Effects Of Protein Synthesis Inhibitors Research Articles

A meta-analysis of the effect of protein synthesis inhibitors on rodent fear conditioning

A meta-analysis of the effect of protein synthesis inhibitors on rodent fear conditioning

Preimplantation embryo gene expression: 56 years of discovery, and counting.

The biology of preimplantation embryo gene expression began 56 years ago with studies of the effects of protein synthesis inhibition and discovery of changes in embryo metabolism and related enzyme activities. The field accelerated rapidly with the emergence of embryo culture systems and progressively evolving methodologies that have allowed early questions to be re-addressed in new ways and in greater detail, leading to deeper understanding and progressively more targeted studies to discover ever more fine details. The advent of technologies for assisted reproduction, preimplantation genetic testing, stem cell manipulations, artificial gametes, and genetic manipulation, particularly in experimental animal models and livestock species, has further elevated the desire to understand preimplantation development in greater detail. The questions that drove enquiry from the earliest years of the field remain drivers of enquiry today. Our understanding of the crucial roles of oocyte-expressed RNA and proteins in early embryos, temporal patterns of embryonic gene expression, and mechanisms controlling embryonic gene expression has increased exponentially over the past five and a half decades as new analytical methods emerged. This review combines early and recent discoveries on gene regulation and expression in mature oocytes and preimplantation stage embryos to provide a comprehensive understanding of preimplantation embryo biology and to anticipate exciting future advances that will build upon and extend what has been discovered so far.

Study of the effect of protein synthesis inhibitors on growing <i>Escherichia coli</i> bacteria using electrochemical sensors

Background. The study of the mechanisms of action of antibiotics requires the integrated use of traditional microbiological and physicochemical methods. The aim. To study the response of Escherichia coli bacteria to the action of four antibiotics, inhibitors of protein synthesis, using combined approach. Methods. Bacteria were grown under aerobic conditions on minimal M9 medium with glucose. Tetracycline, kanamycin, streptomycin and chloramphenicol have been tested. The effect of antibiotics on survival (CFU) and growth rate was determined. Respiratory activity, sulfide production, extracellular potassium, as well as pH and Eh of the medium were measured using electrochemical sensors directly in a growing culture in the “real time”, membrane potential was measured using a DiBAC dye and a Leica DM2000 fluorescent microscope. Results. The tested antibiotics were divided into two groups according to their properties. Tetracycline and chloramphenicol showed a pronounced bacteriostatic effect, growth inhibition began immediately after the addition of antibiotics and occurred at a high rate. Both antibiotics inhibited respiration, stimulated sulfide production and an Eh jump. Bacteria treated with tetracycline and chloramphenicol retained the ability to maintain membrane potential and intracellular potassium better. Inhibition of respiration led to a decrease in glucose catabolism, as evidenced by a lower rate of acidification of the medium compared to the control. Growth inhibition with streptomycin and kanamycin was initiated with a 30-minute delay. Both antibiotics showed a bactericidal effect, did not stimulate sulfide production and Eh jump, did not inhibit respiration, but caused a drop in membrane potential and intracellular potassium. High respiratory activity promoted glucose catabolism, as evidenced by the rapid acidification of the medium. Of interest is the detection of kanamycin-induced sulfide production during E. coli growth on MOPS medium. Conclusion. An analysis of the data obtained indicates that the use of electrochemical sensors in combination with traditional methods is a promising approach to studying the mechanisms of action of antibiotics.

Predator fear memory depends on glucocorticoid receptors and protein synthesis in the basolateral amygdala and ventral hippocampus

Previous studies have suggested that the basolateral amygdala (BLA) and the ventral hippocampus (VH) are critical sites for predator-related fear memory. Predator exposure is an intense emotional experience and should increase plasmatic corticosterone likely to modulate the emotion-related memories. However, it is unclear whether the BLA and VH harbor plastic events underlying predator-related fear memory storage and how molecular and endocrine mechanisms interact to modulate memory to the predatory threat. Here, we first examined the effects of protein synthesis inhibition in the BLA and VH on fear memory to a predatory threat. We next evaluated how exposure to a predatory threat impacts the corticosterone release and how the inhibition of corticosterone synthesis can influence predator-related fear memory. Finally, we examined how predator exposure triggers the activation of glucocorticoid and mineralocorticoid receptors in the BLA and VH and whether the GR antagonist injection affects predator-related fear memory. We showed that predator-related contextual fear is dependent on protein synthesis in the BLA and VH. Moreover, we described the impact of rapid glucocorticoid release during predatory exposure on the formation of contextual fear responses and that GR-induced signaling facilitates memory consolidation within the BLA and VH. The results are relevant in understanding how life-threatening situations such as a predator encounter impact fear memory storage and open exciting perspectives to investigate GR-induced proteins as targets to deciphering and manipulating aversive memories.

Abstract 3903: Rocaglamide A and synthetic analogues sensitize resistant renal carcinoma cells to TRAIL-induced apoptosis and inhibit cell proliferation

Abstract Induction of cancer cell-specific apoptosis via activation of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) signaling has been an attractive goal for cancer therapeutics. However, many tumor cells develop resistance to TRAIL. Therefore, the search for enhancers of TRAIL-induced apoptosis has accelerated over the past several years. A high-throughput screening assay was developed that identified several natural product enhancers of TRAIL-induced apoptosis in TRAIL-resistant renal carcinoma cells. Among these natural products was the protein synthesis inhibitor, rocaglamide A, a cyclopenta[b]benzofuran secondary metabolite from the genus Aglaia, with potent antiproliferative and anti-inflammatory properties. Natural and synthetic rocaglates have been reported to have potent anticancer activities in vitro on various human cancer cell lines and in vivo in mouse models. The mechanism of action involved in the anticancer effects of rocaglamide A is generally thought to be inhibition of translation initiation. However, several other cancer-related cellular effects including cell cycle arrest have been reported in various cancer cell types. In this study, rocaglamide A and its synthetic analogs were assessed for their ability to enhance TRAIL-induced apoptosis, inhibit protein synthesis, and regulate cellular processes associated with TRAIL sensitization in TRAIL-resistant ACHN renal carcinoma cells. Rocaglate treatment enhanced TRAIL signaling resulting in caspase-dependent apoptotic cell death upon addition of TRAIL. Rocaglates also inhibited protein synthesis, which correlates with rapid loss of the antiapoptotic FLICE-inhibitory protein (cFLIP) and MCL-1, which are often overexpressed in TRAIL-resistant cancer cells. On average, the rocaglates were ~4-5-fold more potent than TRAIL sensitizers compared to their protein synthesis inhibitory potency. This difference suggests a possible therapeutic window for induction of TRAIL sensitization while minimizing general effects of protein synthesis inhibition. Rocaglamide treatment alone inhibited cell proliferation leading to cell cycle arrest at G2/M phase but not subsequent apoptosis. These studies suggest that the TRAIL-sensitizing activity of rocaglates and their growth-inhibitory effects as single agents are largely dependent on protein synthesis inhibition. The development of a large number of structurally diverse but mechanistically similar enhancers of TRAIL signaling with a wide range of potencies allows for further understanding of structure-activity relationships regarding both protein synthesis inhibition and TRAIL sensitization for this important family of compounds. Funded (in part) by NCI Contract No. HHSN261200800001E. Citation Format: Ancy D. Nalli, Lauren E. Brown, Cheryl L. Thomas, Thomas J. Sayers, John A. Porco, Curtis J. Henrich. Rocaglamide A and synthetic analogues sensitize resistant renal carcinoma cells to TRAIL-induced apoptosis and inhibit cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3903.

Protein Synthesis Inhibitors Did Not Interfere with Long-Term Depression Induced either Electrically in Juvenile Rats or Chemically in Middle-Aged Rats.

In testing the hypothesis that long-term potentiation (LTP) maintenance depends on triggered protein synthesis, we found no effect of protein synthesis inhibitors (PSIs) on LTP stabilization. Similarly, some studies reported a lack of effect of PSIs on long-term depression (LTD); the lack of effect on LTD has been suggested to be resulting from the short time recordings. If this proposal were true, LTD might exhibit sensitivity to PSIs when the recording intervals were enough long. We firstly induced LTD by a standard protocol involving low frequency stimulation, which is suitable for eliciting NMDAR-LTD in CA1 area of hippocampal slices obtained from juvenile Sprague-Dawley rats. This LTD was persistent for intervals in range of 8–10 h. Treating slices with anisomycin, however, did not interfere with the magnitude and persistence of this form of LTD. The failure of anisomycin to block synaptic-LTD might be relied on the age of animal, the type of protein synthesis inhibitors and/or the inducing protocol. To verify whether those variables altogether were determinant, NMDA or DHPG was used to chemically elicit LTD recorded up to 10 h on hippocampal slices obtained from middle-aged rats. In either form of LTD, cycloheximide did not interfere with LTD stabilization. Furthermore, DHPG application did show an increase in the global protein synthesis as assayed by radiolabeled methodology indicating that though triggered protein synthesis can occur but not necessarily required for LTD expression. The findings confirm that stabilized LTD in either juvenile, or middle-aged rats can be independent of triggered protein synthesis. Although the processes responsible for the independence of LTD stabilization on the triggered protein synthesis are not yet defined, these findings raise the possibility that de novo protein synthesis is not universally necessary.

Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats.

Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of protein synthesis inhibition within this region after experimental stroke. Long-Evans rats were trained to perform a skilled-reaching task (SRT) until they reached plateau performance. A photothrombotic stroke was induced in the forelimb representation of the primary motor cortex (M1) contralateral to the trained paw. The SRT was re-trained after stroke while the protein synthesis inhibitor anisomycin (ANI) or saline were injected into the peri-infarct cortex through implanted cannulas. ANI injections reduced protein synthesis within the peri-infarct cortex by 69% and significantly impaired recovery of reaching performance through re-training. Improvement of motor performance within a single training session remained intact, while improvement between training sessions was impaired. ANI injections did not affect infarct size. Thus, protein synthesis inhibition within the peri-infarct cortex impairs recovery of motor deficits after ischemic stroke by interfering with consolidation of motor memory between training sessions but not short-term improvements within one session.

The differential role of cortical protein synthesis in taste memory formation and persistence.

The current dogma suggests that the formation of long-term memory (LTM) is dependent on protein synthesis but persistence of the memory trace is not. However, many of the studies examining the effect of protein synthesis inhibitors (PSIs) on LTM persistence were performed in the hippocampus, which is known to have a time-dependent role in memory storage, rather than the cortex, which is considered to be the main structure to store long-term memories. Here we studied the effect of PSIs on LTM formation and persistence in male Wistar Hola (n⩾5) rats by infusing the protein synthesis inhibitor, anisomycin (100 μg, 1 μl), into the gustatory cortex (GC) during LTM formation and persistence in conditioned taste aversion (CTA). We found that local anisomycin infusion to the GC before memory acquisition impaired LTM formation (P=8.9E−5), but had no effect on LTM persistence when infused 3 days post acquisition (P=0.94). However, when we extended the time interval between treatment with anisomycin and testing from 3 days to 14 days, LTM persistence was enhanced (P=0.01). The enhancement was on the background of stable and non-declining memory, and was not recapitulated by another amnesic agent, APV (10 μg, 1 μl), an N-methyl-D-aspartate receptor antagonist (P=0.54). In conclusion, CTA LTM remains sensitive to the action of PSIs in the GC even 3 days following memory acquisition. This sensitivity is differentially expressed between the formation and persistence of LTM, suggesting that increased cortical protein synthesis promotes LTM formation, whereas decreased protein synthesis promotes LTM persistence.

Inactivation of the dorsal hippocampus or the medial prefrontal cortex impairs retrieval but has differential effect on spatial memory reconsolidation

Active memories can incorporate new information through reconsolidation. However, the notion that memory retrieval is necessary for reconsolidation has been recently challenged. Non-reinforced retrieval induces hippocampus and medial prefrontal cortex (mPFC)-dependent reconsolidation of spatial memory in the Morris water maze (MWM). We found that the effect of protein synthesis inhibition on this process is abolished when retrieval of the learned spatial preference is hindered through mPFC inactivation but not when it is blocked by deactivation of dorsal CA1. Our results do not fully agree with the hypothesis that retrieval is unneeded for reconsolidation. Instead, they support the idea that a hierarchic interaction between the hippocampus and the mPFC controls spatial memory in the MWM, and indicate that this cortex is sufficient to retrieve the information essential to reconsolidate the spatial memory trace, even when the hippocampus is inactivated.

Contextual Information Drives the Reconsolidation-Dependent Updating of Retrieved Fear Memories.

Stored memories enter a temporary state of vulnerability following retrieval known as 'reconsolidation', a process that can allow memories to be modified to incorporate new information. Although reconsolidation has become an attractive target for treatment of memories related to traumatic past experiences, we still do not know what new information triggers the updating of retrieved memories. Here, we used biochemical markers of synaptic plasticity in combination with a novel behavioral procedure to determine what was learned during memory reconsolidation under normal retrieval conditions. We eliminated new information during retrieval by manipulating animals' training experience and measured changes in proteasome activity and GluR2 expression in the amygdala, two established markers of fear memory lability and reconsolidation. We found that eliminating new contextual information during the retrieval of memories for predictable and unpredictable fear associations prevented changes in proteasome activity and glutamate receptor expression in the amygdala, indicating that this new information drives the reconsolidation of both predictable and unpredictable fear associations on retrieval. Consistent with this, eliminating new contextual information prior to retrieval prevented the memory-impairing effects of protein synthesis inhibitors following retrieval. These results indicate that under normal conditions, reconsolidation updates memories by incorporating new contextual information into the memory trace. Collectively, these results suggest that controlling contextual information present during retrieval may be a useful strategy for improving reconsolidation-based treatments of traumatic memories associated with anxiety disorders such as post-traumatic stress disorder.

Temporal phases of long-term potentiation (LTP): myth or fact?

Long-term potentiation (LTP) remains the most widely accepted model for learning and memory. In accordance with this belief, the temporal differentiation of LTP into early and late phases is accepted as reflecting the differentiation of short-term and long-term memory. Moreover, during the past 30 years, protein synthesis inhibitors have been used to separate the early, protein synthesis-independent (E-LTP) phase and the late, protein synthesis-dependent (L-LTP) phase. However, the role of these proteins has not been formally identified. Additionally, several reports failed to show an effect of protein synthesis inhibitors on LTP. In this review, a detailed analysis of extensive behavioral and electrophysiological data reveals that the presumed correspondence of LTP temporal phases to memory phases is neither experimentally nor theoretically consistent. Moreover, an overview of the time courses of E-LTP in hippocampal slices reveals a wide variability ranging from <1 h to more than 5 h. The existence of all these conflictual findings should lead to a new vision of LTP. We believe that the E-LTP vs. L-LTP distinction, established with protein synthesis inhibitor studies, reflects a false dichotomy. We suggest that the duration of LTP and its dependency on protein synthesis are related to the availability of a set of proteins at synapses and not to the de novo synthesis of plasticity-related proteins. This availability is determined by protein turnover kinetics, which is regulated by previous and ongoing electrical activities and by energy store availability.

146 EFFECT OF PROTEIN SYNTHESIS INHIBITOR ON BOAR SPERM CAPACITATION AND FERTILIZATION IN VITRO

In human, bovine, mouse, and rat sperm, translation of RNA to proteins in the mitochondrial ribosome during capacitation has been reported to be important for fertilization. The objective of this study was to examine effect of protein synthesis inhibitor (ribosome inhibitor) on boar sperm capacitation and IVF. Sperm from an ejaculated sperm-rich fraction of Berkshire boars were washed by centrifugation (1500 rpm for 35 min) in a Percoll gradient (45/90%) and then incubated in modified Medium-199 containing 0.4% BSA and 5 mM caffeine sodium benzoate, supplemented with or without a mitochondrial ribosome-specific (55S ribosome) inhibitor, chloramphenicol (CP; 0.3 mM), or a cytoplasmic ribosome-specific (80S ribosome) inhibitor, cyclohexide (CH; 3.6 mM), in an atmosphere of 5% CO2 in air at 39°C for 45 or 90 min. At 45 and 90 min after culture, sperm viability, motility, and chlortetracyclin-stained patterns (to assess the sperm functional status, capacitation, and acrosome reaction) were examined. Porcine oocytes were matured in vitro for 44 h in porcine oocyte medium supplemented with eCG, hCG, and dibutyryl cyclic adenosine monophosphate for the first 20 h. Matured oocytes after the removal of cumulus cells were co-cultured with sperm (final conc.: 2.5 × 105 cells mL–1) in the absence or presence of CP or CH for 8 h. Sperm penetrability was also determined. Statistical analyses of data from 4 replicated trials were performed by ANOVA. After 45 and 90 min of culture, neither CP nor CH affected sperm viability and motility (P &gt; 0.05). The addition of CP after 45 and 90 min of culture significantly (P &lt; 0.05) decreased capacitated and acrosome-reacted sperm rates, as detected by chlortetracyclin fluorescence assay (capacitated: control 9.6 v. CP 5.6%, control 17.8 v. CP 10.2%; acrosome reacted: control 4.6 v. CP 2.2%, control 9.2 v. CP 4.8%, respectively; P &lt; 0.05). In the presence of CH, IVF rate and number of sperm per penetrated egg were decreased (control 80.8 v. CH 46.8%, 2.2 v. 1.4, respectively; P &lt; 0.05). In the presence of CH, however, the percentage of metaphase II oocytes after co-culture with sperm for 8 h was lower than other 2 groups (control 87.6 v. CP 85.5 v. CH 74.0%; P &lt; 0.05), and the percentage of A/T-II oocytes was higher than in the other 2 groups (control 1.1 v. CP 0 v. CH 9.4%; P &lt; 0.05). From these results, we conclude that mitochondrial ribosome-specific inhibitor, chloramphenicol, affects capacitation and acrosome reaction but not penetration, whereas cytoplasmic ribosome-specific inhibitor, cyclohexide, decreases the number of oocytes that reach metaphase II stage and are penetrated.

Evidence for constitutive protein synthesis in hippocampal LTP stabilization

The notion that blockade of constitutive protein synthesis underlies the effect of protein synthesis inhibitors (PSIs) on long-term potentiation (LTP) stabilization was examined using the rat hippocampal CA3–CA1 synapse. Using a biochemical assay we found protein synthesis rate largely recovered 1h after wash-out of cycloheximide (CHX). Nonetheless, a 4-h CHX application followed by wash-out 1h prior to LTP resulted in a significant decrement of LTP stabilization. Wash-out initiated just prior to LTP, thus extending protein synthesis inhibition well into the post-LTP period, resulted in no further effect on LTP. However, short pre- and continuous post-tetanization application of PSIs failed to influence LTP persistence for up to 7h. Addition of hydrogen peroxide (H2O2) 5–25min following LTP induction resulted in parallel depression of potentiated and non-potentiated inputs, leaving LTP seemingly unaltered. However, in the presence of cyxloheximide the H2O2 application resulted in a significant reduction of LTP. In conclusion: LTP stabilization was impaired by pre-LTP application of protein synthesis inhibition but not by post-LTP application unless the slices were exposed to oxidative stress. We submit that these results favor the notion that constitutive rather than triggered protein synthesis is important for LTP stabilization.

Memory consolidation gets murky

Memory consolidation is widely understood to involve de novo protein synthesis, as the administration of protein synthesis inhibitors such as anisomycin and cyclohexamide prevents the formation of long-term memory following initial learning. A new study shows that in anaesthetized rats, intrahippocampal infusion of anisomycin or cyclohexamide reduces (and can even abolish) overall hippocampal electrical activity. These findings indicate that the reported effects of protein synthesis inhibitors on memory consolidation may have been confounded by their silencing effects on neural activity.

Neurosilence: Profound Suppression of Neural Activity following Intracerebral Administration of the Protein Synthesis Inhibitor Anisomycin

Early in their formation, memories are thought to be labile, requiring a process called consolidation to give them near-permanent stability. Evidence for consolidation as an active and biologically separate mnemonic process has been established through posttraining manipulations of the brain that promote or disrupt subsequent retrieval. Consolidation is thought to be ultimately mediated via protein synthesis since translational inhibitors such as anisomycin disrupt subsequent memory when administered in a critical time window just following initial learning. However, when applied intracerebrally, they may induce additional neural disturbances. Here, we report that intrahippocampal microinfusions of anisomycin in urethane-anesthetized rats at dosages previously used in memory consolidation studies strongly suppressed (and in some cases abolished) spontaneous and evoked local field potentials (and associated extracellular current flow) as well as multiunit activity. These effects were not coupled to the production of pathological electrographic activity nor were they due to cell death. However, the amount of suppression was correlated with the degree of protein synthesis inhibition as measured by autoradiography and was also observed with cycloheximide, another translational inhibitor. Our results suggest that (1) the amnestic effects of protein synthesis inhibitors are confounded by neural silencing and that (2) intact protein synthesis is crucial for neural signaling itself.

Dorsal and ventral striatal protein synthesis inhibition affect reinforcer valuation but not the consolidation of instrumental learning

The evidence for a role of the striatum in the acquisition of uncued instrumental responding is ambiguous. It has been shown that post-session infusions of anisomycin into the core of the nucleus accumbens (NAcc) impaired instrumental acquisition, but pre-training lesions of the NAcc suggest that it is not necessary. Recently, we demonstrated that the infusion of anisomycin into the anterior cingulate cortex impaired instrumental acquisition indirectly through a taste aversion. Thus, we hypothesized that post-session anisomycin infusions into the NAcc affected instrumental acquisition through an effect on reinforcer valuation. For the dorsal striatum, both post-session infusions of anisomycin and pre-training lesion studies suggest that neither the dorsolateral nor the dorsomedial striatum is necessary for the acquisition of instrumental responding. However, it has not been attempted to block plasticity in both regions concurrently, and we hypothesized that both regions independently contribute to acquisition through goal-directed and habitual learning. In the current experiments, we first replicated the effect of unprotected post-session anisomycin infusions into the NAcc on instrumental acquisition. Subsequently, we investigated the effect of protein synthesis inhibition in the NAcc and dorsomedial and dorsolateral striatum concurrently on instrumental acquisition, critically controlling for effects on reinforcer valuation. The anisomycin infusions induced an aversive state, but did not affect instrumental acquisition.

Phosphorylation of JDP2 on threonine-148 by the c-Jun N-terminal kinase targets it for proteosomal degradation

JDP2 (c-Jun dimerization protein 2) is a member of the basic leucine zipper family of transcription factors that is ubiquitously expressed in all examined cell types. JDP2 is phosphorylated on Thr148 by JNK (c-Jun N-terminal kinase) and p38 kinase, although the functional role of its phosphorylation is unknown. In the present paper we show that the JDP2 protein level is dramatically reduced in response to serum stimulation, anisomycin treatment, ultraviolet light irradiation and cycloheximide treatment, all of which activate the JNK pathway. In addition, endogenous and overexpressed JDP2 are phosphorylated in response to these stimuli. Replacement of Thr148 with an alanine residue stabilizes ectopically expressed JDP2 in the presence of the stimuli; conversely, substitution with glutamic acid destabilizes it. Serum-induced phosphorylation and degradation of JDP2 are specific to JNK activation since a JNK inhibitor (SP600125) abolishes these effects, whereas p38 and MEK inhibitors (SB203580 and UO126) have no effect. In the presence of cycloheximide, JDP2 is rapidly phosphorylated and degraded due to the combined effects of protein synthesis inhibition and activation of JNK. Pre-treatment of cells with SP600125 prior to cycloheximide treatment significantly prolongs the half-life of JDP2 that is found mainly in the unphosphorylated form. Lastly, the proteasome inhibitor (MG132) rescues JDP2 degradation following cycloheximide treatment and increases the expression of the JDP2 phospho-mimetic T148E mutant. Collectively, these results suggest that phosphorylation of JDP2 on thr148 by JNK targets it to the proteasome for degradation.

Corticosterone pretreatment suppresses stress-induced hypothalamic-pituitary-adrenal axis activity via multiple actions that vary with time, site of action and de novo protein synthesis

Glucocorticoid regulation of the hypothalamic-pituitary-adrenal (HPA) axis is believed to depend on multiple actions operative within discrete time domains. However, the underlying cellular and molecular mechanisms for those glucocorticoid actions remain undetermined. Moreover, there is absence of in vivo studies examining whether there are multiple glucocorticoid effects on HPA axis-related function within an intermediate feedback time frame (1-3 h after glucocorticoid elevation), and whether those effects depend on de novo protein synthesis. We examined in rats the effects of protein synthesis inhibition on HPA axis response to restraint (15 min) after 1 and 3 h phasic corticosterone (CORT) pretreatment. We measured HPA axis hormones (ACTH and CORT) and gene expression in the paraventricular nucleus (c-fos and crh genes), as well as gene expression in the anterior and intermediate pituitaries (c-fos and pomc genes). Both CORT pretreatment intervals produced inhibition of stress-induced ACTH secretion, but no inhibition was observed in the presence of protein synthesis inhibition. CORT pretreatment produced inhibitory effects on stress-induced gene expression that varied for each gene depending on the anatomical site, pretreatment time, and protein synthesis dependency. Taken together, the ACTH and gene expression patterns support the presence of multiple independent glucocorticoid actions initiated during the intermediate glucocorticoid negative feedback phase. Moreover, we conclude that those effects are exerted predominantly on the intrinsic anatomical elements of the HPA axis, and some of those effects depend on CORT induction of the expression of one or more regulatory gene products.

Citrate transporters play an important role in regulating aluminum-induced citrate secretion in Glycine max

To further understand the process of Al-induced citrate secretion from soybean roots, the effect of protein synthesis inhibitor, anion channel blockers, and citrate carrier inhibitors on Al-induced citrate exudation was investigated in Al-resistant soybean cultivar PI 416937. Citrate exudation from roots increased with the increase of Al concentration from 10 to 50 μM and initiated after 4 h of Al exposure. Protein synthesis inhibitor, cycloheximide (CHM; 25 μM) completely inhibited Al-induced citrate secretion during 12-h exposure, suggesting that novel protein synthesis was necessary in Al-induced citrate efflux. Also both anion channel blocker anthracene-9-carboxylic acid (A-9-C) and citrate carrier inhibitor mersalyl acid (Mersalyl) significantly reduced citrate secretion, suggesting that both anion channels in plasma membrane and citrate carriers in mitochondria membrane were the rate limiting factors of Al dependent citrate release. However, Al-induced citrate secretion was insensitive to anion channel blockers phenylglyoxal (PG), 4,4′-diisothiocyanostibene-2,2′-disulfonat (DIDS) and citrate carrier inhibitor pyridoxal 5′-P (PP).

Cycloheximide produces amnesia for extinction and reconsolidation in an appetitive odor discrimination task in rats

A considerable literature has shown deficits in memory resulting from the administration of protein synthesis inhibitors; however, most of the past literature in this field has focused on acquisition of new memory using aversively-motivated tasks. The effect of protein synthesis inhibition on appetitive learning and memory as well as extinction is less clear. The present study employed an appetitive odor discrimination paradigm to examine the effects of acute cycloheximide administration (1 mg/kg) on reconsolidation and extinction. Male, Long-Evans adult rats were trained to discriminate between two odors (i.e., cocoa and cinnamon) and then received extinction trials following an intraperitoneal injection of cycloheximide or vehicle. Twenty-four hours later, rats were tested via one non-reinforced test trial. Results showed amnesia for extinction as well as original training (i.e., correct odor choice) in cycloheximide-injected rats in this appetitive task, while vehicle-injected controls showed good memory for extinction. These data add to a growing literature showing the importance of protein synthesis inhibition for extinction and reconsolidation in appetitive learning and memory.