Increase In Overall Protein Synthesis Research Articles

Myc coordinates transcription and translation to enhance transformation and suppress invasiveness.

c-Myc is one of the major human proto-oncogenes and is often associated with tumor aggression and poor clinical outcome. Paradoxically, Myc was also reported as a suppressor of cell motility, invasiveness, and metastasis. Among the direct targets of Myc are many components of the protein synthesis machinery whose induction results in an overall increase in protein synthesis that empowers tumor cell growth. At present, it is largely unknown whether beyond the global enhancement of protein synthesis, Myc activation results in translation modulation of specific genes. Here, we measured Myc-induced global changes in gene expression at the transcription, translation, and protein levels and uncovered extensive transcript-specific regulation of protein translation. Particularly, we detected a broad coordination between regulation of transcription and translation upon modulation of Myc activity and showed the connection of these responses to mTOR signaling to enhance oncogenic transformation and to the TGFβ pathway to modulate cell migration and invasiveness. Our results elucidate novel facets of Myc-induced cellular responses and provide a more comprehensive view of the consequences of its activation in cancer cells.

Translational regulation of light-harvesting complex expression during photoacclimation to high-light in Chlamydomonas reinhardtii

When challenged with excess light, the green alga Chlamydomonas reinhardtii responds, in part, by down-regulating light harvesting capacity at Photosystem II while concomitantly reorganising cellular metabolism to increase sink capacity. We examined the role of translational control during different stages of photoacclimation by analysing polysome profiles of two different light-harvesting complex (LHC) genes encoding a major LHCII component ( Lhcbm) and CP29 ( Lhcb4) plus iron superoxide dismutase ( FeSOD), and through measurement of protein synthesis by in vivo labelling. Within two hours following transfer of low-light (LL) acclimated cultures into high-light (HL), Lhcbm transcripts are off-loaded from polysomes indicating a decline in translational initiation. Lhcbm translational repression at two hours was specific since FeSOD mRNA remained associated with polysomes and in vivo labelling showed an increase in overall protein synthesis. After 4 hours HL exposure, however, a global disassembly of polysomes was detected suggesting a general decline in translational initiation. Interestingly, the decrease in polysomes coincides with maximal FeSOD transcript levels emphasizing that transcript profiles do not always accurately reflect gene expression. Within 8 hours after LL to HL shift, polysomes reform and all transcripts examined are loaded back onto polysomes. Disassembly of polysomes was mimicked by hydrogen peroxide application to non-shifted cultures suggesting that production of hydrogen peroxide due to HL-stress may effect the general decline in polysomes observed during HL acclimation.

Changes in Ribosomal Binding Activity of eIF3 Correlate with Increased Translation Rates during Activation of T Lymphocytes

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. The stimulation of translation is due to an increase in the rate of initiation caused by the regulation of initiation factor activities. Here, we focus on eIF3, a large multiprotein complex that plays a central role in the formation of the 40 S initiation complex. Using sucrose density gradient centrifugation to analyze ribosome complexes, we find that most eIF3 is not bound to 40 S ribosomal subunits in unactivated T lymphocytes but becomes ribosome-bound following activation. Immunoblot analyses of sucrose gradient fractions for individual eIF3 subunits show that the small eIF3j subunit is unassociated with the eIF3 complex in quiescent T lymphocytes, but upon activation joins the other eIF3 subunits and binds 40 S ribosomal subunits. Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and then to the 40 S ribosomes. The association of eIF3j with the other eIF3 subunits appears to be inhibited by rapamycin, suggesting a mechanism that lies downstream from the mammalian target of rapamycin kinase. This association requires ionomycin together with a phorbol ester, which also suggests that calcium signaling is involved. We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation.

Induction of protein synthesis in cardiac fibroblasts by cardiotrophin-1: integration of multiple signaling pathways.

Cardiotrophin-1 (CT-1) is a member of the IL-6 family of cytokines and is expressed in various cardiovascular disease states. CT-1 induces cardiomyocyte hypertrophy, and protects myocytes from ischemia reperfusion injury. We sought to elucidate CT-1 signaling in cardiac fibroblasts with respect to initiation of protein synthesis. Cardiac fibroblasts were isolated from the ventricles of 200-g Sprague-Dawley rats and stimulated with CT-1 at specified concentrations with or without inhibitors of cell signaling pathways. Activation of intracellular signaling pathways was determined by Western analysis and immunocytochemistry. Protein synthesis was measured by incorporation of [3H]leucine. CT-1 treatment resulted in activation of the Jak/STAT, MAPK, and Akt pathways in addition to protein synthesis regulatory proteins with resultant increase in overall protein synthesis. Analysis with phospho-specific antibodies revealed that AG490 (Jak inhibitor), PD98059 (MEK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), LY294002 (PI3-K inhibitor) and rapamycin (mTOR inhibitor) act at different levels in the signaling cascade to inhibit CT-1 induced protein synthesis. Cardiotrophin-1 activates the Jak/STAT, PI3K/Akt, p38 and p42/44 MAPK pathways in cardiac fibroblasts. Use of pharmacologic inhibitors reveals that each of these pathways play a role in CT-1 induced protein synthesis.

Glucose Regulates EF-2 Phosphorylation and Protein Translation by a Protein Phosphatase-2A-dependent Mechanism in INS-1-derived 832/13 Cells

The role of elongation factor (EF)-2 phosphorylation in the regulation of pancreatic beta-cell protein synthesis by glucose was investigated in the INS-1-derived cell line 832/13. Incubation of cells in media containing 1 mm glucose resulted in a progressive increase in EF-2 phosphorylation that was maximal by 1-2 h. Readdition of 10 mm glucose promoted a rapid dephosphorylation of EF-2 that was complete in 10 min and maintained over the ensuing 2 h. Similar results were obtained using primary rat islets or Min-6 insulinoma cells. The glucose effect in 832/13 cells was replicated by addition of pyruvate or alpha-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nm insulin, 30 mm potassium chloride, or 0.25 mm diazoxide, indicating that insulin secretion and/or depolarization of beta cells was not required. The locus of the high glucose effect appeared to be protein phosphatase-2A, the principal phosphatase acting on EF-2. Protein phosphatase-2A activity was stimulated by glucose addition to 832/13 cells, but neither protein phosphatase-1 nor calmodulin kinase III (EF-2 kinase) activity was affected under these conditions. The slower rephosphorylation of EF-2 during the transition from high to low glucose may involve effects on EF-2 kinase activity. Addition of 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside in high glucose led to a marked stimulation of EF-2 phosphorylation, consistent with the possibility that increased AMP kinase activity in low glucose stimulates EF-2 kinase. In parallel with the effects on EF-2 dephosphorylation, addition of high glucose to 832/13 cells markedly increased the incorporation of [(35)S]methionine into total protein. Taken together, these results suggest that modulation of extracellular glucose impacts protein translation rate in beta cells at least in part through regulation of the elongation step, via phosphorylation/dephosphorylation of EF-2.

Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1.

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.

Metabolic changes during cell growth inhibition by the IRF-1 system

A genetic approach based on the activation of interferon-regulated-factor-1 (IRF-1) has been applied to arrest BHK cell growth in spinner flasks through the presence of estradiol in the culture medium, leading to the activation of the constitutively expressed IRF-1/estrogen receptor fusion protein (IRF-1-hER). Two days after estradiol addition cell proliferation was inhibited; cell concentration being kept for the next 6 days, although a decrease in cell viability was observed. IRF-1 activation alters the cell energetic metabolism, as there is extra metabolic activity with higher glucose, glutamine and oxygen consumption rates. Although the proteolytic activity is higher than in the control cells, the cell protein content increases significantly after IRF-1 activation, suggesting an overall increase in protein synthesis. A significant increase in lactate dehydrogenase activity and a higher reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyl tetrazolium bromide) were also observed, showing an increase in cell activity during cell growth inhibition. Changes in ATP and in cellular phospholipid content as well in glucose metabolism were detected, in real time by 31P and 13C nuclear magnetic resonance spectroscopy (NMR). The levels of ATP were unaffected but changes in cellular phospholipid content were observed. Although an increase on ADP and AMP content was observed 48 to 72 h after IRF-1 activation, both the ATP content and the energy charge (EC) remained constant. It can be concluded that the IRF-1 activation leads to a significant increase of the cell activity until the cell is no longer able to maintain the same energetic metabolism and starts to lose viability.

Ca2+ Release through Ryanodine Receptors Regulates Skeletal Muscle L-type Ca2+ Channel Expression

Skeletal muscle obtained from mice that lack the type 1 ryanodine receptor (RyR-1), termed dyspedic mice, exhibit a 2-fold reduction in the number of dihydropyridine binding sites (DHPRs) compared with skeletal muscle obtained from wild-type mice (Buck, E. D., Nguyen, H. T., Pessah, I. N., and Allen, P. D. (1997) J. Biol. Chem. 272, 7360-7367 and Fleig, A., Takeshima, H., and Penner, R. (1996) J. Physiol. (Lond.) 496, 339-345). To probe the role of RyR-1 in influencing L-type Ca(2+) channel (L-channel) expression, we have monitored functional L-channel expression in the sarcolemma using the whole-cell patch clamp technique in normal, dyspedic, and RyR-1-expressing dyspedic myotubes. Our results indicate that dyspedic myotubes exhibit a 45% reduction in maximum immobilization-resistant charge movement (Q(max)) and a 90% reduction in peak Ca(2+) current density. Calcium current density was significantly increased in dyspedic myotubes 3 days after injection of cDNA encoding either wild-type RyR-1 or E4032A, a mutant RyR-1 that is unable to restore robust voltage-activated release of Ca(2+) from the sarcoplasmic reticulum (SR) following expression in dyspedic myotubes (O'Brien, J. J., Allen, P. D., Beam, K., and Chen, S. R. W. (1999) Biophys. J. 76, A302 (abstr.)). The increase in L-current density 3 days after expression of either RyR-1 or E4032A occurred in the absence of a change in Q(max). However, Q(max) was increased 85% 6 days after injection of dyspedic myotubes with cDNA encoding the wild-type RyR-1 but not E4032A. Because normal and dyspedic myotubes exhibited a similar density of T-type Ca(2+) current (T-current), the presence of RyR-1 does not appear to cause a general overall increase in protein synthesis. Thus, long-term expression of L-channels in skeletal myotubes is promoted by Ca(2+) released through RyRs occurring either spontaneously or during excitation-contraction coupling.

The response of human dermal microvascular endothelial cells to hypoxia

Dermal microvascular endothelial cells (DMEC) exposed to hypoxic conditions show a rapid induction of several proteins that do not increase in other cell types placed in a similar environment. These DMEC proteins differ from the well-characterized stress proteins that have been observed in a wide variety of cultured cell types. The DMEC proteins are induced rapidly, within 2–4 h, and are expressed transiently. They include a group of acidic proteins ( pI ∼ 5–5.2 ) with molecular weights in the range 100 000–120 000 and at least one glycoprotein ( pI 5.1, M r 57 000 ) that is probably expressed on the cell surface. In some primary DMEC cell strains, this response is accompanied by a transient overall increase in protein synthesis. The oxygen-regulated proteins (ORP) that are induced in most other cell types under hypoxic conditions show little variation in their rate of synthesis in DMEC within the first 24 h. The response of DMEC differs from that of umbilical vein endothelial cells (UVEC) and from spindle-shaped cells derived from DMEC, that show a response to hypoxia that is similar to most other cell types. The changes seen in DMEC proteins take place in the same time scale as ischemia-reperfusion injury and may reflect the specialized change of functions of the microvasculature observed under conditions of hypoxic stress in vivo.

Effect of interleukin 1 on articular cartilage from young and aged horses and comparison with metabolism of osteoarthritic cartilage

Summary The effect of interleukin 1 (il-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of il-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to il-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metallo-proteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The il-1- induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of il-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses. Articular cartilage from affected joints of horses with naturally acquired osteoarthritis did not have metabolic alterations identical to those of il-1-stimulated normal articular cartilage from the same individual, necessitating reevaluation of the validity of the il-1-induced model of osteoarthritis. Osteoar thritis is a common, naturally acquired disease of horses, and tissue from animals of all ages and stages of osteoarthritis is available. The equine model of osteoarthritis may afford an important means of studying the alterations in articular cartilage metabolism as a function of age and disease severity.

Regulation of chicken Hsp70 and Hsp90 family gene expression by transforming growth factor-beta 1.

Transforming growth factor-beta 1 (TGF beta) is a regulator of protein synthesis in cultured chicken embryo cells (CEC). Preceding a gradual increase in overall protein synthesis, members of the Hsp70 family (Hsp70, Hsc70, and Grp78) and the Hsp90 family (90-2 and 90-3) of molecular chaperones are induced rapidly and represent a new class of TGF beta-inducible proteins (I.M. Takenaka and L.E. Hightower, J. Cell. Physiol., 152:568-577, 1992). Herein, 32P-labeled cDNA probes encoding Hsc70 and Hsp90 were used to show that levels of the corresponding mRNAs increased as a fraction of total RNA and in polysomes within five hours of treatment of CEC with TGF beta. This cytokine did not increase rates of hsc70 and hsp90 gene transcription as measured by run-on transcription assays of isolated nuclei. However, the Hsp RNA inductions were inhibited by dactinomycin, indicating a requirement for newly synthesized RNA. Both Hsc70 and Hsp90 mRNAs had relatively short half-lives, measured by Northern blot analyses of dactinomycin chases, which were not altered substantially in TGF beta-treated cells. In contrast, Hsp mRNA half-lives increased in heat shocked CEC exposed to dactinomycin during recovery, revealing a difference in regulation of these genes in stressed cells compared with TGF beta-treated cells. Our results support the conclusion that hsc70 and hsp90 gene expression is regulated posttranscriptionally in TGF beta-treated CEC, and the mechanism likely involves a nuclear event such as increasing the half-lives of nuclear RNA transcripts, processing, or transport into the cytoplasm.

Long-term sensitization training in Aplysia leads to an increase in the expression of BiP, the major protein chaperon of the ER.

Long-term memory for sensitization of the gill- and siphon-withdrawal reflexes in Aplysia californica requires RNA and protein synthesis. These long-term behavioral changes are accompanied by long-term facilitation of the synaptic connections between the gill and siphon sensory and motor neurons, which are similarly dependent on transcription and translation. In addition to showing an increase in over-all protein synthesis, long-term facilitation is associated with changes in the expression of specific early, intermediate, and late proteins, and with the growth of new synaptic connections between the sensory and motor neurons of the reflex. We previously focused on early proteins and have identified four proteins as members of the immunoglobulin family of cell adhesion molecules related to NCAM and fasciclin II. We have now cloned the cDNA corresponding to one of the late proteins, and identified it as the Aplysia homolog of BiP, an ER resident protein involved in the folding and assembly of secretory and membrane proteins. Behavioral training increases the steady-state level of BiP mRNA in the sensory neurons. The increase in the synthesis of BiP protein is first detected 3 h after the onset of facilitation, when the increase in overall protein synthesis reaches its peak and the formation of new synaptic terminals becomes apparent. These findings suggest that the chaperon function of BiP might serve to fold proteins and assemble protein complexes necessary for the structural changes characteristic of long-term memory.

Aedes aegypti: Characterization of hemocyte polypeptide synthesis during wound healing and immune response to inoculated microfilariae

Hemocytes from adult, female Aedes aegypti, intrathoracically inoculated with microfilariae (mf) of the nematode Dirofilaria immitis, were compared to saline-inoculated and uninoculated controls using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 125I-labeling, and wheat germ agglutinin (WGA) binding techniques. Activation of wound healing and/or melanotic encapsulation responses by the inoculation of saline or mf into the host hemocoel induced alterations in the hemocyte activity of these mosquitoes. Protein assays of whole hemocyte lysates revealed that hemocytes from saline- and mf-inoculated mosquitoes had higher protein concentrations than uninoculated controls. Many polypeptides were seen within all three hemocytes preparations when stained with silver nitrate, but there was an overall increase in protein synthesis in hemocytes from inoculated mosquitoes. In addition, a 200-kDa polypeptide was uniquely expressed in hemocytes from inoculated mosquitoes. There were several prominent surface proteins labeled with 125I, and several of these increased dramatically in intensity during wound healing and/or a melanotic encapsulation response. Similar results were seen in two-dimensional separations. A set of basic polypeptides comigrated with an acidic polypeptide resulting in a surface protein of approximately 80–90 kDa that increased in inoculated mosquitoes. Hemocytes from inoculated mosquitoes exhibited a group of three acidic polypeptides, whereas hemocytes from uninoculated mosquitoes exhibited only one of these protein fragments. Three surface polypeptides bound 125I-labeled WGA, and binding of WGA to hemocyte surface polypeptides was successfully inhibited by the incubation of cells with the lectin and its competing sugar.

Cachectin/tumor necrosis factor decreases human adipose tissue lipoprotein lipase mRNA levels, synthesis, and activity.

The effects of the cytokine cachectin/tumor necrosis factor (TNF) on human adipose tissue lipoprotein lipase (LPL) were studied. TNF is produced by activated macrophages and is thought to play a role in mediating hypertriglyceridemia and wasting of adipose tissue triglyceride stores (cachexia) that often accompany infection and malignancy. TNF effects were studied in human adipose tissue fragments maintained in organ culture in the presence of insulin and dexamethasone to induce high LPL activity. Addition of TNF to the culture medium for 20 h caused a dose-dependent inhibition of LPL activity to an average of 37% of controls at 50 U/ml TNF. This inhibition of LPL activity was explained by specific decreases in levels of LPL mRNA (to 40% of controls) and rates of LPL synthesis determined by biosynthetic labeling and immunoprecipitation (to 32% of controls). The decline in LPL synthesis was specific, as it occurred despite a small increase in overall protein synthesis in the presence of TNF. Comparable decreases in LPL activity were observed when TNF was added to adipose tissue cultured solely in the presence of insulin. Thus, similar to results in rodent models, TNF is a potent inhibitor of LPL gene expression in human adipose tissue. TNF may therefore play a role in the disorders of triglyceride catabolism and the pathogenesis of cachexia that occur with stimulation of the immune system in humans.

Growth hormone regulation of DNA replication, but not insulin production, is partly mediated by somatomedin-C/insulin-like growth factor I in isolated pancreatic islets from adult rats.

We have investigated whether the previously demonstrated stimulatory actions of growth hormone on DNA synthesis and (pro)insulin biosynthesis and release of isolated adult rat islets of Langerhans are mediated by an autocrine release of somatomedin-C/insulin-like growth factor I (SM-C/IGF I). In medium containing 1% fetal calf serum, the presence of 16.7 mmol/l glucose, or 2.7 mmol/l glucose supplemented with a concentrate of essential amino acids, caused a significant increase in 3H-thymidine incorporation and insulin release compared to 2.7 mmol/l glucose alone but no increase in SM-C/IGF I release. Further supplementation with 1 microgram/ml growth hormone increased 3H-thymidine incorporation and SM-C/IGF I release within all groups, and insulin release in the 16.7 mmol/l glucose and 2.7 mmol/l plus amino acid groups. The ability of growth hormone to increase 3H-thymidine incorporation in the presence of 16.7 mmol/l glucose, but not its action on insulin release, was partly inhibited by a monoclonal antibody against SM-C/IGF I (control cultures 100%; growth hormone alone 261 +/- 27%, mean +/- SEM; growth hormone + anti-SM-C/IGF I 179 +/- 21%; p less than 0.05, n = 18). Growth hormone, but not 100 ng/ml SM-C/IGF I, increased insulin biosynthesis assessed as immunoprecipitable 3H-labelled insulin by 45%, but this was accompanied by a similar increase in overall protein synthesis. Similarly growth hormone, but not SM-C/IGF I caused a 75% increase in glucose oxidation by islets. Both growth hormone and SM-C/IGF I failed to increase the cellular uptake of alpha-aminoisobutyric acid or 3-O-methyl glucose over a 90 min period.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of chronic dehydration on protein synthesis in neurons of the rat hypothalamus

The rate of methionine incorporation was measured in individual hypothalamic nuclei by a quantitative autoradiographic technique: the l-[ 35S]methionine method. Physiological stimulation of the hypothalamo-neurohypophysial system by two days of water deprivation, a stress that increased the plasma total neurophysins concentration 4–5-fold, resulted in a 70–80% increase in overall protein synthesis in the supraoptic nucleus and in the magnocellular subdivisions of the paraventricular nucleus. The same stimulus had no effect on protein synthesis rate of the suprachiasmatic nucleus and of the other representative cerebral structures examined. This differential effect on protein synthesis was also observed within the paraventricular nucleus.

Lymphocyte blastogenesis. Post-transcriptional controls of protein synthesis

To better understand the regulation of macromolecular synthesis in the response of lymphocytes to a mitogen, we have used two-dimensional electrophoresis to search for specificity in the early increase seen in protein synthesis in human lymphocytes treated with phytohemagglutinin and examined the role of new RNA synthesis in this response. Our results confirm a major increase in overall protein synthesis after 4 h of phytohemagglutinin treatment. A further disproportionate increase in the synthetic rates of certain polypeptides was observed using two-dimensional polyacrylamide gel electrophoresis. While actinomycin-D reduced protein synthesis to a level below that of untreated cells, phytohemagglutinin nonetheless enhanced total protein synthesis even in the presence of actinomycin. Some, but not all, of the disproportionate increases in synthesis seen for certain polypeptides are blocked by actinomycin. These results imply the existence of multiple mechanisms controlling protein synthesis early in the course of lymphocyte stimulation. At least some of these do not require new RNA synthesis and thus operate at a post-transcriptional level.

Messenger RNA content and complexity in normal and overloaded rat heart: a preliminary report.

Cardiac overload is associated with two different changes in gene expression: an increase in overall protein synthesis leading to hypertrophy, and at least two isoenzymic redistributions affecting myosin and creatine kinase. This preliminary report was an attempt to study the cardiac genome in this model. Total RNA (ribonucleic acid) was prepared by a combination of the guanidine-ethanol and phenol procedures. A DNA (deoxyribonucleic acid)-, t(transfer)-RNA-free preparation containing non-degraded 28 and 18 S rRNA and 3% poly A+-m (messenger) RNA was obtained. The poly A+mRNA isolated on an oligo (dT) cellulose column had an average nucleotide length of 1300 to 1800 and its c (complementary) DNA was 600 to 1400 base pairs. The first analysis of the kinetics of hybridation revealed a complex pattern which could be described by division into three components. Northern blot and dot blot using a recombinant cDNA plasmid complementary to myosin heavy chain or to actin mRNA (a gift from A. Weydert and M. Buckingham) indicated the presence of these two mRNA, with the former being frequently degraded. After aortic stenosis, both the content and the concentration of total RNA and poly A+mRNA increased in parallel and peaked by the 4th day at +35%. This is the first suggestion that the control of gene expression is transcriptional in this model.

Effects of triiodothyronine (T3) on the development of rat cerebellar cells in culture

To determine whether or not the effects of thyroxine on the cells of the external granular layer of rat cerebellum are direct or indirect, Purkinje cell-free dissociated cell cultures from 5-day-old rat cerebellum in serum-free medium were treated with triiodothyronine (T3) at concentrations of 20–3000 ng/ml. Cultures were assayed for uptake and synthesis of γ-aminobutyric acid (GABA) after 3 weeks, and for thymidine incorporation at 1, 2, 3 and 4 days. Specific (per mg protein) activity of glutamic acid decarboxylase and of GABA uptake into neurons was reduced by the more physiologic (50 or 500 ng/ml) concentrations of T3, probably due to a non-neuronal increase in overall protein synthesis, since the total per culture values seem unaffected by hormone. Thymidine uptake was significantly increased only at the highest (unphysiologic) concentration of T3 on the second day after treatment. None of the label appeared to be in the nuclei of the neuronal cells at any of the times and concentrations tested.These results are consistent with the hypothesis that T3 does not have a direct effect on the proliferation of neurons derived from the external granular layer, although it does seem to affect some non-neuronal cells. T3 also does not seem to enhance differentiated GABA functions (uptake or synthesis) beyond the levels achieved using insulin, progesterone, putrescine, selenium and transferrin.

Protein synthesis in the uterus of the immature rat: The effects of short exposures to oestradiol-17β

Three methods were used to study the effect of oestrogen on the incorporation of radioactive precursor into uterine protein. Intact tissue was incubated in vitro. Isolated uterine epithelial, stromal and myometrial cells were labelled in vitro. Isolated polysomes were translated in cell free protein synthesising systems. In all of these systems, minor qualitative changes in protein synthesis were detected where the uteri were derived from oestrogen-treated rats. These changes were most dramatic in isolated stromal cells and were accompanied by a marked overall increase in protein synthesis. The translation of mRNA on isolated uterine polysomes revealed a sequence of minor, but reproducible, oestradiol-induced changes. It was difficult, however, to relate these changes to those detected in incubated tissue or cells, possibly because the cell free translation products were not subject to normal post-translational modification and processing.