Effect Of Cycloheximide Research Articles

Dissecting mechanisms of reconsolidation: octopamine reveals differences between appetitive and aversive memories in the crab Chasmagnathus

Ample evidence suggests that, when reactivated by a reminder, a consolidated memory may return to a labile state and needs to be stabilized again in order to persist, a process known as reconsolidation. In a previous study, performed in the crab Chasmagnathus, we found a dual role for the biogenic amine octopamine (OA) during memory consolidation. On the one hand, it was necessary for appetitive memory formation and, on the other, it had a deleterious effect on aversive memory consolidation. Thus, OA could be a good candidate to dissect the neurochemical mechanisms of appetitive and aversive reconsolidation. Here, we initially characterized the reconsolidation of an appetitive memory. Then, we compared appetitive reconsolidation with its aversive counterpart regarding the implication of OA in these processes, and contrasted them with previous findings obtained in the consolidation phase. Our results demonstrate that appetitive reconsolidation takes place when animals are re-exposed to the training context, as shown by the amnesic effect of cycloheximide when applied before the reminder. In addition, the no-reinforcement during the reminder is a necessary condition for appetitive reconsolidation to occur. Remarkably, appetitive reconsolidation is neither impaired by OA receptor antagonists nor facilitated by exogenous OA, whereas aversive reconsolidation can be interfered with by OA administration. Thus, our results indicate that appetitive reconsolidation does not involve OA signaling, while aversive reconsolidation is negatively modulated by OA. All in all, these results could constitute a step towards the identification of particular features of appetitive and aversive reconsolidation.

Texture analysis of poly-adenylated mRNA staining following global brain ischemia and reperfusion

Texture analysis provides a means to quantify complex changes in microscope images. We previously showed that cytoplasmic poly-adenylated mRNAs form mRNA granules in post-ischemic neurons and that these granules correlated with protein synthesis inhibition and hence cell death. Here we utilized the texture analysis software MaZda to quantify mRNA granules in photomicrographs of the pyramidal cell layer of rat hippocampal region CA3 around 1 h of reperfusion after 10 min of normothermic global cerebral ischemia. At 1 h reperfusion, we observed variations in the texture of mRNA granules amongst samples that were readily quantified by texture analysis. Individual sample variation was consistent with the interpretation that animal-to-animal variations in mRNA granules reflected the time-course of mRNA granule formation. We also used texture analysis to quantify the effect of cycloheximide, given either before or after brain ischemia, on mRNA granules. If administered before ischemia, cycloheximide inhibited mRNA granule formation, but if administered after ischemia did not prevent mRNA granulation, indicating mRNA granule formation is dependent on dissociation of polysomes. We conclude that texture analysis is an effective means for quantifying the complex morphological changes induced in neurons by brain ischemia and reperfusion.

Extinction of appetitive learning is disrupted by cycloheximide and propranolol in the sand maze in rats

The present study investigated whether memory for extinction in an appetitive task (the sand maze) could be attenuated by administration of cycloheximide (protein synthesis inhibitor) or propranolol (β-adrenergic receptor antagonist). Ninety-day-old male Long-Evans rats were trained to retrieve a sweet cereal reinforcer from an open container in the sand maze. One day following this non-spatial training, rats received three extinction trials in which they were placed in the maze with the reinforcer present, but unattainable. Thirty minutes prior to the first extinction trial, rats received an intraperitoneal injection of cycloheximide (1mg/kg), propranolol (25mg/kg), or vehicle (1mg/kg distilled water). Twenty-four hours later, rats were tested in the sand maze with the reinforcer again available. Results from the test trial showed that both cycloheximide and propranolol groups found the reinforcer more quickly than controls. Two weeks later, rats were trained on a spatial version of the sand maze in which they had to search for a buried reinforcer using extramaze cues. Cycloheximide and propranolol groups learned this task significantly faster than the control group, demonstrating the long-lasting effect of cycloheximide and propranolol on the blocking of memory for extinction.

17β-Estradiol Augments 18F-FDG Uptake and Glycolysis of T47D Breast Cancer Cells via Membrane-Initiated Rapid PI3K–Akt Activation

Use of (18)F-FDG uptake as a surrogate marker of therapeutic response requires the recognition of biologic factors that influence cancer cell glucose metabolism. Estrogen is a potent stimulator of breast cancer proliferation, a process that requires sufficient energy, which is likely met by increased glycolysis. We thus explored the effect of estrogen on (18)F-FDG uptake in responsive breast cancer cells and investigated the mediating molecular mechanisms. T47D breast cancer cells were stimulated with 17β-estradiol (E(2)) or bovine serum albumin (BSA)-E(2) and measured for (18)F-FDG uptake, lactate release, and mitochondrial hexokinase activity. The effects of antiestrogens, cycloheximide, and major protein kinase inhibitors were investigated. Immunoblots were performed for membrane glucose transporter type 1, phosphorylated phosphatidylinositol 3-kinase (PI3K), and Akt. E(2) augmented T47D cell (18)F-FDG uptake in a dose- and time-dependent manner that preceded and surpassed its proliferative effect. With exposure to 10 nM E(2), protein content-corrected (18)F-FDG uptake reached 172.7% ± 6.6% and 294.4% ± 9.5% of controls by 24 and 48 h, respectively. Lactate release reached 110.9% ± 7.3% and 145.2% ± 10.5% of controls at 24 and 48 h, and mitochondrial hexokinase activity increased to 187.1% ± 31.6% at 24 h. Membrane glucose transporter type 1 expression was unaltered. The effect was absent in estrogen receptor (ER)-negative breast cancer cells and was abrogated by ICI182780, indicating ER dependence. The E(2) effect was not blocked by tamoxifen and was mimicked by membrane-impermeable BSA-E(2), consistent with nongenomic membrane-initiated E(2) action. Inhibition by cycloheximide demonstrated the requirement of a new protein synthesis. Immunoblots displayed rapid phosphorylation of PI3K and Akt within minutes of E(2) treatment, and the specific PI3K inhibitors wortmannin and LY294002 abolished the ability of E(2) to elevate (18)F-FDG uptake. Estrogen augments breast cancer cell (18)F-FDG uptake by stimulating glycolysis and hexokinase activity via membrane-initiated E(2) action that activates the PI3K-Akt pathway. These findings yield important insight into our understanding of the biology of breast cancer metabolism and may have potential implications for (18)F-FDG uptake as a surrogate marker of therapeutic response.

Pulsatile and Sustained Gonadotropin-releasing Hormone (GnRH) Receptor Signaling: DOES THE ERK SIGNALING PATHWAY DECODE GnRH PULSE FREQUENCY?

Gonadotropin-releasing hormone (GnRH) acts via G-protein-coupled receptors on gonadotrophs to stimulate synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. It is secreted in pulses, and its effects depend on pulse frequency, but decoding mechanisms are unknown. Here we have used an extracellular signal regulated kinase-green fluorescent protein (ERK2-GFP) reporter to monitor GnRH signaling. GnRH caused dose-dependent ERK2-GFP translocation to the nucleus, providing a live-cell readout for activation. Pulsatile GnRH caused dose- and frequency-dependent ERK2-GFP translocation. These responses were rapid and transient, showed only digital tracking, and did not desensitize under any condition tested (dose, frequency, and receptor number varied). We also tested for the effects of cycloheximide (to prevent induction of nuclear-inducible MAPK phosphatases) and used GFP fusions containing ERK mutations (D319N, which prevents docking domain-dependent binding to MAPK phosphatases, and K52R, which prevents catalytic activity). These manipulations had little or no effect on the translocation responses, arguing against a role for MAPK phosphatases or ERK-mediated feedback in shaping ERK activation during pulsatile stimulation. GnRH also caused dose- and frequency-dependent activation of the α-gonadotropin subunit-, luteinizing hormone β-, and follicle-stimulating hormone β- luciferase reporters, and the latter response was inhibited by ERK1/2 knockdown. Moreover, GnRH caused frequency-dependent activation of an Egr1-luciferase reporter, but the response was proportional to cumulative pulse duration. Our data suggest that frequency decoding is not due to negative feedback shaping ERK signaling in this model.

Evidence for differential effects of glucose and cycloheximide on mRNA levels of peroxisome proliferator-activated receptor- (PPAR-) machinery members: Superinduction of PPAR-gamma1 and -gamma2 mRNAs.

Quantitative real-time RT-PCR study was conducted to reveal the effects of normal (5 mmol/l) and high (30 mmol/l) glucose without or with oleate (0.3 mmol/l) on mRNA levels of peroxisome proliferator-activated receptor- (PPAR-)alpha, -gamma1, -gamma2, and peroxisome proliferator-activated receptor-gamma coactivator- (PGC-)1alpha and -1beta in commercial human hepatoma-derived HepG2 cells maintained under low-serum condition. Significant decrease in PPAR-gamma1 and PGC-1alpha mRNA levels to about 50 % was observed during the first 4 h incubation period. During the next 4 h period, both PPAR-gamma1 and PGC-1alpha mRNAs were partly but significantly restored in high glucose batches. In this period, the presence of the transcriptional inhibitor actinomycin D revealed a significant protective effect of excess glucose on mature PPAR-gamma1 and PGC-1alpha mRNAs. Furthermore, PPAR-gamma1 and -gamma2 mRNAs were differentially superinduced 1.2-2.5 fold in cells upon the administration of the translational inhibitor cycloheximide. When the cells were co-treated with the combination of cycloheximide and actinomycin D, superinduction was completely suppressed, however. Altogether, the experiments revealed, first, an unexpected protective effect of abundant glucose on PPAR-gamma1 and PGC-1alpha mRNAs in HepG2 cells. Second, we demonstrated cycloheximide-induced, transcription-dependent upregulation of mature PPAR-gamma1 and -gamma2 mRNAs in HepG2 cells associated with preferential expression of the PPAR-gamma2 mRNA variant. The results draw attention to as yet unexplored mechanisms involved in the control of PPAR and PGC genes.

Chlamydia pneumoniae Infection in Polarized Epithelial Cell Lines

We set up a polarized cell culture model to study the pathogenicity of a common respiratory tract pathogen, Chlamydia pneumoniae. Immunofluorescence staining of ZO-1 (a tight junction protein) and Na(+)K(+) ATPase (a protein pump localized at the basolateral membrane in the polarized epithelial cells), as well as TER measurements, suggested that the filter-grown Calu-3 cells, but not the A549 cells, were polarized when grown on collagen-coated membranes. Both the flat and the filter-grown cultures were infected with C. pneumoniae. Infection in the polarized Calu-3 cultures produced more C. pneumoniae genome equivalents than infection in the flat cultures. However, this progeny was not as infective as that in the flat cultures. The maximum amount of C. pneumoniae was detected at 6 days postinfection in the filter-grown A549 cells, indicating a slower developmental cycle than that observed in the flat A549 cultures. The effect of cycloheximide on the growth of C. pneumoniae in the polarized cells was negligible. Furthermore, the infection in the polarized Calu-3 cells was resistant to doxycycline, and several cytokines were released mainly on the apical side of the polarized cells in response to C. pneumoniae infection. These findings indicate that the growth of chlamydiae was altered in the filter-grown epithelial culture system. The diminished production of infective progeny of C. pneumoniae, together with the resistance to doxycycline and polarized secretion of cytokines from the infected Calu-3 cells, suggests that this model is useful for examining epithelial cell responses to C. pneumoniae infection, and it might better resemble in vivo infection in respiratory epithelial cells.

Effect of cycloheximide, iodoacetamide, and antimycin A on inorganic phosphate synthesis in Saccharomyces cerevisiae VKM Y-1173

The effect of inhibitors of protein synthesis (cycloheximide, CHI), glycolysis (iodoacetamide, IAA), and oxidative phosphorylation (antimycin A, ANM) on inorganic phosphate (polyP) synthesis during the first 0.5 h of their hypercompensation in Saccharomyces cerevisiae VKM Y-l173 grown on 2% glucose-containing media at low (hypoxia) or high aeration rates or in the presence of 1 vol % ethanol under high aeration conditions was studied. PolyP accumulation was highest in the medium with glucose under hypoxia; lower, with glucose at high aeration; and lowest, in the medium with ethanol. CHI had a small effect on the total polyP level but significantly stimulated ATP accumulation, irrespective of the culture growth conditions. The low-polymer acid-soluble polyP1 were synthesized most actively by the cells grown on glucose under hypoxia, alkali-soluble polyP3 were synthesized at en hanced aeration, and the most hig-molecular fraction, polyP5, was actively accumulated along with polyP3 at cultivation on ethanol. Regardless of the growth conditions, CHI inhibited accumulation of polyP4, the synthesis of which is associated with the synthesis of mannoproteins. IAA and ANM largely inhibited synthesis of all fractions at yeast growth under hypoxia and on ethanol, respectively. The results as a whole demonstrate the dependence of polyP formation on the main energy-generating cell processes and, at the same time, the absence of direct dependence of their synthesis on ATP concentration in Saccharomyces cerevisiae VKM Y-l 173.

Stored and neosynthesized mRNA in Arabidopsis seeds: effects of cycloheximide and controlled deterioration treatment on the resumption of transcription during imbibition

Dry seeds accumulate translatable mRNAs as well as functional proteins for transcription and translation. They are possibly involved in early physiological responses after imbibition, however, their functions remain poorly understood. The aim of this study is to investigate the function of seed stored transcriptional machinery in resumption of gene expression after the onset of imbibition in Arabidopsis thaliana. First, we examined the characters of stored mRNAs in A. thaliana dry seeds using microarray data from non-dormant Columbia (Col) and dormant Cape Verde Islands (Cvi) accessions. Transcriptomes of Col and Cvi dry seeds resembled one another, suggesting that patterns of stored mRNA do not reflect either the degree of dormancy or germination potential, but rather reflect the developmental context, such as seed maturation. Upon imbibition, changes in mRNA abundance of many genes were initiated between 1- and 2-h after the onset of imbibition. RT-PCR expression analysis of imbibition-responsive genes indicates that early induction was not altered by treatment of cycloheximide. This suggests that de novo protein synthesis is not required for gene expression during early imbibition stages. Moreover, controlled deterioration treatment (CDT), which causes artificial damages on dry seeds, disrupted gene expression specifically during the first 3 h after the start of imbibition, suggesting that seed stored transcription factors play a pivotal role in gene expression during early imbibition periods. Furthermore, the negligible effect of CDT on germination indicates that early imbibition response is dispensable and de novo synthesized proteins compensate for the function of stored proteins for germination.

Systemic treatment with protein synthesis inhibitors attenuates the expression of cocaine memory

It has been proposed that a memory trace enters a labile phase each time it is retrieved. A reactivated memory relies on de novo protein synthesis to be faithfully reconsolidated and restored. Thus, in theory, a long-lasting and pathological memory associated with drug use may be disrupted by inhibiting its reconsolidation through use of protein synthesis inhibitors administered immediately following the memory retrieval. However, effective and efficient strategies to reactivate drug memory remained elusive. This study was undertaken to examine the effects of systemic cycloheximide and anisomycin treatment on the reconsolidation and maintenance of a reactivated cocaine-conditioned place preference (CPP) in mice using several strategies designed to reactivate the previously acquired memory. We found that anisomycin (50 mg/kg/injection) and cycloheximide (15 mg/kg/injection) administered immediately after the reactivation of cocaine-CPP ameliorated subsequent expression and maintenance of this memory. Likewise, when anisomycin and cycloheximide were administered immediately after additional cocaine and saline conditioning trials, the reactivated memory engendered by those extra training trials was also diminished. However, a similar anisomycin dosing regimen failed to affect subsequent expression of cocaine-CPP when additional cocaine conditioning trial was used in the absence of additional saline trial. Finally, cocaine and saline administration to mice in their home cages with or without anisomycin treatment had no effect on later cocaine-CPP expression. Taken together, these findings suggest that systemic treatment with protein synthesis inhibitors immediately after the reactivation of cocaine-CPP effectively diminished the reconsolidation and maintenance of such a cocaine memory. More importantly, reactivation of cocaine-CPP could be achieved by presentation of cocaine-conditioned cues as well as by administering additional cocaine and saline conditioning trials in a balanced fashion.

Microbial community structure and function during abnormal curve development of substrate-induced respiration measurements

Soil respiration measurements are an established method to test the abundance, activity and vitality of the soil microorganisms. However, abnormal progressions of soil respiration curves impede a clear interpretation of the data. The aim of this study was to investigate the changes in the microbial structure during the formation of phenomena like double peaks and terraces by analysis of the PLFA composition (phospholipid fatty acid composition). Moreover, 13C labeled glucose was used as substrate; therefore it was possible to measure δ 13C values both within the PLFA fraction as well as within the carbon dioxide evolved during respiration. As contaminants trinitrotoluene, cycloheximide, and hexadecane were used. The results showed that the appearance of double peaks was mainly related to the growth of fungi with the marker 18:2 δ9,12 due to a toxic effect of trinitrotoluene and cycloheximide. In contrast, the phenomenon of terrace formation was related to the utilization of hexadecane as a carbon source mainly by bacteria.

Neurochemical Mechanisms of Consolidation of Associative Aversive Training to Food in the Common Snail

The effects of the protein synthesis inhibitor cycloheximide and serotonin and NMDA glutamate receptor antagonists on the processes of consolidation of an associative skill consisting of refusing a particular foodstuff were studied in the common snail. When animals were trained on the background of cycloheximide, the skill was not acquired. Repeat training of "amnestic" snails to refuse the same food without the inhibitor also failed to produce the skill. Training of snails on the background of the nonselective serotonin receptor antagonist methiothepin or the NMDA glutamate receptor antagonist MK-801 (dizocilpine maleate) did not lead to acquisition of the conditioned reflex to food. However, on repeat training, the skill was formed more quickly. The studies included the first observation that using a single type of training, treatments addressing different molecular mechanisms evoke reversible or irreversible impairments to the mechanisms of consolidation of long-term memory. It is suggested that the reversible effect is associated with suppression of the processes of reproduction, while the irreversible effect is linked with impairment to engram storage.

Chitosan-induced programmed cell death in plants.

Chitosan, CN(-), or H(2)O(2) caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H(2)O(2), or chitosan + H(2)O(2) on EC. H(2)O(2) formation in EC and GC mitochondria that was determined from 2',7'-dichlorofluorescein fluorescence was inhibited by CN(-) and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN(-)-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN(-)-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H(2)O(2) on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H(2)O(2) as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.

Spreading of mesothelioma cells is rapamycin‐sensitive and requires continuing translation

The interaction of cancer cells with extracellular matrix (ECM) is important in metastasization. Here we identified the molecules of the ECM expressed by sarcomatous malignant mesothelioma, and their effect on adhesion and spreading. In addition, by analyzing the relationship between translation and attachment to matrix, we found that mesothelioma cells rely on continuing translation to efficiently attach to matrix, and rapamycin inhibition affects spreading and migration of cancer cells. Specifically, we found that sarcomatous cells produce high amounts of fibronectin, able to support the spreading of mesothelioma cells. Spreading of cancer cells on fibronectin does not require de novo transcription but is sensitive to cycloheximide, an inhibitor of protein synthesis. Next, we analyzed the involvement of the mammalian target of rapamycin (mTOR) pathway, a major pathway controlling translation. Cancer cells have a constitutively active mTOR pathway; surprisingly, inhibition of mTOR complex 1 (mTORC1) by rapamycin barely affects the global rate of translation and of initiation of translation, but deeply inhibits mesothelioma spreading on ECM. The effects of rapamycin and cycloheximide on spreading were observed in several mesothelioma cell lines, although with different magnitude. Overall, data suggest that adhesion and spreading of mesothelioma cells on ECM require the translation of pre-synthesized mRNAs, and mTORC1 activity. We speculate that mTORC1 activity is required either for the translation of specific mRNAs or for the direct modulation of cytoskeletal remodeling.

Neurochemical Mechanisms of Consolidation of Associative Aversive Learning to Food in the Common Snail

Studies of the effects of the protein synthesis inhibitor cycloheximide and serotonin and NMDA glutamate receptor antagonists on the processes of consolidation of the associative skill of rejecting particular foodstuffs were performed in the common snail. The skill was not acquired when animals were trained on the background of cycloheximide. Repeated training of "amnestic" snails to reject the same foodstuff in the absence of the inhibitor also failed to produce learning. Training of snails on the background of the nonselective serotonin receptor antagonist methiothepin or the NMDA glutamate receptor antagonist MK-801 (dizocilpine maleate) did not result in the acquisition of the conditioned reflex to food. However, repeated training led to the rapid formation of the skill. These experiments provide the first evidence that interventions influencing different molecular mechanisms during a single type of training produce either reversible or irreversible impairment of the mechanisms of consolidation of long-term memory. It is suggested that the reversible effect is associated with suppression of reproduction processes, while irreversible impairment was associated with impairments to engram storage.

Effects of cycloheximide or 6-dimethyl aminopurine on the parthenogenetic activation of pig oocytes using pulsatile treatment with nitric oxide donor

Pig oocytes matured <I>in vitro</I> were parthenogenetically activated using nitric oxide donor SNAP (2mM). Continuous treatment successfully activated the oocytes only after more than 12 hours of exposure. Pulsatile treatments during which oocytes were repeatedly exposed to 2mM SNAP for a short time (10, 20 or 30 minutes) were more efficient with regard to the activation rate, even when the total exposure time did not exceed 4 hours. Parthenogenetic development was very limited after continuous treatment with 2mM SNAP. A significantly higher proportion of developing parthenogenetic embryos was observed after the pulsatile treatment (development to the morula stage 0 vs. 18%; development to the blastocyst 0 vs. 7%; <I>P</I> < 0.05). However, this developmental rate was significantly lower (<I>P</I> < 0.05) than the development induced by conventional activation treatment with calcium ionophore (development to the morula stage, 23%; development to the blastocyst stage, 18%). When we combined pulsatile SNAP-treatment with the effect of protein kinase inhibitor 6-dimethyl aminopurine (6-DMAP) (2mM 6-DMAP for 2 hours) or with the inhibitor of protein synthesis cycloheximide (CHX) (10 µM CHX for 2 hours), we observed a significant increase (<I>P</I> < 0.05) in the activation rate when compared to the respective pulsatile SNAP-treatment without 6-DMAP or CHX (63 vs. 78% of activated oocytes for 6-DMAP; 63 vs. 83% of activated oocytes for CHX). However, the development of parthenogenetic embryos was not enhanced when the pulsatile SNAP-treatment was combined with 6-DMAP or with CHX.

A Switch from Cycloheximide-Resistant Consolidated Memory to Cycloheximide-Sensitive Reconsolidation and Extinction inDrosophila

It is generally accepted that, after learning, memories stabilize over time and integrate into long-term memory (LTM) through the process of consolidation, which depends on de novo protein synthesis. Besides, studies on several species have shown that reactivation of already stabilized LTM can either make this memory labile and then restabilize it (a process called reconsolidation) or inhibit it (extinction). However, the identity of both processes and their interactions with consolidation are still under debate. Regarding memory stabilization, Drosophila offers a striking exception since, in this species, LTM is not the sole stable form of memory. Under specific learning conditions, anesthesia-resistant memory (ARM) can be formed through processes yet unknown but that are resistant to cycloheximide, a classical protein synthesis inhibitor that impairs LTM. Here, we took advantage of this dichotomy to ask whether both ARM and LTM could be extinguished and/or reconsolidated. We also studied whether two forms of memory extinction and reconsolidation exist in flies, as for memory stabilization. We show that either reconsolidation or extinction can be induced after olfactory conditioning in Drosophila, depending on the number of reactivations as in other species. Furthermore, regarding the effect of cycloheximide, the ARM/LTM dichotomy for stabilization does not apply to extinction and reconsolidation. Blocking protein synthesis interfered with both processes regardless of whether initial stabilization was sensitive (LTM) or not (ARM) to cycloheximide. These results thus show that Drosophila is a useful model to tackle these questions and that reconsolidation is not necessarily a mere repetition of consolidation.

EIF5A has a function in the elongation step of translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved throughout evolution. Here, we describe genetic interactions between an eIF5A mutant and a translation initiation mutant (eIF4E) or a translation elongation mutant (eEF2). Polysome profile analysis of single and double mutants revealed that mutation in eIF5A reduces polysome run-off, contrarily to translation initiation mutants. Moreover, the polysome profile of an eIF5A mutant alone is very similar to that of a translation elongation mutant. Furthermore, depletion of eIF5A causes a significant decrease in total protein synthesis and an increase of the average ribosome transit time. Finally, we demonstrate that the formation of P bodies is inhibited in an eIF5A mutant, similarly to the effect of the translation elongation inhibitor cycloheximide. Taken together, these results not only reinforce a role for eIF5A in translation but also strongly support a function for eIF5A in the elongation step of protein synthesis.

Enhanced inhibitory avoidance learning prevents the long-term memory-impairing effects of cycloheximide, a protein synthesis inhibitor

Interference with activity of numerous cerebral structures produces memory deficiencies; in many instances, however, when animals are over-trained such interference becomes innocuous. Systemic administration of protein synthesis inhibitors impairs long-term retention; this effect has been interpreted to mean that protein synthesis is required for memory consolidation, though little is known about the effect of protein synthesis inhibitors on memory of enhanced learning in the rat. To further analyze the protective effect of enhanced learning against amnesic treatments, groups of Wistar rats were trained in a one-trial step-through inhibitory avoidance task, using different intensities of foot-shock during training. Cycloheximide (CXM; 2.8 mg/kg), an inhibitor of protein synthesis, was injected either 30 min before training or immediately after training. Twenty-four hours after training retention latencies were recorded. Our data showed that both pre- and post-training administration of CXM produced amnesia in those groups that had been trained with relatively low foot-shock intensities, but no impairment in retention was observed when relatively high intensities of foot-shock were administered. These and similar results lead us to conclude that protein synthesis inhibitors may interfere with memory consolidation, but their effect disappears when animals are submitted to an enhanced learning experience, calling into question the idea that protein synthesis is required for memory consolidation.

Monitoring "De Novo"APP synthesis by taking advantage of the reversible effect of cycloheximide.

By blocking "de novo" protein synthesis using cycloheximide, we previously described a dynamic model system to monitor turnover of a specific population of the Alzheimer's amyloid precursor protein. Here we show that cycloheximide is nontoxic and its effect is reversible, allowing protein synthesis to reinitiate. Upon cycloheximide removal protein synthesis restarted and by 1 hour the amyloid precursor protein- green fluorescent protein could be clearly detected, permitting the monitoring of amyloid precursor protein anterograde transport, particularly the secretory pathway. The consensus NPTY motif in amyloid precursor protein, typically associated with endocytosis, was mutated to NPTF or NPTE to mimic a constitutively dephosphorylated or phosphorylated residue, respectively. Our data reveal that disruption of this motif affects amyloid precursor protein endocytosis, as shown previously, but also its incorporation into trans-Golgi network budding vesicles. Thus, cycloheximide can be a useful tool to study both anterograde and retrograde "in vivo'' protein transport.