ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.     

Dramatic increase in readthrough acetylcholinesterase in a cellular model of oxidative stress

Moderate, transient oxidative stress is achieved in SH-SY5Y cells using tertiary butylhydroperoxide as oxidant. Over a recovery period of 24 h, the enzymatic activity and protein levels of the acetylcholinesterase (AChE) splice variants tailed AChE (AChE-T) and readthrough AChE (AChE-R) are monitored. Their time-dependent correlation to pro- and anti-apoptotic factors, namely caspase 3 and Bcl-2, respectively, as well as lactate dehydrogenase release as a measure of cell viability is assessed. A distinctly different expression pattern of AChE-T as compared with AChE-R is recorded, in that AChE-T shows only a very slight increase over a 6 h time period. In contrast, AChE-R rises continuously during the recovery period, reaching peak intracellular levels that are up to six times higher than control levels 3-4 h post-stress, and is released from cells in substantial amounts. Moreover, anti-apoptotic Bcl-2 increases significantly, peaking 2-3 h after this AChE-R peak has occurred. We believe this study presents the first work that demonstrates - without relying on techniques of over-expression - the time-dependent correlation between apoptotic processes and related rescue mechanisms involving AChE isoforms in a neuronal cell line.     

Alternate AChE-R variants facilitate cellular metabolic activity and resistance to genotoxic stress through enolase and RACK1 interactions

Tumorogenic transformation is a multifaceted cellular process involving combinatorial protein-protein interactions that modulate different cellular functions. Here, we report apparent involvement in two independent tumorogenic processes by distinct partner protein interactions of the stress-induced acetylcholinesterase AChE-R and N-AChE-R variants. Human testicular tumors showed elevated levels of N-terminally extended N-AChE-R compared with healthy tissue, indicating alternate promoter usage in the transformed cells. Two-hybrid screens demonstrate that the C-terminus common to both N-AChE-R and AChE-R interacts either with the glycolytic enzyme enolase or with the scaffold protein RACK1. In vitro, the AChE-R C-terminal peptide ARP elevated enolase's activity by 12%, suggesting physiological relevance for this interaction. Correspondingly, CHO cells expressing either human AChE-R or N-AChE-R but not AChE-S showed a 25% increase in cellular ATP levels, indicating metabolic significance for this upregulation of enolase activity. ATP levels could be reduced by AChE-targeted siRNA in CHO cells expressing AChE-R but not AChE-S, attributing this elevation to the AChE-R C-terminus. Additionally, transfected CHO cells expressing AChE-R but not N-AChE-R showed resistance to up to 60muM of the common chemotherapeutic agent, cis-platinum, indicating AChE-R involvement in another molecular pathway. cis-Platinum elevates the expression of the apoptosis-regulator p53-like protein, p73, which is inactivated by interaction with the scaffold protein RACK1. In co-transfected cells, AChE-R competed with endogenous RACK1 for p73 interaction. Moreover, AChE-R-transfected CHO cells presented higher levels than control cells of the pro-apoptotic TAp73 as well as the anti-apoptotic dominant negative DeltaNp73 protein, leading to an overall decrease in the proportion of pro-apoptotic p73. Together, these findings are compatible with the hypothesis that in cancer cells, both AChE-R and N-AChE-R elevate cellular ATP levels and that AChE-R modifies p73 gene expression by facilitating two independent cellular pathways, thus conferring both a selective metabolic advantage and a genotoxic resistance.     

Readthrough acetylcholinesterase in inflammation-associated neuropathies

The cholinergic control over inflammatory reactions calls for deciphering the corresponding protein partners. An example is blood–nerve barrier disruption allowing penetration of inflammatory factors, which is notably involved in various neuropathies due to yet unknown molecular mechanism(s). In rats, lipopolysaccharide (LPS) administration followed by intra-neural (i.n.) saline injection inducing a focal blood–nerve disruption leads to systemic inflammatory reaction accompanied by transient conduction impairment in the sciatic nerve. Here, we provide evidence compatible with the hypothesis that ARP, the naturally cleavable C-terminal peptide of the stress-induced “readthrough” acetylcholinesterase variant (AChE-R), is causally involved in the emergence of this LPS-induced conduction impairment. Intra-neural injection to naïve rats of conditioned medium from cultured splenocytes exposed to LPS in vitro (reactive splenocyte medium) induced a transient conduction impairment that was accompanied by facilitated accumulation of cleaved intra-neural ARP. Protein kinase C (PKC) βII, known to interact with ARP, was significantly elevated in the LPS-exposed sciatic nerve preparations. Moreover, direct i.n. injection of synthetic ARP30, bearing the mouse AChE-R C-terminal sequence, similarly induced PKCβII expression and conduction impairment. The induction of neural conduction impairment by ARP, possibly through its interaction with PKCβII, suggests a role for AChE-R expression in inflammation-associated neuropathies.     

Long-lasting acetylcholinesterase splice variations in anticholinesterase-treated Alzheimer's disease patients

Protein levels of different acetylcholinesterase (AChE) splice variants were explored by a combination of immunoblot techniques, using two different antibodies, directed against the C-terminus of the AChE-R splice variant or the core domain common to all variants. Both AChE-R and AChE-S splice variants as well as several heavier AChE complexes were detected in brain homogenates from the parietal cortex of patients with or without Alzheimer's disease (AD) as well as the cerebrospinal fluid (CSF) of AD patients, compatible with the assumption that CSF AChEs might originate from CNS neurons. Long-term changes in the composition of CSF AChE variants were further pursued in AD patients treated with rivastigmine (n = 11) or tacrine (n = 17) in comparison to untreated AD patients (n = 5). In untreated patients, AChE-R was markedly reduced as compared with the baseline level (37%), whereas the medium size AChE-S complex was increased by 32%. Intriguingly, tacrine produced a general and profound up-regulation of all detected AChE variants (up to 117%), whereas rivastigmine treatment caused a mild and selective up-regulation of AChE-R ( approximately 10%, p < 0.05). Moreover, the change in the ratio of AChE-R to AChE-S (R/S-ratio) strongly and positively correlated with sustained cognition at 12 months (p < 0.0001). Thus, evaluation of changes in the composition of CSF AChE variants may yield important information referring to the therapeutic efficacy and/or development of drug tolerance in AD patients treated with anti-cholinesterases.     

Transgenic and antisense manipulations of impairments in cholinergic balance

Inhibitors of acetylcholinesterase (AChE) catalysis are increasingly used as insecticides as well as therapeutic and prophylactic agents. However, the long-term consequences of their use are not yet known. To investigate this topic, we used cultured neurons and transgenic mouse pedigrees that over-express natural variants of human AChE. Following exposure to an anti-AChE, alternative splicing of the pre-mRNA from the single ACHE gene produces the relatively rare AChE-R mRNA variant in mice and in cultured cerebellar neurons. This promotes a rapid (minutes) yet long-lasting (weeks) translocation of AChE-R mRNA into neurites that is associated with extreme neuronal hypersensitivity to both anti-AChEs and atropine (Meshorer et al. 2002, Science , 295 , 508-512). In transgenic mice, over-expression of AChE-R causes stress-induced irregular bursts of increased locomotor activity and failure of short-term memory (Cohen et al. Mol. Psych. , in press). Nanomolar concentrations of an antisense oligonucleotide selectively suppress AChE-R mRNA levels in both hyperproducing cultured neurons and mouse brain. Moreover, this treatment reverses the behavioral and cognitive deficits of the mice (effect apparent for > 24 h, vs. < 45 min for tacrine). The efficacy of our antisense experiments raises the possibility of eventually using such agents to achieve AChE variant-specific suppression, long-lasting effect, and, presumably, fewer side-effects than is offered by conventional anticholinesterase therapy.     

Stress induced IL-10 does not seem to be essential for early monocyte deactivation following cardiac surgery

An increase in circulating levels of IL-10 is believed to contribute to immunosuppression caused by major surgery. Cortisol and catecholamines have been shown to be important costimulatory factors for IL-10 secretion in humans. As thoracic epidural block (TEB) should blunt the perioperative increases in cortisol and catecholamines we investigated whether IL-10 secretion is influenced by TEB. Twenty-six patients undergoing coronary artery bypass graft surgery using cardiopulmonary bypass were randomized to receive either general anesthesia (GA) or GA plus TEB. Sensory and pain levels were measured to demonstrate clinical effectiveness. Plasma concentrations of epinephrine, norepinephrine, cortisol, IL-6 and IL-10 as well as monocyte surface expression of HLA-DR and their ex vivo capacity to release TNF-alpha after LPS stimulation were measured perioperatively. TEB was clinically effective and patients receiving TEB showed decreased circulating levels of IL-10. However, this decrease was independent of decreased levels of cortisol or epinephrine. No influence of TEB on IL-6 levels, monocyte capacity to ex vivo release TNF-alpha upon LPS stimulation or their expression of HLA-DR was found. In conclusion, high TEB reduces antiinflammatory immune suppressing mediators including IL-10 and stress mediators. At least in cardiac surgery patients the monocyte functional depression is not related to systemic release of IL-10 and the influence of cortisol or epinephrine is less important for early monocyte deactivation than what in vitro and animal models have suggested.     

Endotoxin tolerance: regulation of cytokine production and cellular changes in response to endotoxin application in cancer patients.

Endotoxin (lipopolysaccharide, LPS) has the property of inducing tolerance to its own biological effects. This phenomenon has been extensively studied in animal models but only few studies exist on the regulation in humans. Here we describe experiments designed to determine the cytokine regulation and cellular changes in humans during induction of LPS tolerance after repeated LPS injections. Intravenous administration of purified LPS Salmonella abortus equi to cancer patients induces high amounts of circulating tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF). Repeated injections of LPS at daily intervals resulted in a marked downregulation of the cytokine response and in the case of TNF-alpha, IL-8, G-CSF, and M-CSF the cytokine response was reduced to baseline levels. In contrast, significant increases in serum IL-6 were detected up to day 5 of repeated LPS injections. Hematological changes included transient decreases in WBCs affecting granulocytes, monocytes, and lymphocytes, followed by a marked granulocytosis. The drop in WBCs remained unaltered throughout the 5 day course of repeated LPS injections whereas the granulocyte overshoot recovery diminished gradually. When PBMCs of the cancer patients were restimulated ex vivo a marked enhancement of the capacity to produce TNF-alpha, IL-113, and IL-6 occurred, which is in contrast to the decreasing TNF-alpha serum levels obtained in vivo. In parallel, a shift in monocyte subpopulations from CD14+/CD16- to CD14+/CD16+ cells was observed. The data provide evidence that different mechanisms are implicated in the cytokine downregulation following repeated LPS injections to cancer patients. Furthermore, PBMCs from LPS tolerant patients do not demonstrate a reduction in their capacity to produce cytokines.     

Cortisol synthesis in epidermis is induced by IL-1 and tissue injury

Glucocorticoids (GCs) are known inhibitors of wound healing. In this study we report the novel finding that both keratinocytes in vitro and epidermis in vivo synthesize cortisol and how this synthesis regulates wound healing. We show that epidermis expresses enzymes essential for cortisol synthesis, including steroid 11 β-hydroxylase (CYP11B1), and an enzyme that controls negative feedback mechanism, 11β-hydroxysteroid dehydrogenase 2 (11βHSD2). We also found that cortisol synthesis in keratinocytes and skin can be stimulated by ACTH and inhibited by metyrapone (CYP11B1 enzyme inhibitor). Interestingly, IL-1β, the first epidermal signal of tissue injury, induces the expression of CYP11B1 and increases cortisol production by keratinocytes. Additionally, we found induction of CYP11B1 increased production of cortisol and activation of GR pathway during wound healing ex vivo and in vivo using human and porcine wound models, respectively. Conversely, inhibition of cortisol synthesis during wound healing increases IL-1β production, suggesting that cortisol synthesis in epidermis may serve as a local negative feedback to proinflammatory cytokines. Local GCs synthesis, therefore, may provide control of the initial proinflammatory response, preventing excessive inflammation upon tissue injury. Inhibition of GC synthesis accelerated wound closure in vivo, providing the evidence that modulation of cortisol synthesis in epidermis may be an important regulatory mechanism during wound healing.     

Staurosporine-induced apoptosis presents with unexpected cholinergic effects in a differentiated neuroblastoma cell line

Apoptosis of cholinergic neurons is one of the core hallmarks of Alzheimer’s disease. SH-SY5Y neuroblastoma cells differentiated to the cholinergic phenotype were exposed to 100 nM staurosporine. Over a treatment period of 24 h, the pro- and anti-apoptotic factors, caspase-3 and Bcl-2, as well as LDH release as a measure of cell viability, were assessed in conjunction with the number of apoptotic cells by means of fluorescence-activated cell sorting. Caspase-3 activity and LDH release increased by 30% and 20% over controls, respectively, while Bcl-2 levels rose by 200% over controls. Furthermore, staurosporine treatment resulted in decreased acetylcholinesterase (AChE) enzymatic activity and decreased protein levels of the AChE splice variant tailed AChE (AChE-T). Only a slight increase in levels of readthrough AChE (AChE-R) was observed. Likewise, staurosporine reduced levels and activity of the cholinergic players choline acetyltransferase and high affinity choline uptake. The present study demonstrates that treatment with staurosporine leads to apoptotic events, which, however, are not reflected in the increased AChE activity and the alterations of AChE isoforms expression that are usually seen in apoptotic conditions. The effects of various additional phosphorylation inhibitors on AChE activity suggest that these unexpected cholinergic effects, firstly, are linked to the impact of staurosporine on phosphorylation and, secondly, reveal themselves in a first phase of cellular adaption that precedes neurotoxicity and subsequent cell death.     

Cortisol response and immune-related effects of Atlantic salmon (Salmo salar Linnaeus) subjected to short- and long-term stress

It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon (Salmo salar Linnaeus) the effects of both short- (a single 15s out of water) and long-term (4 weeks of daily handling 15s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1beta (IL-1beta) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6h post stress. Constitutive expression of IL-1beta in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1beta in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1 week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1beta expression in macrophages isolated from stressed fish over the first 2 weeks. At weeks 1, 2 and 3 the magnitude of IL-1beta response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4 weeks there was no significant difference in inducible IL-1beta expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1beta, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased leukocyte survival when exposed to A. salmonicida. The implications of these changes in the immune system will be discussed with respect to the use of classical indicators of stress to predict possible effects on the immune system of fish.     

Physiological stress response of Mountain Whitefish (<Emphasis Type="Italic">Prosopium williamsoni</Emphasis>) and White Sucker (<Emphasis Type="Italic">Catostomus commersoni)</Emphasis> sampled along a gradient of temperature and agrichemicals in the Oldman River,Alberta

Species differences in tolerance to environmental stressors can contribute to differences in species distribution and abundance along river gradients. Climate change and intensive agriculture are likely to have major effects on fish populations in temperate zones, yet understanding of the interactions between temperature and chemical stressors on fish physiology is limited. The objective of this study was to compare the stress responses of the Mountain Whitefish, (Prosopium williamsoni, a cold-water fish) and White Sucker (Catostomus commersoni, a cool-water fish), along a temperature and pesticide gradient in the Oldman River, Southern Alberta in spring and summer. Fish were seined, placed into an enclosure, and plasma cortisol, glucose, liver glycogen, and condition factor were measured. Plasma acetylcholinesterase (AChE) activity was used as an indicator of exposure to organophosphate and carbamates pesticides. Whitefish had lower plasma AChE activity and lower liver glycogen reserves compared to suckers at all sites and all sampling times but the differences in plasma cortisol were not species-specific and there were no differences in plasma glucose levels, except at one site. Plasma cortisol increased, and plasma glucose decreased along a downstream river gradient in whitefish in both spring and summer; in sucker only plasma cortisol fluctuated and only in the summer. Liver glycogen decreased along the river gradient in both species at both seasons. Our study detected important species-specific differences in AChE activities and responses of the physiological stress axis, suggesting that whitefish are more sensitive to temperature and pesticide stress than suckers.     

Modified testicular expression of stress-associated "readthrough" acetylcholinesterase predicts male infertility.

Male infertility is often attributed to stress. However, the protein or proteins that mediate stress-related infertility are not yet known. Overexpression of the "readthrough" variant of acetylcholinesterase (AChE-R) is involved in the cellular stress response in a variety of mammalian tissues. Here, we report testicular overexpression of AChE-R in heads, but not tails, of postmeiotic spermatozoa from mice subjected to a transient psychological stress compared with age-matched control mice. Transgenic mice overexpressing AChE-R displayed reduced sperm counts, decreased seminal gland weight, and impaired sperm motility compared with age-matched nontransgenic controls. AChE-R was prominent in meiotic phase spermatocytes and in tails, but not heads, of testicular spermatozoa from AChE-R transgenic mice. Head-localized AChE-R was characteristic of human sperm from fertile donors. In contrast, sperm head AChE-R staining was conspicuously reduced in samples from human couples for whom the cause of infertility could not be determined, similar to the pattern found in transgenic mice. These findings indicate AChE-R involvement in impaired sperm quality, which suggests that it is a molecular marker for stress-related infertility.     

Granulocyte colony-stimulating factor modulation of cytokine receptors on murine bone marrow cells. In vivo and in vitro studies.

Human recombinant granulocyte CSF (G-CSF) modulation of cytokine receptors on murine bone marrow cells (BMC) in vivo and in vitro was investigated. In vivo, G-CSF reduced 125I-G-CSF binding to BMC by greater than 95% within 30 min, with return to base line after 48 h. Human rCSF-1 binding was reduced greater than 85% after 30 min and failed to recover even after 48 h. Murine rTNF-alpha or recombinant granulocyte/macrophage CSF binding was not significantly altered. However, human rIL-1 alpha binding increased greater than 1.5-fold after 3 h, was elevated greater than 5-fold between 6 and 12 h, and declined to base line after 48 h. In vitro, G-CSF induced a greater than 1.5-fold increase in IL-1 binding to BMC after 8 h, suggesting that up-modulation of IL-1 binding in vivo required G-CSF and other influences. Further studies indicated that BMC responded to glucocorticoids and G-CSF with a synergistic increase of IL-1 binding. This synergistic IL-1R modulation was a time- and dose-dependent process and was inhibited by cycloheximide or actinomycin D in a dose-dependent manner. Binding studies further revealed that the synergistic stimulation of IL-1R expression on BMC was probably due to increased receptor number, rather than increased receptor affinity. In addition, this phenomenon was also observed in other hematopoietic cells. Our results demonstrated that G-CSF was capable of stimulating IL-1R expression on BMC both in vivo and in vitro and G-CSF in combination with glucocorticoids synergistically up-modulated IL-1 binding to BMC in vitro. Inasmuch as IL-1 induces the secretion of G-CSF and glucocorticoids in vivo, this synergistic induction may play an important, as yet unknown, role in the inflammatory cascade.     

Acetylcholinesterase-R increases germ cell apoptosis but enhances sperm motility

Changes in protein subdomains through alternative splicing often modify protein-protein interactions, altering biological processes. A relevant example is that of the stress-induced up-regulation of the acetylcholinesterase (AChE-R) splice variant, a common response in various tissues. In germ cells of male transgenic TgR mice, AChE-R excess associates with reduced sperm differentiation and sperm counts. To explore the mechanism(s) by which AChE-R up-regulation affects spermatogenesis, we identified AChE-R's protein partners through a yeast two-hybrid screen. In meiotic spermatocytes from TgR mice, we detected AChE-R interaction with the scaffold protein RACK1 and elevated apoptosis. This correlated with reduced scavenging by RACK1 of the pro-apoptotic TAp73, an outcome compatible with the increased apoptosis. In contrast, at later stages in sperm development, AChE-R's interaction with the glycolytic enzyme enolase-alpha elevates enolase activity. In transfected cells, enforced AChE-R excess increased glucose uptake and adenosine tri-phosphate (ATP) levels. Correspondingly, TgR sperm cells display elevated ATP levels, mitochondrial hyperactivity and increased motility. In human donors' sperm, we found direct association of sperm motility with AChE-R expression. Interchanging interactions with RACK1 and enolase-alpha may hence enable AChE-R to affect both sperm differentiation and function by participating in independent cellular pathways.     

Interleukin-1-induced granulocytopenia and pulmonary leukostasis in rabbits

Pulmonary leukostasis is a postulated prerequisite lesion for acute lung injury. Interleukin-1 (IL-1) mediates components of the acute-phase response, stimulates granulocyte metabolism and secretion, and augments endothelial adhesiveness. We studied the effects of murine IL-1 infusion on circulating granulocytes, their sequestration within the pulmonary microvasculature, lung water, and bronchoalveolar lavage fluid (BALF) protein concentration in rabbits at 3 and 24 h after infusion. IL-1 administration induced significant (P less than 0.01) granulocytopenia compared with saline-injected controls and at 3 h induced significant increases in both mean alveolar septal wall granulocytes per high power field (HPF) (P less than 0.001) and mean myeloperoxidase (MPO) activity per gram lung tissue (P less than 0.001). At 24 h, IL-1 induced a marked granulocytosis and again significantly increased both mean alveolar septal wall granulocytes per HPF (P less than 0.001) and lung MPO (P less than 0.01). Increased lung water or BALF protein concentration could not be demonstrated in animals killed at either 3 or 24 h after IL-1 administration. Therefore, IL-1 can induce an early profound granulocytopenia followed by later granulocytosis, as well as sustained pulmonary leukostasis in the absence of detectable pulmonary edema formation or an alveolar-capillary leak.     

GSK3beta mediates the induced expression of synaptic acetylcholinesterase during apoptosis

Besides its role in terminating acetylcholine-mediated neurotransmission, acetylcholinesterase (AChE) is found to be expressed and participate in the process of apoptosis in various cell types. However, the mechanisms underlying AChE up-regulation in neuronal cells remain elusive. Herein we demonstrated that glycogen synthase kinase-3β (GSK3β) mediates induced AChE-S expression during apoptosis. In this study, A23187 and thapsigargin (TG) were employed to induce apoptosis in neuroendocrine PC12 cells. The results showed that exposure of PC12 cells to A23187 and TG up-regulated AChE activity significantly. The same treatment also led to activation of GSK3β. Two different inhibitors of GSK3β (lithium and GSK3β-specific inhibitor VIII) could block A23187- or TG-induced up-regulation of AChE activity, AChE-S mRNA level and protein expression. However, lithium could not inhibit the induction of AChE-R mRNA and protein under similar conditions. Taken together, our results show that GSK3β is specifically involved in the induction of AChE-S expression in PC12 cells during apoptosis.     

Circulating hematopoietic progenitor cells in runners.

Because endurance exercise causes release of mediators and growth factors active on the bone marrow, we asked whether it might affect circulating hematopoietic progenitor cells (HPCs) in amateur runners [n = 16, age: 41.8 +/- 13.5 (SD) yr, training: 93.8 +/- 31.8 km/wk] compared with sedentary controls (n = 9, age: 39.4 +/- 10.2 yr). HPCs, plasma cortisol, interleukin (IL)-6, granulocyte colony-stimulating factor (G-CSF), and the growth factor fms-like tyrosine kinase-3 (flt3)-ligand were measured at rest and after a marathon (M; n = 8) or half-marathon (HM; n = 8). Circulating HPC counts (i.e., CD34(+) cells and their subpopulations) were three- to fourfold higher in runners than in controls at baseline. They were unaffected by HM or M acutely but decreased the morning postrace. Baseline cortisol, flt3-ligand, IL-6, and G-CSF levels were similar in runners and controls. IL-6 and G-CSF increased to higher levels after M compared with HM, whereas cortisol and flt3-ligand increased similarly postrace. Our data suggest that increased HPCs reflect an adaptation response to recurrent, exercise-associated release of neutrophils and stress and inflammatory mediators, indicating modulation of bone marrow activity by habitual running.     

Cloning of the glucocorticoid receptor (GR) in gilthead seabream (Sparus aurata). Differential expression of GR and immune genes in gilthead seabream after an immune challenge

In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1beta, TNF-alpha, Mx protein, cathepsin D and PPAR-gamma). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.