Protein Synthesis-dependent Mechanisms Research Articles

Effects of Probiotics, Prebiotics and Synbiotic Supplementation on Cognitive Impairment: A Review

Learning and memory formation are the two essential terms widely used in the field of cognition. Learning can be defined as acquiring new information or skills. Memory is formed due to changes in the neuronal system as a result of continuous stimulus exposure. Both learning and memory are fundamental processes that occur in all living organisms. Memory is broadly categorized into two different categories such as short-term memory (STM) and long-term memory (LTM). Compared to STM, LTM plays an essential role in the day-to-day activities of different living organisms. LTM requires RNA and protein synthesis-dependent mechanisms for memory storage, which lasts up to their lifetime. LTM formation is initiated when the neurotransmitters are released from the presynaptic neuron; further released neurotransmitters bind with their respective receptors present in the postsynaptic neuron and initiate the calcium influx. Calcium influx results in the further activation of molecules involved in the neuronal signaling pathway and results in memory formation. Present review reports the outcome of recent studies which showed that probiotic supplement is responsible for the retrieval of memory in case of memory impairment and its uses in the treatment of neurodegenerative disorders like mild cognitive impairment (MCI), Alzheimer's disease (AD). Recent research studies were shown that probiotic microorganisms may positively regulate neurotransmitter release and increase the calcium influx, brain derived neurotrophic factor (BDNF), and N-methyl-D-aspartate receptor (NMDAR) and plays a pivotal role in the LTM formation in gut-dysbiosed & memory-impaired animal models.

BDNF‐dependent phrenic motor facilitation: role of protein synthesis

Moderate acute intermittent hypoxia (mAIH) induces a form of respiratory motor plasticity known as phrenic long‐term facilitation (pLTF) — a persistent increase in inspiratory phrenic motor output that requires cervical spinal serotonin receptor activation and new synthesis of brain‐derived neurotrophic factor (BDNF). One fundamental difference between short‐term (minutes) and long‐term (days) synaptic plasticity is the reliance on transcription versus translation of genes required for protein synthesis. Although it is known that mAIH‐induced pLTF requires new synthesis of BDNF protein (Baker‐Herman et al. 2004), it is unknown if this form of plasticity also requires gene transcription. In turn, neurotrophins such as BDNF regulate synaptic transmission via rapid protein synthesis‐independent versus delayed protein synthesis‐dependent mechanisms. Although BDNF protein synthesis is necessary for mAIH‐induced pLTF, and cervical spinal application of exogenous BDNF is sufficient to elicit phrenic motor facilitation, it is unknown if phrenic motor facilitation requires new‐protein synthesis downstream from BDNF/TrkB signaling. Thus, we tested the hypotheses that: 1) mAIH‐induced pLTF requires transcriptional regulation of new protein synthesis, and 2) if translational regulation of new protein synthesis is necessary for BDNF‐induced phrenic motor facilitation. Experiments were performed on anesthetized, mechanically ventilated adult male Sprague Dawley rats. To inhibit spinal transcription, rats were pre‐treated with cervical spinal intrathecal injections of the transcription inhibitor triptolide, and then exposed to mAIH (3, 5‐min hypoxic episodes, PaO2 35–55 mmHg; 5 min intervals). To explore the requirement for new protein synthesis downstream from BDNF/TrkB signaling, a widely used translation inhibitor (emetine), was injected into the intrathecal space prior to BDNF injections. Intrathecal cervical spinal triptolide injections had no discernible effect on mAIH‐induced pLTF, demonstrating that pLTF does not require gene transcription. On the other hand, pre‐treatment with emetine blocked mAIH‐induced pLTF, confirming that pLTF requires new protein synthesis. Whereas cervical spinal BDNF injections induced phrenic motor facilitation similar to pLTF, its magnitude was unaffected by emetine pre‐treatment. Thus, BDNF/TrkB elicits phrenic motor facilitation via a mechanism that does not require additional protein synthesis. Collectively, our findings suggest that pLTF requires new synthesis of BDNF protein via translation‐dependent, transcription‐independent mechanisms, whereas additional new protein synthesis is not required downstream from BDNF/TrkB signaling. Thus, BDNF‐induced phrenic motor facilitation likely occurs exclusively via covalent modifications of existing proteins, presumably phosphorylation events mediated via BDNF/TrkB dependent activation of PKC‐q (Agosto‐Marlin & Mitchell, 2017).Support or Funding InformationNIH HL69064, HL111598 and the UF McKnight Brain InstituteThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of the visual experience-dependent nascent proteome in neuronal plasticity.

Experience-dependent synaptic plasticity refines brain circuits during development. To identify novel protein synthesis-dependent mechanisms contributing to experience-dependent plasticity, we conducted a quantitative proteomic screen of the nascent proteome in response to visual experience in Xenopus optic tectum using bio-orthogonal metabolic labeling (BONCAT). We identified 83 differentially synthesized candidate plasticity proteins (CPPs). The CPPs form strongly interconnected networks and are annotated to a variety of biological functions, including RNA splicing, protein translation, and chromatin remodeling. Functional analysis of select CPPs revealed the requirement for eukaryotic initiation factor three subunit A (eIF3A), fused in sarcoma (FUS), and ribosomal protein s17 (RPS17) in experience-dependent structural plasticity in tectal neurons and behavioral plasticity in tadpoles. These results demonstrate that the nascent proteome is dynamic in response to visual experience and that de novo synthesis of machinery that regulates RNA splicing and protein translation is required for experience-dependent plasticity.

Differential effects of dopamine signalling on long-term memory formation and consolidation in rodent brain.

BackgroundUsing auditory discrimination learning in gerbils, we have previously shown that activation of auditory-cortical D1/D5 dopamine receptors facilitates mTOR-mediated, protein synthesis-dependent mechanisms of memory consolidation and anterograde memory formation. To understand molecular mechanisms of this facilitatory effect, we tested the impact of local pharmacological activation of different D1/D5 dopamine receptor signalling modes in the auditory cortex. To this end, protein patterns in soluble and synaptic protein-enriched fractions from cortical, hippocampal and striatal brain regions of ligand- and vehicle-treated gerbils were analysed by 2D gel electrophoresis and mass spectrometry 24 h after intervention.ResultsAfter auditory-cortical injection of SKF38393 – a D1/D5 dopamine receptor-selective agonist reported to activate the downstream effectors adenylyl cyclase and phospholipase C – prominent proteomic alterations compared to vehicle-treated controls appeared in the auditory cortex, striatum, and hippocampus, whereas only minor changes were detectable in the frontal cortex. In contrast, auditory-cortical injection of SKF83959 – a D1/D5 agonist reported to preferentially stimulate phospholipase C – induced pronounced changes in the frontal cortex. At the molecular level, we detected altered regulation of cytoskeletal and scaffolding proteins, changes in proteins with functions in energy metabolism, local protein synthesis, and synaptic signalling. Interestingly, abundance and/or subcellular localisation of the predominantly presynaptic protein α-synuclein displayed dopaminergic regulation. To assess the role of α-synuclein for dopaminergic mechanisms of memory modulation, we tested the impact of post-conditioning systemic pharmacological activation of different D1/D5 dopamine receptor signalling modes on auditory discrimination learning in α-synuclein-mutant mice. In C57BL/6JOlaHsd mice, bearing a spontaneous deletion of the α-synuclein-encoding gene, but not in the related substrains C57BL/6JCrl and C57BL/6JRccHsd, adenylyl cyclase-mediated signalling affected acquisition rates over future learning episodes, whereas phospholipase C-mediated signalling affected final memory performance.ConclusionsDopamine signalling modes via D1/D5 receptors in the auditory cortex differentially impact protein profiles related to rearrangement of cytomatrices, energy metabolism, and synaptic neurotransmission in cortical, hippocampal, and basal brain structures. Altered dopamine neurotransmission in α-synuclein-deficient mice revealed that distinct D1/D5 receptor signalling modes may control different aspects of memory consolidation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0069-2) contains supplementary material, which is available to authorized users.

Dopaminergic Modulation of Auditory Cortex-Dependent Memory Consolidation through mTOR

Previous studies in the auditory cortex of Mongolian gerbils on discrimination learning of the direction of frequency-modulated tones (FMs) revealed that long-term memory formation involves activation of the dopaminergic system, activity of the protein kinase mammalian target of rapamycin (mTOR), and protein synthesis. This led to the hypothesis that the dopaminergic system might modulate memory formation via regulation of mTOR, which is implicated in translational control. Here, we report that the D1/D5 dopamine receptor agonist SKF-38393 substantially improved gerbils’ FM discrimination learning when administered systemically or locally into the auditory cortex shortly before, shortly after, or 1 day before conditioning. Although acquisition performance during initial training was normal, the discrimination of FMs was enhanced during retraining performed hours or days after agonist injection compared with vehicle-injected controls. The D1/D5 receptor antagonist SCH-23390, the mTOR inhibitor rapamycin, and the protein synthesis blocker anisomycin suppressed this effect. By immunohistochemistry, D1 dopamine receptors were identified in the gerbil auditory cortex predominantly in the infragranular layers. Together, these findings suggest that in the gerbil auditory cortex dopaminergic inputs regulate mTOR-mediated, protein synthesis-dependent mechanisms, thus controlling for hours or days the consolidation of memory required for the discrimination of complex auditory stimuli.

Decrease of effectivity of ‘competing’ synaptic input requires protein synthesis

We describe that tetanus-induced long-term increase of the response amplitude in one input to an identified neuron is accompanied by a decrease of synaptic responses in another, non-tetanized input. Induction of the decrease of responses in non-tetanized inputs was prevented by bath application of protein synthesis blockers (anisomycin or cycloheximide). The decrease of responses also did not occur in experiments when tetanization was substituted by short-term serotonin applications or extracellular stimulation of serotonergic cells known to potentiate investigated synaptic inputs. The results suggest that the heterosynaptically evoked decrease of synaptic response amplitude may be due to involvement of postsynaptic protein synthesis-dependent mechanisms.

Signal transduction events in Chinese hamster ovary cells expressing human CD14; effect of endotoxin desensitization.

Previous studies suggest that endotoxin (LPS) stimulation of CD14 receptors may be coupled to heterotrimeric G proteins. However, characterization of the G protein-coupled signaling pathways is incomplete. Also, specific changes in the transduction pathways occur in a phenomenon known as LPS tolerance or desensitization induced by prior exposure to LPS. In the present study, we examined potential CD14-dependent G protein-coupled signaling events in response to LPS, and changes in signaling in these pathways during LPS desensitization in Chinese Hamster Ovary (CHO) cells. LPS stimulated inhibitory kappa B alpha (IkappaB alpha) degradation and p38 phosphorylation in CHO cells transfected with human CD14 receptor (CHO-CD14), but not in CHO cells transfected with vector only. However, activation of these signaling events diverged early in the signal transduction pathways. Pretreatment with pertussis toxin, which inactivates inhibitor G protein (G alpha i) function, significantly inhibited LPS-induced p38 phosphorylation, but not LPS-induced IkappaB alpha degradation. Mastoparan, a putative G alpha i agonist, synergized with LPS to induce p38 phosphorylation. Thus, LPS stimulation of p38 phosphorylation is, in part, G alpha i coupled, whereas IkappaB alpha degradation is not. In subsequent studies, CHO-CD14 cells were desensitized by prior LPS exposure. LPS-desensitized cells exhibited augmented IkappaB alpha content and were refractory to LPS-induced IkappaB alpha degradation and p38 phosphorylation. Pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the effect of LPS desensitization on augmenting cellular IkappaB alpha content and its refractoriness to LPS-induced degradation. However, cycloheximide pretreatment did not prevent impaired p38 phosphorylation in desensitized cells. IkappaB alpha upregulation in LPS tolerance may occur through increased synthesis and/or induction of protein that suppress IkappaB alpha degradation. The latter protein synthesis-dependent mechanisms may be distinct from mechanismis inhibiting p38 phosphorylation in tolerance. These findings suggest that LPS tolerance induces CD14-dependent signaling alterations in G alpha i-coupled pathways leading to mitogen-activated (MAP) kinase activation as well as G alpha i-independent pathways inducing IkappaB alpha degradation.

Sphingomyelinase and phospholipase A2 regulate type I deiodinase expression in FRTL-5 cells.

We have previously reported (Mol. Cell. Endocrinol. (1994) 101, R31-R35) that the proinflammatory cytokines, tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma), have a marked inhibitory effect on the expression and activity of type I iodothyronine deiodinase (D1) in FRTL-5 rat thyroid cells, while the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta 1) had no effect. These three proinflammatory cytokines utilize a number of intracellular second messenger systems including the pathways beginning with activation of sphingomyelinase and phospholipase A2. We have studied the time-dependent and dose-dependent effects of sphingomyelinase, ceramide, phospholipase A2 (PLA2), and arachidonic acid on the expression and activity of D1 in FRTL-5 cells. Sphingomyelinase (0.3 U/mL) inhibited D1 activity 55% and reduced D1 mRNA levels 70% to 90% by 8 hours. Similar treatment with 10 U/mL PLA2 inhibited D1 activity 54%. Treatment with 15 microM 5, 8, 11-eicosatriynoic acid (ETI), a nonmetabolizable analog of arachidonic acid, or 15 microM ceramide for 3 hours reduced D1 activity with a half-time of disappearance (t1/2) of 4.2 hours and 3.7 hours, respectively, but ETI and ceramide did not alter the D1 immunoreactivity or mRNA levels. Treatment for 8 hours with cycloheximide (5 or 10 micrograms/mL) had no effect on the D1 mRNA level, but blocked the TNF-induced reduction of this mRNA. We conclude that proinflammatory cytokines inhibit D1 expression and activity in FRTL-5 cells, in part, by activation of sphingomyelinase and PLA2 that results in (1) competitive inhibition of D1 activity by the enzymatic products ceramide and arachidonic acid and (2) reduction of D1 mRNA stability by protein synthesis-dependent mechanisms.

Dexamethasone inhibition of acute opioid physical dependence in vitro is reverted by anti-lipocortin-1 and mimicked by anti-type II extracellular PLA 2 antibodies

Dexamethasone has been shown to inhibit opiate withdrawal, in an in vitro model. In this respect, we suggested that dexamethasone could reduce opiate withdrawal by blocking the release of prostaglandins' precursor, arachidonic acid through protein synthesis dependent-mechanisms (1). Since arachidonic acid is released by the enzyme phospholipase A 2 (PLA 2) in the present paper we evaluate whether dexamethasone effect may by related to inhibition of PLA 2 activity. Therefore, the effect of a neutralizing anti-lipocortin-1 antibody and a polyclonal anti-type II extracellular phospholipase A 2 antibody on the opiate withdrawal in vitro was considered. Following a 4 min in vitro exposure to morphine a strong contracture of guinea-pig isolated ileum was observed after the addition of naloxone. Dexamethasone at concentration of 5x10 −5 M reduces of 50% morphine withdrawal and the polyclonal anti-type II extracellular PLA 2 antibody (in a dilution 1:1000) mimicked dexamethasone inhibitory effect. Incubation of the ileum preparation with a neutralizing anti-lipocortin-1 antibody (at a dilution of 1:10.000) 30 min before dexamethasone reverted the steroid effects. These results suggest that dexamethasone inhibition of opiate withdrawal is due to extracellular type II PLA 2 inhibition through lipocortin-1.

Effects of phorbol ester on expression of CNTF-mRNA in cultured astrocytes from rat olfactory bulb

Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine which has various functions, such as survival promoting effect on both peripheral and central neurons, promotion of cholinergic differentiation, and participation in differentiation of Type-2 astrocytes (reviewed in ref. [30]). However, the regulatory mechanism of the CNTF expression is largely unknown. In this study, we analyzed the effects of phorbol 12-myristate 13-acetate (PMA), an activator of PKC, on the expression of CNTF-mRNA in cultured astrocytes from neonatal rat olfactory bulb. PMA induced a transient decrease of CNTF-mRNA levels which was followed by a persistent increase of the mRNA up to 4-fold of the control level at 24 h after the addition of the compound. Both the PMA-induced decrease and increase of the CNTF-mRNA levels were canceled by treatment with cycloheximide, an inhibitor of protein synthesis, suggesting that protein synthesis-dependent mechanisms participate in both the PMA-induced decrease and increase of CNTF-mRNA levels. On the other hand, PMA induced expressions of mRNAs of several subunit members of the AP-1 complex, such as c fos, c- jun and jun-B. Furthermore, dexamethasone, a synthetic glucocorticoid which is known to inhibit the AP-l complex-mediated transcription [14,27,36], canceled the PMA-induced decrease of the CNTF-mRNA levels. These results suggested that the AP-1 complex participates in the regulatory mechanism of the CNTF expression in the cultured astrocytes treated with PMA.

Adenosine 3′,5′-cyclic monophosphate and the self-priming effect of gonadotrophin-releasing hormone

An in vitro perifusion system was used to ascertain the role of cAMP in the genesis of the self-priming effect of gonadotrophin-releasing hormone (GnRH) in rat pituitaries. Ten-minute pulses of 20 nmol/l GnRH administered 150 min apart resulted in the manifestation of the self-priming effect, an effect which was inhibited by 5 μmol/l cycloheximide. Forskolin (1 μmol/l) which does not stimulate luteinizing hormone (LH) secretion or affect the initial LH response to GnRH significantly potentiated the second response through protein synthesis-dependent mechanisms. Additionally, an initial 10-min pulse of forskolin alone was sufficient to prime the pituitary to a subsequent pulse of GnRH 150 min later. Interestingly, similar amounts of LH were secreted in response to forskolin + GnRH or GnRH administered 150 min after forskolin. Flufenamate, an inhibitor of GnRH-stimulated increases in cAMP production prevented the manifestation of the self-priming effect of GnRH. Forskolin which bypasses the inhibitory effects of flufenamate on cAMP production reversed the flufenamate-induced inhibition of the self-priming effect of GnRH through protein synthesis-dependent processes. These results suggest that cAMP does not mediate the LH response to an initial exposure of GnRH, but does play a pivotal role in the genesis of the self-priming effect of GnRH through the stimulation of de novo protein synthesis. Once the newly synthesized proteins are available, the nucleotide is not required for the manifestation of the phenomenon.

Calcium mobilization is a prerequisite for the expression of phorbol ester-stimulated luteinizing hormone secretion from pituitaries of male and acutely ovariectomized rats

Ionomycin, which mobilizes Ca2+, and phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), were used to compare the effects/interactions of Ca2+ and PKC on luteinizing hormone (LH) secretion from pituitaries of intact male and acutely ovariectomized (72 h) rats. Quartered pituitaries from donor animals were perifused at 0.25 ml/min and sequential effluent fractions were collected every 10 min. Continuous administration (4 h) of 1 nmol of gonadotropin-releasing hormone (GnRH) resulted in an increase in LH secretion. Cycloheximide (5 mumol) dissociated the GnRH-stimulated LH responses into protein synthesis-independent and -dependent components. While ionomycin (10 mumol) stimulated LH secretion from pituitaries of both sexes by protein synthesis-independent mechanisms, PMA (1 mumol) and the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (PDD), were ineffective secretagogues. However, PMA (but not PDD) interacted synergistically with ionomycin and GnRH to augment LH secretion by protein synthesis-dependent mechanisms. These results suggest a similarity in the effects/interactions of Ca2+ and PKC in gonadotropes from male and ovariectomized rats. If the effects of PMA can be attributed to PKC activation, then it also appears that Ca2+ mobilization is necessary for the manifestation of PKC as a mediator of LH secretion from these gonadotropes. While PKC activity can be divorced from the protein synthesis-independent component of LH release (this component appears to be mediated by Ca2+ mobilization), the enzyme might be involved in amplifying the response to Ca2+ mobilization through synergistic protein synthesis-dependent mechanisms.

Epidermal growth factor induces prolactin mRNA in GH 4C 1 cells via a protein synthesis-dependent pathway

Prolactin (PRL) gene expression is regulated through a complex network of signal transduction pathways activated by various hormones and growth factors. Estrogens regulate PRL gene transcription in vivo through both direct and indirect, protein synthesis-dependent, mechanisms. Therefore, we hypothesized that other stimulators of PRL gene transcription might also act via protein synthesis-dependent mechanisms. To test this hypothesis, we examined, in GH 4C 1rat pituitary tumor cells, the effects of protein synthesis inhibitors on the induction of PRL mRNA by known stimulators of PRL gene transcription. Whereas induction by epidermal growth factor (EGF) was abolished by cycloheximide and puromycin, increases in PRL mRNA caused by thyrotropin releasing hormone, 12- O-tetradecanoylphorbol 13-acetate, forskolin, or dibutyryl cyclic AMP were unaffected. These data suggest that the induction of PRL mRNA by EGF may require the induced synthesis of an intermediary regulatory protein.

Estradiol modulation of PMA - and ionomycin-stimulated LH secretion from pituitaries of castrated rats

Quartered pituitaries from castrated (72 h) ± estradiol (E 2)-treated (24 h) rats were used in a perifusion system to investigate estradiol modulation of ionomycin and ionomycin + PMA stimulated LH secretion. Estradiol enhanced the LH responses to GnRH (1 nM) and ionomycin (10 μM), and was necessary for the manifestation of PMA-stimulated (1 μM) LH secretion. Cycloheximide (5 μM) inhibited the E 2-enhanced responses to GnRH, ionomycin and PMA. The protein synthesis inhibitor also partially suppressed the GnRH response from pituitaries of castrates, but was totally ineffective against the ionomycin-induced LH secretion. Protein synthesis-dependent, synergistic interactions between PMA and ionomycin were evident from pituitaries of castrates (even though PMA alone was an ineffective secretagogue). Synergistic interactions were not apparent from pituitaries of castrated + E 2-treated rats. These results indicate that: (i) estradiol enhances the responsiveness of male gonadotropes to ionomycin and PMA by protein synthesis-dependent mechanisms which appear to mask their synergistic interactions; and (ii) increases in cytoplasmic Ca 2+ might be a prerequisite for an expression of the involvement of PKC as a mediator of LH secretion in the absence of high concentrations of estradiol.

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 > hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not ‘pure’ antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.

Endothelin-1 gene expression and regulation of endothelin mRNA and protein biosynthesis in avascular human amnion. Potential source of amniotic fluid endothelin.

We demonstrated previously that preproendothelin mRNA is present in avascular human amnion tissue and in human amnion cells maintained in primary monolayer culture. In this investigation we sought to identify the specific endothelin (ET) gene that is expressed in amnion and to determine whether endothelin is produced by amnion. Using oligonucleotides specific for ET-1, ET-2, and ET-3 mRNA, we identified preproET-1 mRNA in human amnion tissue. By radioimmunoassay of ET we found that human amnion tissue explants and amnion cells in culture secrete immunoreactive ET into the medium. PreproET mRNA levels and immunoreactive ET production by human amnion cells in monolayer culture are increased in response to treatment with agents that are known to be present in human amniotic fluid, i.e. epidermal growth factor, interleukin-1, and tumor necrosis factor-alpha. We found that the level of preproET mRNA in amnion cells was low compared with that in human umbilical endothelial cells; treatment with cycloheximide together with a stimulus of ET-1 gene transcription led to a striking increase in the level of preproET mRNA in amnion cells compared with a much weaker response in endothelial cells. These findings suggest that protein synthesis-dependent mechanisms may be of great importance in maintaining low levels of preproET mRNA in amnion tissue and in regulating the amount of preproET mRNA in amnion exposed to stimuli of ET-1 transcription. In addition, we demonstrated that immunoreactive ET is present in human amniotic fluid at the midtrimester of pregnancy and at term. Thus, it is likely that the avascular fetal amnion is one tissue site of origin of ET in amniotic fluid during human pregnancy.

Dependency of phorbol ester-induced gonadotropin secretion on estradiol

Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC) was used to investigate the estradiol dependency of PKC-stimulated luteinizing hormone (LH) secretion from perifused anterior pituitaries. Infusions of PMA stimulated LH secretion from diestrous II, ovariectomized + estradiol-treated, and orchidectomized + estradiol-treated quartered pituitaries, by protein synthesis-dependent mechanisms. In contrast, pituitaries from intact, orchidectomized males, or ovariectomized females were unresponsive to PMA. Interestingly, dispersed male pituitary cells differed from male pituitary tissue blocks, in that the dispersed cells responded to PMA with increased LH secretion. These results indicate that PKC's ability to directly stimulate LH secretion is dependent on de novo protein synthesis and estradiol. Moreover, the effects of estradiol on PKC-stimulated secretion form at least one basis for the estradiol-induced increased responsiveness of gonadotrophs to GnRH. Additionally, it appears that dispersed pituitary cells may not respond to activators of PKC in a physiological manner.

Expression of c-myc proto-oncogene in normal human lymphocytes. Regulation by transcriptional and posttranscriptional mechanisms.

Aberrant expression of the c-myc gene results from nonrandom chromosomal translocations involving the transcriptionally active antigen receptor gene loci, in particular lymphocytic leukemias and lymphomas, and is believed to contribute to the etiology of these neoplasms. In addition to its expression in abnormal lymphocytes, increased accumulation of c-myc mRNA occurs rapidly in normal B- and T-lymphocytes after stimulation with appropriate mitogens. The mechanisms that mediate these mitogen-induced elevations in c-myc mRNA levels, however, have not been determined for normal B and T cells. By using enriched populations of B- and T-lymphocytes obtained from freshly isolated human tonsils and stimulated with Staphylococcus-A or with phytohemagglutinin, respectively, we observed marked elevations (20-40-fold) in the steady state levels of accumulated c-myc messenger RNA (mRNA) within 1 h of exposure of cells to mitogens; modest increases (three- to fivefold) in the relative rate of transcription of the c-myc gene through protein synthesis-independent (cycloheximide-insensitive) mechanisms; and rapid rates of degradation of mature c-myc mRNAs through protein synthesis-dependent (cycloheximide-sensitive) mechanisms. These findings corroborate previous studies in other cell types and provide evidence for both transcriptional and posttranscriptional control of c-myc proto-oncogene expression in normal human lymphocytes.

Dissociation of rapid eye movement (REM) sleep features: possible implications for REM triggering mechanisms.

Neurophysiological and biochemical mechanisms regulating REM sleep episodes were studied in behaving cats with chronic electrodes. Push-pull perfusion of the midbrain or pontine reticular formation (RF) by a protein synthesis inhibitor, chloramphenicol (CAP), reduced neuronal unit discharge, particularly discharge bursts, within the perfused area, and reduced the incidence of sustained REM periods. During transitions to "aborted" as compared to sustained REM episodes. RF unit discharge was reduced, under all conditions. In 72-hour sleep deprived cats, systemic injections of both atropine (ATR) and CAP attenuated REM rebound, but with different patterns. CAP reduced REM episode frequency. ATR reduced REM duration. ATR also reduced PGO wave frequency. Combined CAP and ATR treatment produced additive REM depressant effects. These data suggest that cholinergic mechanisms regulate PGO activity and maintenance of REM episodes and that protein synthesis-dependent mechanisms regulate RF unit discharge bursts, and REM triggering.