Protein Synthesis-dependent Process Research Articles

DNA methyltransferase activity in the basolateral amygdala is critical for reconsolidation of a heroin reward memory.

The persistence of drug memory contributes to relapse to drug seeking. The association between repeated drug exposure and drug-related cues leads to cravings triggered by drug-paired cues. The erasure of drug memories has been considered a promising way to inhibit cravings and prevent relapse. The re-exposure to drug-related cues destabilizes well-consolidated drug memories, during which a de novo protein synthesis-dependent process termed “reconsolidation” occurs to restabilize the reactivated drug memory. Disrupting reconsolidation of drug memories leads to the attenuation of drug-seeking behavior in both animal models and people with addictions. Additionally, epigenetic mechanisms regulated by DNA methyltransferase (DNMT) are involved in the reconsolidation of fear and cocaine reward memory. In the present study, we investigated the role of DNMT in the reconsolidation of heroin reward memory. In the heroin self-administration model in rats, we tested the effects of DNMT inhibition during the reconsolidation process on cue-induced reinstatement, heroin-priming-induced reinstatement, and spontaneous recovery of heroin-seeking behavior. We found that the bilateral infusion of 5-azacytidine (5-AZA) inhibiting DNMT into the basolateral amygdala (BLA) immediately after heroin reward memory retrieval, but not delayed 6 h after retrieval or without retrieval, decreased subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior. These findings demonstrate that inhibiting the activity of DNMT in BLA during the reconsolidation of heroin reward memory attenuates heroin-seeking behavior, which may provide a potential strategy for the therapeutic of heroin addiction.

Temporal dynamic of the hippocampal structural plasticity associated with the fear memory destabilization/reconsolidation process

Reconsolidation of a contextual fear memory is a protein synthesis-dependent process in which a previously destabilized memory returns to a stable state. This process has become the subject of many studies due to its importance in memory processing, maintenance and updating, and its potential role as a therapeutical target in fear memory disorders such as phobias and post-traumatic stress disorder. In this sense, understanding the underlying mechanisms of memory reconsolidation is paramount in developing potential treatments for such memory dysfunctions. In the present work, we studied the interaction between two key neural structures involved in the reconsolidation process: the basolateral amygdala complex of the amygdala (BLA) and the dorsal hippocampus (DH). Our results show changes in the structural plasticity of the CA1 region of the DH in the form of dendritic spines density changes associated with the destabilization/reconsolidation process. Furthermore, we demonstrate a modulatory role of BLA over such structural plasticity by infusing different drugs such as ifenprodil, a destabilization blocker, and propranolol, a reconsolidation disruptor, in this brain structure. Altogether our work shows a particular temporal dynamic in the CA1 region of DH that accompanies the destabilization/reconsolidation process and aims to provide new information on the underlying mechanisms of this process that potentially contributes for a better understanding of memory storage, maintenance, expression and updating, and its potential medical applications.

Electroconvulsive Shock Does Not Impair the Reconsolidation of Cued and Contextual Pavlovian Threat Memory

Existing memories, when retrieved under certain circumstances, can undergo modification through the protein synthesis-dependent process of reconsolidation. Disruption of this process can lead to the weakening of a memory trace, an approach which is being examined as a potential treatment for disorders characterized by pathological memories, such as Post-Traumatic Stress Disorder. The success of this approach relies upon the ability to robustly attenuate reconsolidation; however, the available literature brings into question the reliability of the various drugs used to achieve such a blockade. The identification of a drug or intervention that can reliably disrupt reconsolidation without requiring intracranial access for administration would be extremely useful. Electroconvulsive shock (ECS) delivered after memory retrieval has been demonstrated in some studies to disrupt memory reconsolidation; however, there exists a paucity of literature characterizing its effects on Pavlovian fear memory. Considering this, we chose to examine ECS as an inexpensive and facile means to impair reconsolidation in rats. Here we show that electroconvulsive seizure induction, when administered after memory retrieval, (immediately, after 30 min, or after 1 h), does not impair the reconsolidation of cued or contextual Pavlovian fear memories. On the contrary, ECS administration immediately after extinction training may modestly impair the consolidation of fear extinction memory.

Molecular Mechanisms of Reconsolidation-Dependent Memory Updating.

Memory is not a stable record of experience, but instead is an ongoing process that allows existing memories to be modified with new information through a reconsolidation-dependent updating process. For a previously stable memory to be updated, the memory must first become labile through a process called destabilization. Destabilization is a protein degradation-dependent process that occurs when new information is presented. Following destabilization, a memory becomes stable again through a protein synthesis-dependent process called restabilization. Much work remains to fully characterize the mechanisms that underlie both destabilization and subsequent restabilization, however. In this article, we briefly review the discovery of reconsolidation as a potential mechanism for memory updating. We then discuss the behavioral paradigms that have been used to identify the molecular mechanisms of reconsolidation-dependent memory updating. Finally, we outline what is known about the molecular mechanisms that support the memory updating process. Understanding the molecular mechanisms underlying reconsolidation-dependent memory updating is an important step toward leveraging this process in a therapeutic setting to modify maladaptive memories and to improve memory when it fails.

New learning while consolidating memory during sleep is actively blocked by a protein synthesis dependent process.

Brief experiences while a memory is consolidated may capture the consolidation, perhaps producing a maladaptive memory, or may interrupt the consolidation. Since consolidation occurs during sleep, even fleeting experiences when animals are awakened may produce maladaptive long-term memory, or may interrupt consolidation. In a learning paradigm affecting Aplysia feeding, when animals were trained after being awakened from sleep, interactions between new experiences and consolidation were prevented by blocking long-term memory arising from the new experiences. Inhibiting protein synthesis eliminated the block and allowed even a brief, generally ineffective training to produce long-term memory. Memory formation depended on consolidative proteins already expressed before training. After effective training, long term memory required subsequent transcription and translation. Memory formation during the sleep phase was correlated with increased CREB1 transcription, but not CREB2 transcription. Increased C/EBP transcription was a correlate of both effective and ineffective training and of treatments not producing memory.

Epigenetic regulation of neuronal immediate early genes is associated with decline in their expression and memory consolidation in scopolamine-induced amnesic mice.

Recently, we reported a correlation of scopolamine mediated decline in memory consolidation with increase in the expression of DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) in the mouse hippocampus. Memory consolidation is a protein synthesis-dependent process which involves the expression of synaptic plasticity genes, particularly neuronal immediate early genes (IEGs). However, the mechanism of regulation of these genes during decline in memory is poorly understood. Therefore, we have studied the epigenetic regulation of expression of neuronal IEGs in scopolamine-induced amnesic mice. Scopolamine significantly impaired memory consolidation as tested by radial arm maze, and the expression of neuronal IEGs was downregulated in the hippocampus as revealed by qRT-PCR and Western blotting. Further, methylated DNA immunoprecipitation (MeDIP) analysis showed increase in DNA methylation, while chromatin immunoprecipitation (ChIP) revealed decrease in H3K9/14 acetylation at the promoter of neuronal IEGs. Taken together, the present study shows that increased DNA methylation and decreased histone acetylation at the promoter of neuronal IEGs are associated with decline in their expression and memory consolidation during scopolamine-induced amnesia. These findings suggest that the epigenetic regulation through altered DNA methylation and histone acetylation might be explored further to develop potential therapeutic interventions for amnesia.

Activation of NF-κB in Basolateral Amygdala Is Required for Memory Reconsolidation in Auditory Fear Conditioning

Posttraumatic stress disorder (PTSD) is characterized by acute and chronic changes in the stress response, manifested as conditioned fear memory. Previously formed memories that are susceptible to disruption immediately after retrieval undergo a protein synthesis-dependent process to become persistent, termed reconsolidation, a process that is regulated by many distinct molecular mechanisms that control gene expression. Increasing evidence supports the participation of the transcription factor NF-κB in the different phases of memory. Here, we demonstrate that inhibition of NF-κB in the basolateral amygdala (BLA), but not central nucleus of the amygdala, after memory reactivation impairs the retention of amygdala-dependent auditory fear conditioning (AFC). We used two independent pharmacological strategies to disrupt the reconsolidation of AFC. Bilateral intra-BLA infusion of sulfasalazine, an inhibitor of IκB kinase that activates NF-κB, and bilateral intra-BLA infusion of SN50, a direct inhibitor of the NF-κB DNA-binding complex, immediately after retrieval disrupted the reconsolidation of AFC. We also found that systemic pretreatment with sodium butyrate, a histone deacetylase inhibitor that enhances histone acetylation, in the amygdala rescued the disruption of reconsolidation induced by NF-κB inhibition in the BLA. These findings indicate that NF-κB activity in the BLA is required for memory reconsolidation in AFC, suggesting that NF-κB might be a potential pharmacotherapy target for posttraumatic stress disorder.

Protein synthesis and degradation are required for the incorporation of modified information into the pre-existing object-location memory

Although some reports indicate that protein synthesis dependent process may be induced by updating information, the role of protein synthesis and degradation in changing the content of pre-existing memory is yet unclear. In this study, we utilized an object rearrangement task, in which partial information related to a pre-existing memory is changed, promoting memory modification. Inhibitors of both protein synthesis and protein degradation impaired adequate incorporation of the altered information, each in a distinctive way. These results indicate that protein synthesis and degradation play key roles in memory modification.

Temporal requirement of C/EBPβ in the amygdala following reactivation but not acquisition of inhibitory avoidance

Following learning, a memory is fragile and undergoes a protein synthesis-dependent consolidation process in order to become stable. Established memories can again become transiently sensitive to disruption if reactivated and require another protein synthesis-dependent process, known as reconsolidation, in order to persist. Here, we show that, in the basolateral amygdala (BLA), protein synthesis is necessary for both consolidation and reconsolidation of inhibitory avoidance (IA) memory, while the expression of the transcription factor CCAAT enhancer binding protein beta (C/EBPbeta) is essential only for the reconsolidation process. Moreover, the critical roles of both protein synthesis and C/EBPbeta following IA reactivation are temporally restricted, as they are necessary only for recent but not old IA memories. These results, together with previous findings showing that in the hippocampus both protein synthesis and C/EBPbeta expression are required for consolidation but not reconsolidation of IA indicate that the stabilization process that takes place either after training or memory retrieval engages distinct neural circuits. Within these circuits, the C/EBPbeta-dependent molecular pathway appears to be differentially recruited.

On the participation of hippocampal PKC in acquisition, consolidation and reconsolidation of spatial memory

Memory consolidation involves a sequence of temporally defined and highly regulated changes in the activation state of several signaling pathways that leads to the lasting storage of an initially labile trace. Despite appearances, consolidation does not make memories permanent. It is now known that upon retrieval well-consolidated memories can become again vulnerable to the action of amnesic agents and in order to persist must undergo a protein synthesis–dependent process named reconsolidation. Experiments with genetically modified animals suggest that some PKC isoforms are important for spatial memory and earlier studies indicate that several PKC substrates are activated following spatial learning. Nevertheless, none of the reports published so far analyzed pharmacologically the role played by PKC during spatial memory processing. Using the conventional PKC and PKCμ inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrollo[3,4-c]carbazole (Gö6976) we found that the activity of these kinases is required in the CA1 region of the rat dorsal hippocampus for acquisition and consolidation of spatial memory in the Morris water maze learning task. Our results also show that when infused into dorsal CA1 after non-reinforced retrieval, Gö6976 produces a long-lasting amnesia that is independent of the strength of the memory trace, suggesting that post-retrieval activation of hippocampal PKC is essential for persistence of spatial memory.

Protein synthesis and the mechanisms of lasting change in anxiety induced by severe stress

Brief, unprotected exposure of rats to cats (predator stress) may be lastingly anxiogenic in a variety of tests of rodent anxiety. Recent findings suggest that predator stress induced plasticity in neural circuitry implicated in fear learning underlies some of these anxiogenic effects. In addition, recent work implicates a consolidation-like process in the impact of predator stress on anxiety in that effects of predator stress may be interrupted by immediate post stressor pharmacological interventions. The present study tested whether “consolidation” of the anxiogenic effects of predator stress were dependent on protein synthesis. In addition, the study examined whether a protein synthesis dependent reconsolidation-like process was at work when rats were exposed to a cat twice. Anisomycin (210 mg/kg) or vehicle (Tween 80 in saline) was injected subcutaneously 1 min after a single cat exposure (consolidation test paradigm) or a 1 min after a second cat exposure (reconsolidation test paradigm) and behavior tested 7–8 days after predator stress. In the consolidation test paradigm, anisomycin blocked the anxiogenic effects of predator stress in the elevated plus maze (EPM) measured with open arm exploration. Moreover, anisomycin blocked the potentiation of startle by predator stress when rats were startled in the light, but not when startled in the dark. In contrast, the delay of habituation of startle produced by predator stress was unaffected by anisomycin. Suppression of risk assessment in the EPM by predator stress was not affected by anisomycin either. In startle testing, vehicle injection 1 min after predator stress led to a lasting suppression, rather than enhancement of startle response. Vehicle plus predator stress enhanced and prolonged corticosterone level changes sampled over 30–180 min after treatment when compared to handled or predator stressed only rats. In addition, predator stress plus vehicle suppression of startle was blocked by a benzodiazepine anxiolytic (chloradiazepoxide) or the glucorticoid receptor (GR) blocker RU486. Both drugs returned startle to the predator stressed only heightened levels. It is argued that an added anxiogenic effect of vehicle injection plus predator stress leads to a suppression, rather than enhancement of startle. Startle suppression appears to be mediated, in part, by activation of GR by corticosterone which engages a protein synthesis dependent process, since anisomycin blocked the startle suppressive effects of vehicle. Startle suppression also appeared to be independent of the startle enhancing effect of predator stress and in competition with it. Since predator stress may model aspects of hyperarousal associated with post traumatic stress disorder (PTSD), implications of these findings for understanding of mechanisms of initiation of the disorder and for treatment are discussed.

Effects of aerobic versus anoxic conditions on glutamine synthetase activity in eelgrass ( Zostera marina L.) roots: regulation of ammonium assimilation potential

Root glutamine synthetase (GS; EC 6.3.1.2) activity was measured daily (0 to 4 days) for eelgrass ( Zostera marina L.) plants held under continuous darkness rooted in sediments, continuous darkness without sediments, continuous light without sediments, and control light/dark cycle (Control L/D). Roots experiencing prolonged aerobiosis exhibited lower activity in vitro than controls, whereas roots experiencing prolonged anoxia exhibited increased activity. Plants held in darkness without sediments had activity intermediate between controls and anoxic roots. One-hour pretreatment of root extracts with ATP slightly reduced in vitro glutamine synthetase activity, whereas pretreatment with ADP and AMP increased activity ≈50%. While glutamine synthetase activity increased with higher adenylate energy charge (AEC) in the reaction mixture, pretreatment of enzyme extracts at high adenylate energy charges decreased subsequent activity relative to pretreatment at lower energy charges. One-hour pretreatment with l-alanine (Ala) had little effect on enzyme activity. Pretreatment with l-glutamine (Gln), l-glutamate (Glu), and γ-amino butyric acid (GABA) increased activity ≈75%. Incubation of excised roots under anoxic conditions for 24 h nearly doubled enzyme activity. However, addition of cycloheximide to anoxic root incubations lessened or prevented the increase in activity. It appears that enhanced glutamine synthetase activity following periods of root anoxia results from interactions with metabolites that fluctuate between aerobic and anoxic conditions, particularly adenylates, and from de novo synthesis of glutamine synthetase or some other protein synthesis-dependent process.

Extracellular signal-regulated kinase 1/2 is required for the induction of group I metabotropic glutamate receptor-mediated epileptiform discharges.

Transient stimulation of group I metabotropic glutamate receptors (mGluRs) induces persistent prolonged epileptiform discharges in hippocampal slices via a protein synthesis-dependent process. At present, the signaling process underlying the induction of these epileptiform discharges remains unknown. We examined the possible role of extracellular signal-regulated kinases (ERK1 and ERK2) because these kinases can be activated by group I mGluRs, and their activation may regulate gene expression and alter protein synthesis. The group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 50 microm) induced activation of ERK1/2 in hippocampal slices. 2-(2-Diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059) (50 microm) a specific inhibitor of mitogen-activated protein kinase kinase (MEK), suppressed ERK1/2 activation by DHPG. PD98059 or another MEK inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (10 microm), also prevented the induction of the prolonged epileptiform discharges by DHPG. In the presence of ionotropic glutamate receptor inhibitors and tetrodotoxin (blockers), DHPG-induced epileptiform discharges were suppressed, whereas ERK1/2 activation persisted. Protein kinase C inhibitors (2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide, 1 microm; or chelerythrine, 10 microm) did not prevent the generation of DHPG-induced epileptiform discharges, nor did they suppress the activation of ERK1/2 by DHPG in slices pretreated with the blockers. Genistein (30 microm), a broad-spectrum tyrosine kinase inhibitor, suppressed the DHPG-induced epileptiform discharges and the ERK1/2 activation in the presence of blockers. Induction of DHPG-mediated epileptiform discharges was also suppressed by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (10 microm), an Src-family tyrosine kinase inhibitor. The study shows that group I mGluRs activate ERK1/2 through a tyrosine kinase-dependent process and that this activation of ERK1/2 is necessary for the induction of prolonged epileptiform discharges in the hippocampus.

TGFβ inhibits LPS-induced chemokine mRNA stabilization

AbstractThe mechanisms involved in anti-inflammatory action of transforming growth factor β (TGFβ) have been examined by evaluating its effect on chemokine gene expression in mouse macrophages. Lipopolysaccharide (LPS)–stimulated expression of the CXC chemokines KC and MIP-2 was selectively reduced by TGFβ in a time- and protein synthesis–dependent process. While TGFβ had a modest effect on transcription of the KC and MIP-2 mRNAs as measured by nuclear run-on, it had no effect on LPS-stimulated luciferase expression driven by the KC promoter nor on the activation of nuclear factor κB (NFκB) DNA-binding activity and transactivation function. Interestingly, KC mRNA levels were markedly reduced by TGFβ treatment in cells transfected with KC genomic or cDNA constructs driven from either the KC or cytomegalovirus (CMV) promoters, demonstrating the importance of sequences within the mature mRNA and suggesting that suppression may involve a posttranscriptional mechanism. In support of this possibility, LPS stimulation prolonged the half-life of KC mRNA and this stabilization response was blocked in cells treated with TGFβ. Examination of KC mRNA expressed under control of a tetracycline-responsive promoter demonstrated that TGFβ prevented stabilization of KC mRNA, in response to LPS but did not alter KC mRNA half-life directly. KC mRNA stabilization by LPS was dependent on activation of p38 mitogen-activated protein kinase (MAPK) activity, and TGFβ treatment inhibited p38 MAPK activation. These findings support the hypothesis that TGFβ-mediated suppression of chemokine gene expression involves antagonism of LPS-stimulated KC mRNA stabilization via inhibition of p38 MAPK.

Various phosphorylation pathways, depending on agonist and antagonist binding to endogenous estrogen receptor alpha (ERalpha), differentially affect ERalpha extractability, proteasome-mediated stability, and transcriptional activity in human breast cancer cells.

Estrogen receptor-alpha (ER) is down-regulated in the presence of its cognate ligand, estradiol (E2), as well as in the presence of antiestrogens, through the ubiquitin proteasome pathway. Here, we show that, at pharmacological concentrations, the degradation rate of pure antagonist/endogenous ER complexes from human breast cancer MCF-7 cells is 10 times faster than that of ER-E2 complexes, while 4-hydroxy-tamoxifen (4-OH-T)-ER complexes are stable. Whereas pure antagonist-ER complexes are firmly bound to a nuclear compartment from which they are not extractable, the 4-OH-T-ER accumulates in a soluble cell compartment. No difference was observed in the fate of ER whether bound to pure antiestrogens ICI 182,780 or RU 58668. Cycloheximide experiments showed that, while the proteasome-mediated destruction of E2-ER (unlike that of RU 58668- and ICI 182,780-ER) complexes could implicate (or not) a protein synthesis-dependent process, both MAPKs (p38 and ERKs p44 and p42) are activated. By using a panel of kinase inhibitors/activators to study the impact of phosphorylation pathways on ER degradation, we found that protein kinase C is an enhancer of proteasome-mediated degradation of both ligand-free and ER bound to either E2, 4-OH-T, and pure antagonists. On the contrary, protein kinase A, MAPKs, and phosphatidyl-inositol-3 kinase all impede proteasome-mediated destruction of ligand free and E2-bound ER while only MAPKs inhibit the degradation of pure antiestrogens/ER species. In addition, no correlation was found between the capacity of kinase inhibitors to affect ER stability and the basal or E2-induced transcription. These results suggest that, in MCF-7 breast cancer cells, ER turnover, localization, and activity are maintained by an equilibrium between various phosphorylation pathways, which are differently modulated by ER ligands and protein kinases.

Dynamical aspects of in vitro conditioning in Hermissenda type B photoreceptor.

Dynamics of changes in physiology and morphology were studied in Hermissenda photoreceptors after in vitro conditioning with paired light and vibration. An increase in input resistance of the type B photoreceptor was observed following 5 paired presentations of light and vibration. It peaked at 10 min after in vitro conditioning, then decreased to a level twice the pre-conditioning level for more than 60 min. Contraction of the terminal branches along centro-lateral direction was initiated 5 min after conditioning and reached its final state at 10 min after conditioning. The pairing specific contraction of the axon terminal was not observed in ASW containing anisomycin. The dynamics in physiology and morphology were completely parallel 30 min after conditioning. These findings suggested that in vitro conditioning induced contraction was dependent on protein synthesis dependent process initiated within 5 min after training trials and that the change of cell morphology is a form of short-term synaptic plasticity that involves changes in macromolecular synthesis. Present findings that functional remodeling at the terminal branch of the type B photoreceptor occurred within 10 min after conditioning was the fastest modification process reported so far.

Survival of hippocampal neurons in culture upon hypoxia: effect of erythropoietin.

The potential of erythropoietin (EPO) to reduce hypoxia-induced cell death has been investigated in 5-day-old primary cultures of rat postnatal hippocampal neurons. Application of EPO (100 pM) at the start of hypoxia resulted in a significant reduction of neuronal death (33.0 +/- 7.5% in cells incubated with EPO vs 56.75 +/- 7.3% in non-treated cells; n = 4, p < 0.021). Similiar results were obtained upon application of cycloheximide (CHX; 1 microM) simultaneously with hypoxia (34.75 +/- 5.6% vs 56.75 +/- 7.3% with and without CHX, respectively, n = 4, p < 0.035), indicating that hypoxia-induced neuronal death is an active, protein synthesis-dependent process. Both, EPO and EPO receptor (EPOR) were found to be expressed after hypoxia in hippocampal neurons in vitro and in vivo. These results demonstrate for the first time that EPO can reverse hypoxia-induced neuronal death when applied simultaneously with the hypoxic stimulus.

The tryptophan catabolite picolinic acid selectively induces the chemokines macrophage inflammatory protein-1 alpha and -1 beta in macrophages.

We previously found that the tryptophan catabolite picolinic acid (PA) is a costimulus for the activation of macrophage effector functions. In this study, we have investigated the ability of PA to modulate the expression of chemokines in macrophages. We demonstrate that PA is a potent activator of the inflammatory chemokines MIP (macrophage inflammatory protein)-1 alpha and MIP-1 beta (MIPs) mRNA expression in mouse macrophages in a dose- and time-dependent fashion and through a de novo protein synthesis-dependent process. The induction by PA occurred within 3 h of treatment and reached a peak in 12 h. The stimulatory effects of PA were selective for MIPs because other chemokines, including monocyte chemoattractant protein-1, RANTES, IFN-gamma-inducible protein-10, MIP-2, and macrophage-derived chemokine, were not induced under the same experimental conditions and were not an epiphenomenon of macrophage activation because IFN-gamma did not affect MIPs expression. Induction of both MIP-1 alpha and MIP-1 beta by PA was associated with transcriptional activation and mRNA stabilization, suggesting a dual molecular mechanism of control. Iron chelation could be involved in MIPs induction by PA because iron sulfate inhibited the process and the iron-chelating agent, desferrioxamine, induced MIPs expression. We propose the existence of a new pathway leading to inflammation initiated by tryptophan catabolism that can communicate with the immune system through the production of PA, followed by secretion of chemokines by macrophages. These results establish the importance of PA as an activator of macrophage proinflammatory functions, providing the first evidence that this molecule can be biologically active without the need for a costimulatory agent.

Identification of immediate early genes during TPA-induced human myeloblastic leukemia ML-1 cell differentiation.

Human myeloblastic ML-1 can be induced to differentiate into monocytes/macrophages by 12-0-tetradecanoylphorbol-13-acetate (TPA). In order to understand the molecular mechanism regulating ML-1 cell differentiation, we focused on the characterization of immediate early genes activated by TPA using the mRNA differentiation display polymerase chain reaction (DD-PCR) and Northern analyses. A modified procedure, the reverse dot slot, was developed to confirm upregulated genes during the early stages of TPA-induced ML-1 cell differentiation. DNA sequencing analyses of 10 subcloned cDNA fragments, selected on the basis of the outcome of the reverse dot slot procedure, revealed that eight were derived from distinct genes. Among these clones, one was a novel gene (G07-5), another (A02-1) was highly homologous to the sequence of a fetal brain cDNA fragment, and the remaining six corresponded to jun-D, rantes, ssat, CD 14, ferritin heavy chain (fhc) and transposons Tn10-like transcript, respectively. Although these genes were all upregulated by TPA, the peak time of mRNA expression varied. jun-D, ssat and A02-1 expressions were superinduced in the presence of cycloheximide, which indicates that they belong to the immediate early gene family. On the other hand, TPA-induced rantes expression was not superinduced by cycloheximide, suggesting a protein synthesis-dependent process. As there are no previous reports of expression of these genes in TPA-induced ML-1 cells, little or no information is available concerning their function in mediating myeloblastic cell differentiation. Thus, this study illuminates new avenues of research for elucidating the function of genes regulating terminal differentiation of myeloid progenitors. 1998 Elsevier Science B.V.

Bradykinin B 1 receptors in human umbilical vein

The present study was undertaken to demonstrate the presence of bradykinin B 1 receptors mediating contraction of human umbilical vein. The bradykinin B 1 receptor selective agonist, des-Arg 9-bradykinin, produced a dose-dependent contractile response of human umbilical vein rings. Furthermore, des-Arg 9-bradykinin-mediated response increased in a time-dependent manner in vitro. The maximal response to des-Arg 9-bradykinin, expressed as percentage of the maximum elicited by serotonin, was: 10 ± 2 at 15 min, 55 ± 5 at 120 min and 80 ± 3 at 300 min. Des-Arg 9-bradykinin-mediated contractions were inhibited by the specific bradykinin B 1 receptor antagonist des-Arg 9-[Leu 8]bradykinin which produced parallel shifts in the dose-response curve to the selective bradykinin B 1 receptor agonist. Schild regression analysis of data established a pA 2 value of 6.16 ± 0.06. Kinin-induced contraction was not modified by pre-treatment with indomethacin (10 μM), a cyclo-oxygenase inhibitor. On the other hand, continuous exposure to the anti-inflammatory steroid dexamethasone (100 μM) or to the protein synthesis inhibitor cycloheximide (70 μM) largely prevented the sensitization to des-Arg 9-bradykinin in incubated human umbilical vein rings. These results confirm the presence of bradykinin B 1 receptors which mediate contraction in isolated human umbilical vein. These responses are up-regulated in a time- and protein synthesis-dependent process.