Recovery Of Protein Synthesis Research Articles

Protein synthesis and protein phosphorylation during heat stress, recovery, and adaptation.

Incubating cells at elevated temperatures causes an inhibition of protein synthesis. Mild heat stress at 41-42 degrees C inhibits the fraction of active, polysomal ribosomes from greater than 60% (preheating) to less than 30%. A return to 37 degrees C leads to an increase in protein synthesis, termed "recovery." Continuous incubation at 41-42 degrees C also leads to a gradual restoration of protein synthesis (greater than 70% of ribosomes reactivated by 2-4 h), termed "adaptation". Protein synthesis inhibition and reactivation is prestressed, recovered cells that contain elevated levels of the heat stress proteins occur to the same extent and at the same rate as in "naive" cells. The adaptation response requires transcription of new RNA whereas recovery does not. A large number of phosphorylation changes are induced by severe heat stress and occur with kinetics similar to the inhibition of protein synthesis. These include phosphorylation of eukaryotic protein synthesis initiation factor (eIF)-2 alpha and dephosphorylation of eIF-4B and eIF-4Fp25 (eIF-4E). However, the extent to which the modification occurs is proportional to the severity of the stress, and, under mild (41-42 degrees C) heat stress conditions, these initiation factor phosphorylation changes do not occur. Similarly, under conditions of severe heat stress eIF-2 alpha and eIF-4B frequently recover to their prestress phosphorylation state before the recovery of protein synthesis. eIF-4E dephosphorylation likewise does not occur under mild heat stress conditions. Therefore, these changes in phosphorylation states, which are thought to be sufficient cause, are not necessary for the inhibition of protein synthesis observed.

Regulation of the inducible heat shock 71 genes in early neural development of cultured rat embryos.

Activation of the inducible heat shock 71 genes and their role in the heat shock response was studied in vitro in 9.5 day-old rat embryos at neural tube closure. The transcriptional response of a 71 kilodalton (kD) heat shock gene induced after various regimes of heat shock and acquired thermotolerance was investigated. Expression and accumulation of the heat shock (hs) 71 mRNA in the neuroectoderm was studied by Northern and dot blot analysis. Specific expression in various cell types and regions of the neuroectoderm were examined by in situ hybridization. Exposure of embryos to a heat shock at 43 degrees C for 7.5 min caused high levels of hs mRNA 71 accumulation in the neuroectoderm, pronounced protein synthesis inhibition, and regulated recovery. Specific neuroectoderm cell death followed, resulting in major developmental defects of the eye and forebrain region. A mild heat shock of 42 degrees C for 10 min induced the heat shock response, hsp synthesis, and cell recovery, but produced no cell death or deformities. Preheating the embryo at 42 degrees C resulted in acquired thermotolerance to an otherwise teratogenic 43 degrees C heat shock. Thermotolerance was associated with a rapid recovery of protein synthesis associated with hs 71 mRNA expression. Dot blot analysis showed that after a 42 degrees C heat shock, 71 mRNA was rapidly transcribed and transported into the cytoplasm where it was degraded within 2 hr of the initial response. The results suggest that the heat shock protein (hsp) 71 gene may have a protective rather than a rescuing function.

Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance.

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total RNA, and rRNA synthesis. Increasing exposure of CHO cells to 45 degrees C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotolerance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance. The involvement of rRNA synthesis was further investigated by using actinomycin D at 0.1 microgram m1(-1), a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was approximately 3-fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cells following heat shock.

Responses of the Circadian System in the Aplysia Eye to Inhibitors of Protein Synthesis

Protein synthesis seems to be a general requirement for circadian timing. Defining the time period when inhibition of protein synthesis changes the phase of the biological clock may help identify proteins that are involved in the molecular mechanism of circadian timing.Rothman and Strumwasser (1976), Jacklet (1977), and Lotshaw and Jacklet (1986) gen erated phase response curves (PRCs) for relatively long pulses (6 hr) of anisomycin and puromycin administered to Aplysia eyes. Using somewhat different conditions, we generated a 4-hr anisomycin PRC from Aplysia eyes and found that our anisomycin PRC was similar to that previously described by Lotshaw and Jacklet (1986). We studied recovery of protein synthesis after 1-hr and 6-hr anisomycin treatments and found recovery to be very slow; from 8 to 12 hr appeared to be required for full recovery after anisomycin. Slow recovery occurred when eyes were treated either in buffered artificial seawater or in enriched culture media. Because of the slow recovery after anisomycin, it is difficult to infer accurately from the anisomycin PRC when protein synthesis is important.To identify an inhibitor whose effect reverses quickly, we studied recovery from inhibi tion of protein synthesis after emetine, L-O-methylthreonine, and cycloheximide. Both eme tine and L- O-methylthreonine seemed to reverse no faster than anisomycin, but cycloheximide reversed faster than all the other inhibitors. Cycloheximide (10 mM, 1 hr) produced 89% inhibition of [3H] leucine incorporation, and within 3 hr after removal of cycloheximide, the recovery was 85%.A PRC was obtained using 1-hr treatments of cycloheximide (10 mM). Cycloheximide did not significantly phase-shift from circadian time (CT) 8 to CT 20, and cycloheximide delayed (by about 1 hr or less) the circadian rhythm from CT 20 to CT 8. The cycloheximide PRC was not due to different kinetics of recovery at different phases, as evidenced by similar recovery times when recovery from inhibition by cycloheximide was measured at two phases (a phase when cycloheximide produced no phase shift and a phase when cycloheximide delayed the rhythm).

Regional differences in inhibition and recovery of protein synthesis after transient hindbrain ischaemia of gerbils.

Regional protein synthesis was estimated autoradiographically in a model of transient hindbrain ischaemia of gerbils. In studies of 5 min ischaemia followed by 5 min recirculation, incorporation of [14C]valine into the TCA-insoluble protein fraction was not affected. In studies of 15 min ischaemia followed by 5 min recirculation, incorporation of the tracer into the protein fraction was severely depressed in the ischaemic lesion of the brain stem and cerebellum. However, the granular layer of the cerebellar cortex had partially preserved protein synthesis. When recirculation time was extended to 2 h after 30 min ischaemia, protein synthesis of the cerebellar cortex almost recovered to the full level of the control. However, in the pontine grey matter, inferior colliculus and vestibular nucleus, a significant reduction in protein synthesis persisted. These results indicate that protein synthesis in the pontine grey matter, inferior colliculus and vestibular nucleus are selectively inhibited by ischaemia. Further its recovery after recirculation is slow. The cerebellar cortex is less vulnerable to ischaemia, and recovery is relatively fast. The regional heterogeneity of protein synthesis is neither due to the degree of ischaemia in this model nor the extent of postischaemic hypoperfusion. Factors that influence protein synthesis in this ischaemic model are discussed.

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63 Ag8 cells

Using the suspension cell line P3X63Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0 degrees C. Subsequent resealing of the pores was completed after a 5-min incubation at 37 degrees C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37 degrees C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K+, Mg2+, and Ca2+) and resealing (in the presence of K+, Mg2+, and Ca2+), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.

Focal protein synthesis inhibition in a model of neonatal hypoxic-ischemic brain injury

Cerebral hypoxia-ischemia was produced in 1-week-old rats by exposing them to 8% O 2-92% N 2 commencing 4 to 6 h after unilateral carotid artery ligation. Protein synthesis rates were measured in cerebral cortex, caudate-putamen, thalamus, and lateral septal nuclei during 2 h of hypoxia and at three times (15 min, 10 h, and 18 h) after a 3.5-h hypoxic exposure. Protein synthesis was inhibited in the ipsilateral but not contralateral forebrain during the brief hypoxic exposure, and in both hemispheres during early recovery after a 3.5 h exposure, which was sufficient to produce brain injury in the ipsilateral hemisphere. At 10 after hypoxia, protein synthesis rates in the contralateral forebrain had recovered to control values, but in vulnerable structures of the ipsilateral forebrain, recovery of protein synthesis was transient and incomplete (cerebral cortex) or remained at the low values found immediately after hypoxia (caudate-putamen). The early development of abnormal patterns of protein synthesis in vulnerable brain regions during hypoxia and its persistence in some rats during recovery when irreversible cell injury becomes manifest suggests a possible role for abnormal protein metabolism in the evolution of irreversible brain cell damage.

Role of uptake of [ 14C]valine into protein in the development of tolerance to diisopropylphosphorofluoridate (DFP) toxicity

In a subchronic toxicity study male Sprague-Dawley rats were daily treated with diisopropylphosphorofluoridate (DFP) (0.5 mg/kg, sc) for 14 days. Maximum signs of anticholinesterase toxicity were observed during Days 4 and 5 comparable to those seen 10–15 min following a single sublethal dosage (1.5 mg DFP/kg, sc). Signs disappeared after Days 6–7 of exposure and rats became apparently normal during the remainder of the treatment period. Significant hypothermia was seen following the second to fifth doses with maximum effect after the fifth injection. Subsequent injections of DFP did not cause any reduction in temperature. Incorporation of [ 14C]valine was measured 24 hr after the 5th and 14th injections of DFP, at a time when body temperature had recovered co control values. The rate of in vivo incorporation of [ 14C]valine was measured 0.5, 1.0, and 2.0 hr after a subcutaneous injection of l-[1- 14C]valine at a dose of 5 μCi/mmol/100 g body wt. After five injections the rate of l-[1- 14C]valine uptake into the free amino acid pool and the incorporation into the protein bound pool was significantly ( p < 0.01) reduced in discrete brain regions, liver, kidney, and skeletal muscles. At the end of the 14-day treatment, protein synthesis in all the skeletal muscles tested had recovered completely ( p > 0.01) to the values of nontreated control animals. In brain, liver, and kidney, however, no recovery was seen during this period. The recovery of protein synthesis in skeletal muscle may be one of the mechanisms that lead to tolerance development during prolonged administration of subacute concentrations of DFP.

Cerebral protein synthesis during long-term recovery from severe hypoglycemia.

Regional protein synthesis was investigated in the rat brain during long-term recovery from insulin-induced hypoglycemia with 30 min of cerebral electrical silence. At various time intervals up to 14 days after glucose replenishment, animals received a single dose of L-[3,5-3H]tyrosine and were killed 30 min later. Brains were processed for autoradiography using the stripping film technique. Although hypoglycemia sufficiently severe to cause cessation of EEG activity leads to almost complete inhibition of amino acid incorporation in all "vulnerable" forebrain structures (cerebral cortex, hippocampus, caudoputamen), autoradiographs revealed a very specialized sequence with differential posthypoglycemic restoration of biosynthetic activity in certain neuronal cell types. Three major subpopulations could be distinguished: Neurons that fully regained their protein synthetic capacity within 6 h following hypoglycemia (cortical neurons of layer III-VI, large neurons in the caudoputamen, CA3 and CA4 pyramidal neurons, the majority of granule cells of the dentate gyrus) seemed to escape neuronal necrosis. Prolonged impairment of protein synthesis with only partial restoration during the early posthypoglycemic recovery period (CA1 neurons, most small- to medium-sized neurons of the caudoputamen) carried an increased risk of permanent cell damage. The large majority of these neurons, however, showed full recovery of protein synthesis as late as 7 days after hypoglycemia. Neurons with complete lack of amino acid incorporation after 6 h of recovery (granule cells at the crest of the dentate gyrus, small neurons of the dorsolateral caudoputamen) never resumed protein synthesis, regressed, and died. These studies in conjunction with morphological analysis indicate that the sequential recovery of protein synthesis reflects the extent to which neuronal populations are at risk during severe hypoglycemia.

Recovery of Monkey Brain after Prolonged Ischemia. II. Protein Synthesis and Morphological Alterations

Recovery of protein synthesis following 1 h of complete ischemia of the monkey brain was assessed by 3H-labeled amino acid incorporation in vivo at various postischemic periods between 1.5 and 24 h. The regional autoradiographic patterns obtained were compared on the basis of precursor-product relationships determined biochemically at the end of the tracer incorporation studies. Shortly after ischemia, protein synthesis was severely inhibited, but it gradually recovered with increasing recirculation times. In the cerebellum it returned to almost normal levels within 3 h and in the cortex within 24 h. Hippocampal and thalamic regions, however, did not recover control levels of protein synthesis at 24 h. Histoautoradiographic evaluation of amino acid incorporation in individual neurons revealed recovery of pyramidal neurons in the CA1 and CA3 sectors of the hippocampus within 6 h of recirculation, which, however, was followed by secondary inhibition after longer recirculation. Neurons in cortical layer 5 steadily recovered to near control within 24 h, with the exception of those located in arterial border zones, which returned to only 50% of control at 24 h. Incomplete recovery was also observed in thalamic neurons and Purkinje cells. The regional and histoautoradiographic pattern of protein synthesis correlated with the morphological appearance of cells. Ischemic cell changes (mainly of the dark type with microvacuolization and perineuronal glial swelling) were marked after short recirculation times but gradually disappeared in parallel with the return of protein synthesis in most regions of the brain. Only in pyramidal cells of the hippocampus, thalamic neurons, and Purkinje cells were changes not reversed during the observation period. The results obtained corroborate the electrophysiological observations reported in the first part of this investigation and support the notion that the majority of the neurons of monkey brain survive complete cerebrocirculatory arrest of 1 h for at least 1 day.

Activation of hemin-regulated initiation factor-2 kinase in heat-shocked HeLa cells.

Protein synthesis was drastically inhibited in HeLa cells incubated for 5 min at 42.5 degrees C, but it resumed after 20 min at a rate about 50% that of control cells. After 10 min of heat shock, the binding of Met-tRNAf to 40 S ribosomal subunits was greatly reduced and a polypeptide identified by immunoprecipitation with the alpha subunit of eukaryotic initiation factor-2 (eIF-2) was phosphorylated. Extracts prepared from control and heat-shocked cells were assayed for in vitro protein synthesis. Both extracts were active when supplemented with hemin, but the extract from heat-shocked cells had little initiation activity without this addition. A Mr 90,000 polypeptide and eIF-2 alpha were phosphorylated in this extract, but hemin or an antibody which inhibits the protein kinase designated heme-controlled repressor reduced this phosphorylation. These findings implicated heme-controlled repressor as the kinase at least in part responsible for eIF-2 alpha phosphorylation. Furthermore, the initial inhibition of protein synthesis and eIF-2 alpha phosphorylation after heat shock were reduced by adding hemin to intact HeLa cells. These cells synthesized heat-shock proteins with some delay relative to cells without added hemin. The binding of Met-tRNAf to 40 S ribosomal subunits was inhibited by about 50% in extracts prepared from cells heat-shocked for 40 min, and eIF-2 alpha phosphorylation was increased in these cells. These results suggest that heme-controlled repressor is activated in heat-shocked cells and that eIF-2 alpha phosphorylation limits mRNA translation even after partial recovery of protein synthesis.

Effect of external K+ on protein and DNA synthesis during and after heat shock in rat hepatoma cells.

The effects of extracellular K+ concentrations on protein and DNA synthesis after non-lethal heat shock were studied in the hepatoma cell lines Reuber H35 and HTC. Elevation of the extracellular K+ concentration by equimolar replacement of Na+ by K+ in growth media of Reuber H35 and HTC cells caused an increase of the intracellular K+ content in both cell lines. This property was subsequently used to study the effect of elevated intracellular K+ concentrations on protein and DNA synthesis after hyperthermic treatment at 42 degrees C for 30 min. In normal K+ medium, protein and DNA synthesis were inhibited rapidly after the start of the hyperthermic treatment in both Reuber H35 and HTC cells. Increasing the external K+ concentration of the medium did not influence the inhibition and subsequent recovery of protein synthesis after heat shock in both cell lines. In contrast, in media with elevated K+ concentrations, DNA synthesis after heat-shock was inhibited less in Reuber H35 cells than in cells incubated in normal K+ medium and, furthermore, showed no inhibition in HTC cells. The protective effect of external K+ on DNA synthesis after heat shock was maximal between 50 and 70 mM in the temperature range 42-44 degrees C.

Recovery of protein synthesis and RNA synthesis after rewetting of desiccated leaves of the angiosperm Xerophyta villosa

Summary When desiccated leaves of the desiccation tolerant angiosperm X. villosa are rehydrated, [ 14 C]-uridine incorporation increases steadily during the first 8 h. Incorporation of [ 14 C]-amino acids into protein is detectable at 2h rewetting, and increases rapidly at 6–8 h. Protein synthesis during the first 4 h of rehydration is dependent mainly on new RNA synthesis including mRNA, since RNA synthesis inhibitor 3′ deoxyadenosine strongly inhibits both the incorporation of [ 3 H]-uridine into poly(A)-RNA including total RNA and also the incorporation of [ 14 C]-amino acids into protein.

Regulation of actin mRNA levels and translation responds to changes in cell configuration.

The role of cell configuration in regulating cell metabolism has been studied, using a system in which cell shape and surface contact can easily be manipulated. The suspension of anchorage-dependent mouse fibroblasts in Methocel results in a coordinate decrease of DNA, RNA, and protein synthesis. These processes are restored upon reattachment of cells to a solid surface. This recovery process has two or more components: a rapid recovery of protein synthesis requiring only surface contact, and a slower restoration of nuclear events which is dependent upon extensive cell spreading (A. Ben-Ze'ev, S.R. Farmer, and S. Penman, Cell 21:365-372, 1980). In the present study, we examined 3T3 cells while in suspension culture and after attachment to a tissue culture dish surface to study cell configuration-dependent expression of specific cytoskeleton protein genes. The 3T3 line of fibroblasts used here shows these responses much more dramatically compared with 3T6 cells previously studied. We demonstrate that whereas total protein synthesis was strongly inhibited upon suspension, actin synthesis was preferentially inhibited, decreasing from 12% of total protein synthesis in control cells to 6% in suspended cells. This occurred apparently at the level of translation of actin mRNA, since the amount of actin mRNA sequences in the cytoplasm was unchanged. Reattachment initiated the rapid recovery of overall protein synthesis which was accompanied by a dramatic, preferential increase in actin synthesis reaching peak values of 20 to 25% of total protein synthesis 4 to 6 h later, but then declining to control values by 24 h. Translation in vitro and hybridization of mRNA to a cloned actin cDNA probe revealed that the induction of actin synthesis was due to increased levels of translatable mRNA sequences in the cytoplasm. These results imply a close relationship among cell cytoarchitecture, expression of a specific cytoskeletal protein gene, and growth control. The expression of the actin gene appears to be regulated at both the level of translation (during suspension) and mRNA production (during recovery).

Regulation of actin mRNA levels and translation responds to changes in cell configuration.

The role of cell configuration in regulating cell metabolism has been studied, using a system in which cell shape and surface contact can easily be manipulated. The suspension of anchorage-dependent mouse fibroblasts in Methocel results in a coordinate decrease of DNA, RNA, and protein synthesis. These processes are restored upon reattachment of cells to a solid surface. This recovery process has two or more components: a rapid recovery of protein synthesis requiring only surface contact, and a slower restoration of nuclear events which is dependent upon extensive cell spreading (A. Ben-Ze'ev, S.R. Farmer, and S. Penman, Cell 21:365-372, 1980). In the present study, we examined 3T3 cells while in suspension culture and after attachment to a tissue culture dish surface to study cell configuration-dependent expression of specific cytoskeleton protein genes. The 3T3 line of fibroblasts used here shows these responses much more dramatically compared with 3T6 cells previously studied. We demonstrate that whereas total protein synthesis was strongly inhibited upon suspension, actin synthesis was preferentially inhibited, decreasing from 12% of total protein synthesis in control cells to 6% in suspended cells. This occurred apparently at the level of translation of actin mRNA, since the amount of actin mRNA sequences in the cytoplasm was unchanged. Reattachment initiated the rapid recovery of overall protein synthesis which was accompanied by a dramatic, preferential increase in actin synthesis reaching peak values of 20 to 25% of total protein synthesis 4 to 6 h later, but then declining to control values by 24 h. Translation in vitro and hybridization of mRNA to a cloned actin cDNA probe revealed that the induction of actin synthesis was due to increased levels of translatable mRNA sequences in the cytoplasm. These results imply a close relationship among cell cytoarchitecture, expression of a specific cytoskeletal protein gene, and growth control. The expression of the actin gene appears to be regulated at both the level of translation (during suspension) and mRNA production (during recovery).

Expression of a set of fish genes following heat or metal ion exposure.

Elevation of the incubation temperature of Chinook salmon embryo cells from 20 to 24 degrees C or exposure to heavy metals such as CdCl2 (5 microM) or ZnCl2 (100 to 500 microM) induces the reversible expression of a set of heat shock or stress proteins. Continuous exposure of the cells to either metal ions or heat shock results in recovery of protein synthesis to a control-like pattern. Treatment of these cells with either ZnCl2 or CdCl2 also induces the protein metallothionein. Heat shock, however, does not induce metallothionein, suggesting that it does not belong to the common group of heat shock or stress proteins. The induction of these stress proteins can be inhibited by pretreatment with actinomycin D, suggesting that their expression is regulated at the transcriptional level. The major stress proteins are detectable in the products of an in vitro translation system programmed with RNA isolated from heat shock- or metal ion-treated cells. A recombinant DNA probe complementary to Drosophila mRNA coding for the 70,000-dalton heat shock protein was found to hybridize to RNA isolated from heat shock-or metal ion-treated cells but not from control cells. The fish mRNA coding for the heat shock protein with a molecular weight of 70,000 appears to be of similar size to the corresponding Drosophila mRNA.

Initial inhibition and recovery of protein synthesis in cycloheximide-treated hepatocytes

Previous studies conducted with intact rats had demonstrated that protein synthesis was reversibly inhibited by cycloheximide. Polysome aggregation occurred during inhibition with a return to normal during recovery, suggesting that the block of translational activity involved termination and release of polypeptides. This study involving freshly isolated hepatocytes was undertaken to clarify the mechanism of the biphasic response to cycloheximide. Cycloheximide at 1 μM inhibited [ 3H]leucine incorporation into both cellular and secreted proteins by at least 86%, without having deleterious effects on membrane integrity as indicated by trypan blue uptake and lactate dehydrogenase (LDH) (EC 1.1.1.27) release. After removal of cycloheximide, incorporation of labeled amino acids into cellular protein and protein secreted into the medium returned to control levels. Kinetically, incorporation into secreted protein exhibited a lag of 30–45 min, indicating that a longer recovery period for restoration of proteosynthetic ability is required for membrane-bound polysomes. During the first 100 min of the recovery period, 30% of the cellular protein, which had been prelabeled during cycloheximide inhibition, was secreted into the medium; treated cells, however, secreted prelabeled protein at a lower initial rate. To elucidate the mechanism of action of cycloheximide, the content of the cytoplasmic ribo-nucleoprotein complexes (RFC), polysome size classes, and the distribution of radioactivity among the various ribosome classes were determined during inhibition and recovery. Larger size class polysomes (7+) were increased by cycloheximide treatment and remained increased during recovery. During inhibition, there was enhanced [ 3H]leucine labeling with increasing polysome size, implicating termination as the rate-limiting step, whereas during the recovery phase the labeled nascent polypeptides were removed from the ribonucleoprotein complex at a 3- to 4-fold greater rate than control, indicating an accelerated release of completed proteins.

The role of amino acids in the regulation of protein synthesis in perfused rat liver. I. Reduction in rates of synthesis resulting from amino acid deprivation and recovery during flow-through perfusion.

The role of perfusate amino acid concentrations in regulating rates of protein synthesis was investigated using the perfused rat liver. Livers from fed rats were perfused with a nonrecirculating medium and the incorporation of [3H]leucine into albumin and total protein was determined under conditions where the leucyl-tRNALeu and perfusate leucine specific activities were equal and constant. During perfusions of less than 1 h, rates of total protein synthesis were sensitive to the concentrations of amino acids in the perfusate. When no exogenous amino acids were provided, rates of synthesis of albumin and total protein were 40% of the maximal rates which were achieved when the medium was supplemented with 5 times the normal plasma concentrations of amino acids. However, rates of synthesis in livers perfused with amino acid-deficient medium rose with extension of the duration of perfusion to 95 min. The defect induced by amino acid deficiency did not appear to result from reductions in the charging of tRNA since no change in the quantities of amino acids bound to tRNA occurred in the amino acid-deficient perfusion. The recovery of protein synthesis with time was prevented by inhibitors of proteolysis suggesting a role for protein degradation in this phenomenon.

Induced thermal tolerance and heat shock protein synthesis in Chinese hamster ovary cells

We have performed experiments to determine the kinetics of induction of thermal tolerance in Chinese hamster HA-1 cells, and the effects of heat treatments on the recovery of protein synthesis, with particular attention to whether heat induces specific proteins, perhaps the heat shock proteins (HSP). The kinetics of the development of thermal tolerance were measured by increases in cellular survival. In parallel experiments, the effects of heat treatment on the recovery of protein synthesis in HA-1 cells were examined. After heating (45°, 20 minutes), some of these cells were immediately labeled with 35S-methionine (10 μCi/ml) for 1 hour at 37°, while the others were incubated at 37° for 1–8 hours and then labeled. The cell samples were prepared for electrophoresis on a gradient SDS gel. The incorporation of label into HA-1 cell proteins was drastically inhibited by the 45° heat treatment, but recovered gradually during the 8-hour incubation period at 37°C. A comparison of the proteins synthesized following heat shock with those synthesized by non-heated cells showed that the levels of synthesis of certain proteins were greatly enhanced following the 45° treatment. By 8 hours, it was qualitatively apparent that three proteins, with molecular weights of 59K, 70K and 87K, were synthesized in greater amounts than in untreated cells. The kinetics of HSP synthesis were compared to the kinetics of thermal tolerance; these showed good correlation. Overall protein synthesis also increased during this time, although at a rate slower than the synthesis of the HSP. The question of whether the HSP play a causative role in the development of thermal tolerance and if so, what that role might be, has not been answered.

Effect of interferon on transient shut-off of cellular RNA and protein synthesis induced by mengo virus infection

Infection of mouse L929 cells with Mengo virus resulted in a rapid shut-off of cellular RNA synthesis followed within the first hours post infection by a gradual decrease in host protein synthesis. Pretreatment of the cells with high doses of interferon, blocking viral multiplication, did not affect the virus-induced shut-off of host macromolecular synthesis. In these interferon-treated cells the 2′,5′A-activated nuclease may account for the degradation of viral RNA, soon after its replication. However, the inhibition of host protein synthesis could not be explained by this mechanism. Poly(A)-containing RNA, present in interferon-treated and infected cells, amounted to as much as 70% of that present in interferon-treated, non-infected cells. On the other hand, extracted cytoplasmic RNA was efficiently translated in a reticulocyte lysate, showing that extensive mRNA degradation was not involved in the inhibition of host protein synthesis. In the continued presence of interferon, the virus-induced shut-off was found to be transient. Late in infection, RNA synthesis was found to recover, followed by recovery of protein synthesis and survival of the cells.