General Inhibition Of Protein Synthesis Research Articles

Cellular oxygen sensing need in CNS function: physiological and pathological implications.

Structural and functional integrity of brain function profoundly depends on a regular oxygen and glucose supply. Any disturbance of this supply becomes life threatening and may result in severe loss of brain function. In particular, reductions in oxygen availability (hypoxia) caused by systemic or local blood circulation irregularities cannot be tolerated for longer periods due to an insufficient energy supply to the brain by anaerobic glycolysis. Hypoxia has been implicated in central nervous system pathology in a number of disorders including stroke, head trauma, neoplasia and neurodegenerative disease. Complex cellular oxygen sensing systems have evolved for tight regulation of oxygen homeostasis in the brain. In response to variations in oxygen partial pressure (P(O(2))) these induce adaptive mechanisms to avoid or at least minimize brain damage. A significant advance in our understanding of the hypoxia response stems from the discovery of the hypoxia inducible factors (HIF), which act as key regulators of hypoxia-induced gene expression. Depending on the duration and severity of the oxygen deprivation, cellular oxygen-sensor responses activate a variety of short- and long-term energy saving and cellular protection mechanisms. Hypoxic adaptation encompasses an immediate depolarization block by changing potassium, sodium and chloride ion fluxes across the cellular membrane, a general inhibition of protein synthesis, and HIF-mediated upregulation of gene expression of enzymes or growth factors inducing angiogenesis, anaerobic glycolysis, cell survival or neural stem cell growth. However, sustained and prolonged activation of the HIF pathway may lead to a transition from neuroprotective to cell death responses. This is reflected by the dual features of the HIF system that include both anti- and proapoptotic components. These various responses might be based on a range of oxygen-sensing signal cascades, including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low P(O(2)) affinity cytochrome c oxidase (aa(3)) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF prolyl-hydroxylase (PHD) and HIF asparaginyl hydroxylase, known as factor-inhibiting HIF (FIH-1). Thus specific oxygen-sensing cascades, by means of their different oxygen sensitivities, cell-specific and subcellular localization, may help to tailor various adaptive responses according to differences in tissue oxygen availability.

Translational Thermotolerance Provided by Small Heat Shock Proteins Is Limited to Cap-dependent Initiation and Inhibited by 2-Aminopurine

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.

Expression of constitutively active 4EBP-1 enhances p27Kip1 expression and inhibits proliferation of MCF7 breast cancer cells

BackgroundEukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells.ResultsA constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1.ConclusionsExpression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.

PROTEIN SERINE/THREONINE PHOSPHATASE 2A ACTIVITY IS INHIBITED BY cAMP IN MA-10 CELLS

PP1 and PP2A are members of the protein serine/threonine phosphatases (PPs) family and their activities have been proposed as a requirement for hormone- and cAMP-regulated steroid synthesis. These findings raise the question whether the PPs activity is increased by hormonal action in steroidogenic systems. Thus, the aim of the study was to evaluate the action of cAMP on the activity of PP1 and PP2A in MA-10 Leydig cells. Our results demonstrate that 8Br-cAMP stimulation produces a transient inhibition of PP2A activity. In contrast, PP1 activity remains unchangeable. As reported in other steroidogenic cells, cAMP-induced steroidogenesis in MA-10 cells is reduced by Cantharidin (Can) and also by Calyculin A (CA), two chemically unrelated PP1/PP2A inhibitors (data not shown). Taking into account the inhibitory effect of cAMP treatment on PP2A activity, the latest findings result paradoxical. Therefore, we next evaluated the action of these compounds on total protein synthesis. Can 10−5 M and CA 10−7 M markedly reduced total protein synthesis (35 and 50% respectively) in MA-10 cells, measured by 35S-methonine incorporation. These results suggest that hormone-dependent steroidogenesis is working through inhibition of PP2A-dependent dephosphorylation and the effect of PP1/PP2A inhibitors on steroidogenesis may be due to a general inhibition of protein synthesis rather than to a specific action on StAR protein induction.

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.

Translational inhibition of E-selectin expression stimulates P-selectin-dependent neutrophil recruitment.

Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E-selectin-dependent rolling similar to that of tumor necrosis factor-alpha, suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E-selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation. Cycloheximide and thrombin (4 h) induced sufficient P-selectin-dependent rolling to recruit as many neutrophils as were recruited with 4 h of stimulation with thrombin alone. Histamine in the presence of cycloheximide or cycloheximide alone did not evoke the P-selectin response at 4 h, suggesting that this was not due to direct cycloheximide induction of P-selectin. Treatment of endothelium with tumor necrosis factor-alpha (an E-selectin inducer) and cycloheximide also eliminated E-selectin expression but, much like thrombin, induced P-selectin expression and neutrophil recruitment. In conclusion, inhibition of E-selectin via protein synthesis inhibition activates the protein synthesis-independent pathway of P-selectin expression to support adequate leukocyte recruitment.

Inhibition of S6 Kinase by Rapamycin Blocks Maturation ofRana dybowskiiOocytes

Studies were carried out to define the hormone-induced signal transduction pathway during maturation ofRana dybowskiioocytes. Rapamycin, a specific inhibitor of S6 kinase, blocked progesterone-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner indicating that S6 kinase is required for meiotic maturation ofRanaoocytes. Addition of rapamycin within 3 h, but not 6 h, of progesterone treatment inhibited GVBD. In contrast, cycloheximide, a general protein synthesis inhibitor, blocked GVBD even when added 9 h after progesterone addition. A twofold increase in S6 kinase activity occurred within 1 h of progesterone stimulation and rapamycin inhibited this activity. Rapamycin also suppressed, in a dose-dependent manner, progesterone-induced protein synthesis during the first 12 h of culture but less effectively later. Histone H1 kinase activity (maturation-promoting factor, MPF) was observed in oocyte extracts at two different times (between 6 and 9 h and at 24 h) following progesterone stimulation. Rapamycin blocked H1 kinase activity between 6 and 9 h of culture but not that observed at 24 h. In contrast, cycloheximide suppressed progesterone-induced H1 kinase activity as well as protein synthesis throughout the course of incubation. Such results indicate that rapamycin and cycloheximide have common and unique effects on oocyte maturation and suggest that progesterone-induced S6 kinase activity is closely associated with induction of protein synthesis and activation of MPF during oocyte maturation. Results inRanacontrast with those obtained inXenopuswhere rapamycin inhibited S6 kinase but failed to inhibit GVBD or protein synthesis. Differences in the response ofRanaandXenopusoocytes to rapamycin are discussed in relation to seasonal, biochemical, and species variations.

Effect of Lead on the Synthesis of Tissue Plasminogen Activator by Vascular Endothelial Cells in Culture

To investigate the effect of lead on the synthesis of fibrinolytic proteins by vascular endothelial cells, cells obtained from human umbilical vein were exposed to the metal in a culture system. It was found that the secretion of tissue plasminogen activator (t-PA) is inhibited by lead but that of plasminogen activator inhibitor type 1 (PAI-1) was unaffected by the metal. However, gene expression of t-PA as well as PAI-1 was unaffected by lead when evaluated by quantitative reverse transcription-polymerase chain reaction. The inhibition of t-PA secretion by lead was observed even in the presence of actinomycin D, an inhibitor of mRNA synthesis. The inhibition of t-PA secretion by lead was also observed in the presence of the adenylate cyclase activator forskolin but was suppressed and disappeared in the presence of either 8-bromo adenosine 3', 5'-cyclic monophosphate that is a congener of adenosine 3', 5'-cyclic monophosphate which is resistant to degradation by phosphodiesterase or the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. On the other hand, both the incorporation of [ 14C] leucine into the acid-insoluble fraction of endothelial cells and the leakage of lactate dehydrogenase from the cells were unchanged by lead, indicating that the metal did not cause inhibition of general protein synthesis and nonspecific cell damage. The present data suggest that lead selectively inhibits t-PA synthesis by vascular endothelial cells at the post-transcriptional level and the inhibition is mediated by activation of the cyclic AMP-dependent pathway as a result of a lower activity of phosphodiesterase.

Microplitis croceipes teratocytes: in vitro culture and biological activity of teratocyte secreted protein

Teratocytes originate from the dissociation of the extraembryonic serosal membrane in some Braconidae and Scelionidae. Methods used to culture teratocytes in vitro are described and the yield of teratocyte secreted proteins (TSP) was measured. Although 90% are viable after 6 days, in vitro teratocytes reached only half the diameter (32 μm) of the same age teratocytes obtained in vivo. Teratocytes cultured in vitro secrete as much as 0.7 μg of protein per day per larval equivalent (≈900 cells). Presence of parasitoid larvae enhanced teratocyte viability while periodic exchange of medium did not. However, medium exchange significantly increased the total amount of protein secreted. Size and viability were improved with the addition of 10% FBS to the Ex-cell 400 culture medium. Non-denaturing PAGE showed at least 15 proteins with molecular sizes estimated to be between 24 to 347 kDa in medium containing teratocytes. An in vitro fat body assay was developed to measure the effect of TSP on protein synthesis and juvenile hormone esterase (JHE) activity. Crude TSP inhibited in vitro incorporation of [ 35S]-methionine into protein synthesized by the fat body. The amount of JHE released from in vitro fat body treated with crude TSP was significantly less than controls, most likely caused by the inhibition of general protein synthesis. The active fraction of TSP passed through a 30 kDa molecular weight cutoff filter but was retained by a 3 kDa filter. SDS-PAGE revealed four proteins with molecular weights between 8 and 20 kDa not present in control medium incubated without teratocytes.

Temporal relationships between de novo protein synthesis, calpain and caspase 3-like protease activation, and DNA fragmentation during apoptosis in septo-hippocampal cultures.

Caspase 3-like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade. However, the influence of upstream events on calpain/caspase activation and the role of calpain/caspase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis-related events after staurosporine-induced apoptosis in mixed glial-neuronal septo-hippocampal cell cultures. Following 3 hr exposure to staurosporine (0.5 microM), calpain and caspase 3-like proteases processed alpha-spectrin to their signature proteolytic fragments prior to endonuclease-mediated DNA fragmentation (not evident until 6 hr), indicating that endonuclease activation is downstream from calpain/caspase activation. Cycloheximide, a general protein synthesis inhibitor, completely prevented processing of alpha-spectrin by calpains and caspase 3-like proteases, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains and caspase 3-like proteases. Calpain inhibitor II and the pan-caspase inhibitor Z-D-DCB each inhibited their respective protease-specific processing of alpha-spectrin and attenuated endonuclease DNA fragmentation and cell death. Thus, activation of calpains and caspase 3-like proteases is an early event in staurosporine-induced apoptosis, and synthesis of, as yet, unknown protein(s) is necessary for their activation.

Temporal relationships between de novo protein synthesis, calpain and caspase 3‐like protease activation, and DNA fragmentation during apoptosis in septo‐hippocampal cultures

Caspase 3-like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade. However, the influence of upstream events on calpain/caspase activation and the role of calpain/caspase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis-related events after staurosporine-induced apoptosis in mixed glial-neuronal septo-hippocampal cell cultures. Following 3 hr exposure to staurosporine (0.5 μM), calpain and caspase 3-like proteases processed α-spectrin to their signature proteolytic fragments prior to endonuclease-mediated DNA fragmentation (not evident until 6 hr), indicating that endonuclease activation is downstream from calpain/caspase activation. Cycloheximide, a general protein synthesis inhibitor, completely prevented processing of α-spectrin by calpains and caspase 3-like proteases, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains and caspase 3-like proteases. Calpain inhibitor II and the pan-caspase inhibitor Z-D-DCB each inhibited their respective protease-specific processing of α-spectrin and attenuated endonuclease DNA fragmentation and cell death. Thus, activation of calpains and caspase 3-like proteases is an early event in staurosporine-induced apoptosis, and synthesis of, as yet, unknown protein(s) is necessary for their activation. J. Neurosci. Res. 52:505–520, 1998. © 1998 Wiley-Liss, Inc.

Long-term changes in excitability induced by protein kinase C activation in Aplysia sensory neurons.

Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. In this report, we demonstrate that PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4beta-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produced a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment was independent of protein synthesis, while it was disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level.

Biochemical analysis of the stress protein response in human oesophageal epithelium

Background—The oesophageal epithelium is exposed routinely to noxious agents in the environment, including gastric acid, thermal stress, and chemical toxins. These epithelial cells have presumably evolved effective protective mechanisms to...

Immunoglobulin Binding Protein (BiP) Function Is Required to Protect Cells from Endoplasmic Reticulum Stress but Is Not Required for the Secretion of Selective Proteins

BiP/GRP78 is a lumenal stress protein of the endoplasmic reticulum (ER) that interacts with polypeptide folding intermediates transiting the secretory compartment. We have studied the secretion and the stress response in Chinese hamster ovary (CHO) cells that overexpress either wild-type immunoglobulin binding protein (BiP) or a BiP deletion molecule (residues 175-201) that can bind peptides and ATP but is defective in ATP hydrolysis and concomitant peptide release. Overexpressed wild-type BiP was localized to the ER and unique vesicles within the nucleus, whereas overexpressed ATPase-defective BiP was localized to the ER and cytoplasmic vesicles but was absent from the nucleus. Compared with wild-type CHO cells, overexpression of ATPase-defective BiP prevented secretion of factor VIII, a coagulation factor that extensively binds BiP in the lumen of the ER. Under these conditions factor VIII was stably associated with the ATPase-defective BiP. In contrast, the secretion of monocyte/macrophage colony stimulating factor, a protein that is not detected in association with BiP, was not affected by overexpression of ATPase-defective BiP. These results show that BiP function is not required for secretion of some proteins and suggest that some proteins do not interact with BiP upon transport through the ER. The presence of unfolded protein in the ER induces transcription of BiP and also elicits a general inhibition of protein synthesis. Overexpression of wild-type BiP prevented the stress-mediated transcriptional induction of BiP in response to either calcium ionophore A23187 treatment or tunicamycin treatment. In contrast, overexpression of ATPase-defective BiP did not prevent the stress induction of BiP, showing that the ATPase activity is required to inhibit transcriptional induction. Overexpression of wild-type BiP, but not ATPase-defective BiP, increased survival of cells treated with A23187. The increased survival mediated by overexpressed wild-type BiP correlated with reduced translation inhibition in response to the stress condition. These results indicate that overexpressed BiP alleviated the stress in the ER to prevent BiP transcriptional induction and permit continued translation of cellular mRNAs.

The antiangiogenic agent linomide inhibits tumor necrosis factor-alpha secretion via inhibition of its synthesis.

We have previously reported that linomide, a quinoline-3-carboxamide, has antitumor effects against prostatic cancers in vivo through its ability to inhibit tumor angiogenesis. Subsequently, we reported that linomide inhibits several steps in the process of angiogenesis, including direct effects on endothelial cell proliferation and their chemotactic migration and invasion. Besides these direct effects, linomide's antiangiogenic activity also involves indirect effects secondary to inhibition of tumor infiltration of macrophages and their ability to secrete the angiogenic factor tumor necrosis factor-alpha (TNF-alpha). The current studies were conducted to gain insight into the mechanism by which linomide inhibits macrophage TNF-alpha secretion. The virally transformed RAW 264.7 mouse macrophage cell line was used as a model system. Chronic in vitro exposure (7 days) to 81-650 microM linomide is cytostatic to RAW cells. Such chronic exposure to linomide significantly decreased (P < 0.05) RAW cells' baseline ability to secrete TNF-alpha and also their ability to up-regulate TNF-alpha secretion in response to lipopolysaccharide (LPS) challenge. Ribonuclease protection assays demonstrated that linomide's ability to inhibit baseline and LPS-challenged TNF-alpha secretion is not functioning at the mRNA level, because steady-state levels of TNF-alpha mRNA do not change in response to linomide. Linomide's ability to inhibit TNF-alpha secretion is not associated with an increase in cell-associated TNF-alpha levels. Immunoprecipitation experiments demonstrated that linomide did not inhibit the normal proteolytic processing of the initial 26 kDa plasma membrane-bound TNF-alpha to the secreted 17 kDa soluble form. These results demonstrate that linomide inhibits TNF-alpha secretion by inhibition of the synthesis of the TNF-alpha protein. Linomide's ability to inhibit TNF-alpha protein synthesis is not due to an inhibition of general protein synthesis or secretion and is not mediated via a change in cyclic adenosine monophosphate levels.

Combined effects of ionizing radiation and cycloheximide on gene expression

We performed experiments to determine the effects of ionizing radiation exposure on expression of genes such as beta-actin, c-fos, histone H4, c-myc, c-jun, Rb, and p53 after exposure of Syrian hamster embryo (SHE) cells to the protein synthesis inhibitor cycloheximide. The purpose of these experiments was to determine the role of a labile protein in the radiation-induced response. The results revealed that when ionizing radiation (either fission-spectrum neutrons or gamma rays) was administered 15 min after cycloheximide treatment of SHE cells, the radiation exposure reduced cycloheximide-mediated gene induction of c-fos, histone H4, and c-jun. In addition, dose-rate differences were found when radiation exposure most significantly inhibited the cycloheximide response. Our results suggest that ionizing radiation does not act as a general protein-synthesis inhibitor and that the presence of a labile protein is required for the maintenance of specific gene transcription and mRNA accumulation after radiation exposure, especially at high dose-rates.

Postsynaptic modifications in long-term facilitation in Aplysia: upregulation of excitatory amino acid receptors

Long-term sensitization of the gill and siphon withdrawal in Aplysia is accompanied by facilitation of sensorimotor synaptic connections that depends on new protein synthesis. This phenomenon has been previously shown to involve presynaptic growth. At the postsynaptic level, a reorganization should occur to parallel the formation of new synaptic contacts. We show here that 24 hr following an application of 5-HT, which produces long-term synaptic facilitation (LTF), the response of the motoneuron to an excitatory amino acid agonist of the synaptic receptors is increased. General inhibition of protein synthesis with anisomycin blocks this enhancement. Inhibiton of protein synthesis limited to the postsynaptic neuron by intracellular injection of gelonin, a ribosome-inactivating toxin, also blocks the increase in the response to the agonist but fails to block 24 hr LTF. These results are compatible with a model of LTF that involves coordinate pre- and postsynaptic changes. The latter may include an upregulation of functional postsynaptic receptors. These may not be initially required for LTF measured at a 24 hr time point, but could become necessary for later stages of LTF. An increase in the number of functional postsynaptic receptors in a reserve pool may also prime the postsynaptic neuron for subsequent learning-associated plasticity.

Effects of ochratoxin A upon early and late events in human T-cell proliferation

We have investigated the effect of ochratoxin A (OA) on both early and late events during activation of human T lymphocytes. As early activation parameters we chose to measure both changes in intracellular Ca 2+ levels and the activity of protein kinase C following the triggering process. Our results demonstrate that concentrations of OA that inhibit overall activation had no effect neither on the absolute levels nor on the duration of the Ca 2+ response. Furthermore, PKC activity as measured by phosphorylation of two specific cytosolic substrate substrate proteins, was also unaffected. However, when the cells were prestimulated for 48 h to measure effects on late events in the activation cascade, addition of graded concentrations of ochratoxin A down to 6.4 μm completely inhibited the DNA synthesis. This shows that ochratoxin A is able to block DNA synthesis efficiently even if the activation process has been running for 48 h in advance. To investigate whether the inhibitory effect on DNA synthesis could be ascribed to inhibition of protein synthesis, we carried out experiments to measure protein synthesis both in resting and activated T-cells. In resting T-cells protein synthesis was nearly abrogated by 12.5 μM ochratoxin A, but only minor effects were observed in stimulated cells. The low impact of ochratoxin A on protein synthesis in activated cells may indicate that other mechanisms than just a general inhibition of protein synthesis are operating.

Inhibition of protein synthesis in insect cells by baculovirus-expressed heme-regulated eIF-2 alpha kinase.

To study further the regulation of the heme-regulated eIF-2 alpha kinase (HRI), we have produced functional wild type HRI using the baculovirus expression system. The amount of recombinant HRI protein expressed in insect cells is approximately 10 times higher than levels in reticulocytes. Baculovirus-expressed HRI (BV-HRI) is indistinguishable from HRI purified from rabbit reticulocytes. It is active both as an autokinase and an eIF-2 alpha kinase. BV-HRI is regulated by heme in vitro as well as in intact insect cells. Coexpression of the wild type HRI with the inactive K199R HRI, S51A eIF-2 alpha, or interleukin-1 beta (IL-1 beta) results in diminished expression of these proteins. Expression of wild type HRI also results in severe inhibition of general protein synthesis in Sf9 cells when compared with cells expressing K199R HRI or IL-beta. In addition, the guanine nucleotide exchange activity of eIF-2B is suppressed in Sf9 cells expressing wild type HRI but not in cells expressing the K199R HRI or IL-1 beta. Furthermore, expression of wild type HRI is increased by coexpression with the nonphosphorylatable S51A eIF-2 alpha or by the addition of hemin, which inhibits HRI activity. These results provide evidence that translational regulation by phosphorylation of eIF-2 alpha and sequestration of eIF-2B can operate in insect cells.

Transient inhibition of protein synthesis accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms

It has been widely believed that bloodstream forms of Trypanosoma brucei must be first transformed into intermediary and/or short-stumpy forms in the bloodstream of the mammalian host before differentiation to the procyclic culture form can occur. In our recent studies, the pleomorphic T. brucei strain TREU667 was found to differentiate directly from the long-slender bloodstream form to the procyclic form in Cunningham's medium at 26°C [7]. In the present investigation, the same was found true for another pleomorphic strain of T. brucei, STIB366D. Four independent monomorphic strains of T. brucei were tested; two, #427 and EATRO164, were found capable of differentiating in vitro directly into procyclic forms, whereas the other two, TREU667/RP-56 and EATRO110, could not. There is thus no correlation between the capability of differentiating in vitro and the ability of being converted from long-slender to intermediary and short-stumpy bloodstream forms. Two additional markers for following differentiation, other than observing morphological changes, were tested. Assays for the emerging phosphoenolpyruvate carboxykinase (PEPCK) by immunoblottings worked well, with results agreeing closely with the morphological change. But immunoblottings of glycosomal phosphoglycerate kinase (gPGK) failed to demonstrate a significant decrease in the protein level upon completion of differentiation. Apparently, gPGK has a rather long half-life and is unsuitable as a marker of differentiation. When temperature was dropped from 37°C to 26°C at the starting point of in vitro differentiation, protein synthetic activity in the pleomorphic T. brucei TREU667 bloodstream form was decreased by 4-fold. When the activity was gradually brought back to and beyond the original level after a day's incubation, the profile of newly synthesized proteins was that of the procyclic form. A monomorphic variant of TREU667, RP-56, which is incapable of differentiating in vitro, has a much higher protein synthetic activity than its pleomorphic parent in the bloodstream form. This high activity and the bloodstream profile of proteins thus synthesized were unaffected by the decreased temperature in Cunningham's medium until cell death. We thus conclude that a general inhibition of protein synthesis in bloodstream forms caused by temperature drop may be among the early events triggering differentiation into the procyclic form.