation were similar (sensitivity to 10(-3)m Mg(2+)) to 70S ribosomes obtained from cells in which initiation of protein synthesis had been prevented by trimethoprim, an inhibitor of formylation. Since N-formyl-methionyl-transfer ribonucleic acid is required for initiation, the 70S ribosomes obtained in both methionine-starved and trimethoprim-treated cells must result from association of 50 and 30S subunits for reasons other than reinitiation. These results suggest that the level of ribonucleic acid synthesis does not influence the distribution of ribosomes in the polysome profile and vice versa.     

Polysome activity in relation to growth and protein starvation in brains and hearts of cultured early chick embryos

In previous studies, brains but not hearts of intact early chick embryos were found to be sensitive to protein starvation. In this study, the in vitro protein synthetic activity of polysomes isolated from brains was found to be greater than those isolated from hearts. Starvation reduced the protein synthetic activity of polysomes in vitro but the extent of the reduction was approximately the same for both brains and hearts. A reduction in the amount of ribosomes as polysomes may have contributed to the lower synthetic activity of polysomes from tissues of starved embryos but not to the differences in synthetic activities between brains and hearts. In addition, neither the stability of isolated polysomes nor ribosome-associated ribonuclease activity appeared responsible for the differences observed in polysome synthetic activities. In direct relationship to the differential sensitivity of brains and hearts to starvation observed in the intact embryo, ribosomes isolated from brains of both growing and starved embryos were more readily degraded during in vitro incubation than those from hearts.     

Direct Test for Altered Gas Exchange Rates in Water stressed Soybean Nodules

Water stress usually lowers the nitrogenase activity of soybeanroot nodules. This loss in activity might result from an increasedbarrier to nodular gas exchange, from a general reduction inbiochemical function, or both. To test for the possibility ofan increased barrier to gas diffusion, we measured the apparentlag time for initiation of acetylene reduction by intact soybeanplants, both before and after water stress. Mild nodular waterloss (i.e. 10% of fresh weight or less) seldom altered the apparentlag time, whereas severe water stress (20–40% f. wt loss)frequently produced a small increase in apparent lag time. Severewater stress also produced a large decrease (24%) in the externaldiameter of the nodules and a loss of the white lenticel traces.Water stress usually caused a decrease in the apparent K_m foracetylene. The data do not suggest a large change in the diffusiveresistance to acetylene of nodules subjected to water stress.Thus, the observed decrease in nitrogenase activity may resultprimarily from biochemical, rather than physical, changes. However,because of the relatively greater importance of gas-phase diffusionfor oxygen entry, we cannot exclude the possibility of a largechange in a small gas pathway that affects oxygen influx morethan acetylene influx. Diffusion, Glycine max, nitrogen fixation, water stress     

Utilization of stored messenger RNA during early aging of Jerusalem artichoke tuber slices

Using dissociation in 0.8 M KCl, it was established that in freshly excised Jerusalem artichoke (Helianthus tuberosus L.) tuber slices less than 8% of the ribosomes were in polysomes. The first hour of aging in water was the period of most rapid polysome accumulation; over 32% of the ribosomes carried nascent polypeptide chains at the end of this time. Thereafter polysome accumulation continued to increase, but more gradually. While synthesis of high-molecular-weight RNA (presumed mRNA) was inhibited more than 95% by -amanitin during the first hour of aging, the inhibitor had no effect on polysome formation. As determined by [³H]polyuridylic acid hybridization, unaged cells contained polyadenylated RNA with a size range of 6–30S. The amount of polyadenylated RNA did not change during the first hour of aging. In control cells in water the in-vivo rate of protein synthesis increased exponentially during the first 4 h of aging without a comparable increase in polysomes. In -amanitintreated tissues a similar increase in protein synthesis was not observed despite the presence of near control levels of polysomes. It is suggested that early polysome formation depends on stored mRNA. Inhibition of mRNA synthesis by -amanitin prevents the normal development of an enhanced rate of protein synthesis which is not directly related to numbers of ribosomes in polysomes.Abbreviations Poly(A) polyadenylic acid - Poly(A)+RNA polyadenylated RNA - Poly(U) polyuridylic acid - TCA trichloroacetic acid     

Ribonuclease Activity Associated With Ribosomes of Zea mays

At pH 6.5, a ribonuclease(s) is associated with ribosomes isolated from corn (Zea mays L.) and cannot be removed by repeated differential centrifugation or by sedimenting through the sucrose gradient. The enzyme is active under conditions favoring the maintenance of integrity of the ribosomes. Little or no latent ribonuclease appears to be present. The activity of the enzyme at pH 5.8 is stimulated by KCl and inhibited by polyvinyl sulfate, zinc, and bentonite. Deoxyribonuclease is also found on the particles.

The enzyme can be removed from ribosomes by adsorption onto bentonite. Ribosomes are also adsorbed but to a much lesser extent at low bentonite concentrations. The enzyme is easily dissociated from ribosomes by raising the pH to 8.5, and readsorbed when the pH is lowered.

The ribonuclease activity on ribosomes shows a sharp increase with cell age that parallels closely the increase in total activity in the homogenate. The ratio of activities of deoxyribonuclease to ribonuclease on ribosomes also changes with cell age and again the changes appear to reflect changes in the homogenate. It is suggested that most of the association of ribonuclease with corn ribosomes may not be meaningful in vivo and occurs only after the cells are ruptured.

     

Action of red and far red light on ribosome formation in cabbage seedlings

The action of light on ribosome formation was examined in the cabbage seedlings, a system extensively used in the studies of anthocyanin synthesis. Ribosomes were extracted 18 h after the beginning of the irradiation and separated by sucrose gradient centrifugation. In the cotyledons of dark-grown cabbage seedlings, a brief red light induces an increase both in total ribosomes and in the fraction present as polysomes; the effect of red light is reversed by far red light, indicating the involvement of phytochrome in polysome formation in cabbage seedlings. Continuous red and continuous far red light are about equally effective in bringing about an increase of total ribosomes and of the polysome fraction. Streptomycin, which inhibits chlorophyll synthesis and chloroplast development, and enhances anthocyanin synthesis in cabbage seedlings, causes a decrease of total ribosomes and of the fraction present as polysomes. In hypocotyls, the red-far red reversibility is evident only for the polysome content and streptomycin does not decrease the polysome/monosomo ratio as it does in cotyledons.     

Use of antibiotics to determine ribosome-messenger RNA interactions necessary for in vivo stability of specific messengers

With the use of inhibitors of individual reactions in protein synthesis, a method has been developed for (a) determining the role of ribosome messenger RNA interactions in specific bacteriophage T4 mRNA stability and (b) localizing the primary site of interaction of messenger ribonuclease (mRNase) on messengers. Antibiotics that freeze ribosomes in or near the initiation site stabilize T4 deoxynucleotide kinase mRNA. In contrast, T4 α-glucosyltransferase mRNA is stable only when the polysome configuration is kept intact. These results indicate the deoxynucleotide kinase mRNA initiation site is most susceptible to mRNase action, whereas the primary site of mRNase action on the α-glucosyltransferase mRNA is distal to the initiation site. Determining the role of ribosome-mRNA interactions in mRNA stability by the use of specific inhibitors of protein synthesis may be applicable to any procaryotic or eucaryotic mRNA that can be translated in vitro.