The role of serine/threonine protein phosphatase type 5 (PP5) in the regulation of stress-induced signaling networks and cancer.

Although the aberrant actions of protein kinases have long been known to contribute to tumor promotion and carcinogenesis, roles for protein phosphatases in the development of human cancer have only emerged in the last decade. In this review, we discuss the data obtained from studies examining the biological and pathological roles of a serine/threonine protein phosphatase, PP5, which suggest that PP5 is a potentially important regulator of both hormone- and stress-induced signaling networks that enable a cell to respond appropriately to genomic stress.

Predominant role of peripheral catecholamines in the stress-induced modulation of CYP1A2 inducibility by benzo(alpha)pyrene.

The potential involvement of catecholamines and in particular of alpha(2)-adrenoceptor-related signalling pathways, in the regulation of drug-metabolizing enzymes by stress was investigated in Wistar rats after exposure to the environmental pollutant benzo(alpha)pyrene. For this purpose, total cytochrome P450 content, the CYP1A2 mRNA levels, 7-methoxyresorufin-O-dealkylase (MROD), 7-pentoxyresorufin-O-dealkylase (PROD) and p-nitrophenol hydroxylase activity levels were determined in the livers of rats exposed to repeated restraint stress after treatment with benzo(alpha)pyrene coupled with pharmacological manipulations of peripheral and/or central catecholamines and alpha(2)-adrenoceptors. The data show that stress is a significant factor in the regulation of CYP1A2 induction and that catecholamines play a central role in the stress-mediated modulation of hepatic CYP1A2 inducibility by benzo(alpha)pyrene. The up-regulating effect of stress on benzo(alpha)pyrene-induced CYP1A2 gene expression was eliminated after a generalized catecholamine depletion with reserpine. Similarly, in a state where only peripheral catecholamines were depleted and central catecholamines remained intact after guanethidine administration, the up-regulating effect of stress was eliminated. It is apparent that stress up-regulates the induction of CYP1A2 by benzo(alpha)pyrene mainly via peripheral catecholamines, while central catecholamines hold a minor role in the regulation. Pharmacological manipulations of alpha(2)-adrenoceptors appear to interfere with the effect of stress on the regulation of CYP1A2 inducibility. Either blockade or stimulation of alpha(2)-adrenoceptors with atipamezole and dexmedetomidine respectively, eliminated the up-regulating effect of stress on CYP1A2 benzo(alpha)pyrene-induced expression, while it enhanced MROD activity. In contrast, stress and pharmacological manipulations of catecholamines and alpha(2)-adrenoceptors did not affect total P450 content, the CYP2B1/2-dependent PROD and the CYP2E1-dependent p-nitrophenol hydroxylase activities. In conclusion, stress is a significant factor in the regulation of the CYP1A2 inducibility by benzo(alpha)pyrene, which in turn is involved in the metabolism of a large spectrum of toxicants, drugs and carcinogenic agents. Although the mechanism underlying the stress effect on CYP1A2 induction has not been clearly elucidated, it appears that peripheral catecholamines hold a predominant role, while central catecholamines and in particular, central noradrenergic pathways hold a minor role.

Expression of sphingomyelin synthase 1 gene in rat brain focal ischemia.

Metabolites of the sphingomyelin cycle are reported to play an important role in neuronal death after ischemia. To elucidate the involvement of the key enzyme of this cycle, sphingomyelin synthase (SMS), in the mechanism underlying cerebral ischemia, we, for the first time, investigated changes in the mRNA expression of the SMS1 gene in rats after focal cerebral ischemia. According to our histological analysis, the damaged area is localized only in the ipsilateral cortex. In the ischemic cortex, the level of SMS1 transcripts was decreased at 3 and 24 h after occlusion, and at 72 h it had returned to the control level. A reduced level of SMS1 mRNA expression in the subcortex of rats with occlusion and sham-operated animals also was appeared during the first 24 h after surgery. This could be attributed to the effect of surgical stress. Seventy-two hours after occlusion, SMS1 mRNA expression in subcortex of ischemic rats was still at a decreased level; this may be considered to be a somewhat distant extended effect. Our results show the early response of the SMS1 gene that can be induced by both ischemia and stress. The results also suggest that inhibition of SMS1 mRNA expression may contribute to ceramide accumulation in a damaged cortex.

Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK-->ERK1/2 pathway.

5'-AMP-activated protein kinase (AMPK) serves as an energy sensor and is at the center of control for a large number of metabolic reactions, thereby playing a crucial role in Type 2 diabetes and other human diseases. AMPK is present in the nucleus and cytoplasm; however, the mechanisms that regulate the intracellular localization of AMPK are poorly understood. We have now identified several factors that control the distribution of AMPK. Environmental stress regulates the intracellular localization of AMPK, and upon recovery from heat shock or oxidant exposure AMPK accumulates in the nuclei. We show that under normal growth conditions AMPK shuttles between the nucleus and the cytoplasm, a process that depends on the nuclear exporter Crm1. However, nucleocytoplasmic shuttling does not take place in high-density cell cultures, for which AMPK is confined to the cytoplasm. Furthermore, we demonstrate that signaling through the mitogen-activated protein kinase kinase (MEK)-->extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK. As such, pharmacological inhibitors that interfere with this pathway alter AMPK distribution under nonstress conditions. Taken together, our studies identify novel links between the physiological state of the cell, the activation of MEK-->ERK1/2 signaling, and the nucleocytoplasmic distribution of AMPK. This sets the stage to develop new strategies to regulate the intracellular localization of AMPK and thereby the modification of targets that are relevant to human disease.

Determinants of the diurnal course of salivary alpha-amylase.

OBJECTIVE: Previous data from our group and others have shown that salivary alpha-amylase activity increases in response to stress. It has been suggested that salivary alpha-amylase may be a marker for adrenergic activity. Less is known about other determinants of salivary alpha-amylase activation. The objective of the current study was to describe the diurnal pattern of salivary amylase and its determinants. METHODS: Saliva samples were collected immediately after waking-up, 30 and 60 min later, and each full hour between 0900 and 2000 h by 76 healthy volunteers (44 women, 32 men). Compliance was controlled by electronic monitors. In order to control factors which might influence the diurnal profile of salivary alpha-amylase (such as momentary stress, mood, food, or body activity), at each sampling time point the subjects filled out a diary examining the activities they had carried out during the previous hour. RESULTS: Salivary alpha-amylase activity shows a distinct diurnal profile pattern with a pronounced decrease within 60 min after awakening and a steady increase of activity during the course of the day. Mixed models showed a relative independence of diurnal salivary alpha-amylase from momentary stress and other factors, but significant associations with chronic stress and mood. CONCLUSIONS: Our results suggest that diurnal profiles of salivary alpha-amylase are relatively robust against momentary influences and therefore may prove useful in the assessment of sympathetic nervous system activity. The findings underscore the need to control for time of day in studies using salivary alpha-amylase as a dependent variable.

H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells.

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.

AMPK alpha subunit gene characterization in Artemia and expression during development and in response to stress.

AMP-activated protein kinase (AMPK) plays a central role in maintaining the energy balance of organisms under physiological and environmental stresses. Here two AMPK alpha subunit gene transcripts (named Afr-AMPKalpha1 and Afr-AMPKalpha2) from Artemia franciscana were isolated and gene expression was characterized by semiquantitive reverse transcription-polymerase chain reaction (RT-PCR). Afr-AMPKalpha1 was differentially expressed during Artemia developmental stages as well as in response to stresses, such as heat-shock, starvation and a hyperosmotic environment. Afr-AMPKalpha1 mRNA expression in adult Artemia decreased under heat shock, but not in a time- and temperature-dependent manner. By contrast, the transcript sharply decreased in heat-shocked cysts in a time-dependent manner. Under hyperosmotic stress, however, the mRNA level in adults first declined and then increased with prolonged exposure. In the case of starvation, the gene expression in adults decreased and was undetectable after day 9. In addition, Afr-AMPKalpha2 mRNA expression was too low to be detected without nested PCR. Southern blot analysis, moreover, indicated AMPK alpha subunit was present in multiple copies in the Artemia genome. Furthermore, our results demonstrate that the Afr-AMPKalpha1 mRNA level sharply decreases in Artemia carrying diapause-destined embryos and this indicates the possibility that Afr-AMPKalpha1 is involved in determining the reproductive mode in Artemia.

Correlation between salivary alpha-amylase activity and pain scale in patients with chronic pain.

BACKGROUND AND OBJECTIVES: The visual analog scale (VAS) is commonly used to assess pain intensity. However, the VAS is of limited value if patients fail to reliably report. Objective assessments are therefore clearly preferable. Previous reports suggest that elevated salivary alpha-amylase may reflect increased physical stress. There is a close association between salivary alpha-amylase and plasma norepinephrine under stressful physical conditions. In this study, we have determined the usefulness of a portable salivary alpha-amylase analyzer as an objective biomarker of stress. METHODS: Thirty patients (male/female = 15/15, age: 60.5 +/- 15.3 years) with chronic low back or leg pain (pain (+) group) and 20 pain-free control patients undergoing elective surgery under general anesthesia with epidural analgesia (pain (-) group) were recruited. Patients received epidural block with 5 to 10 mL 1% lidocaine. VAS, blood pressure, and heart rates were assessed before and 30 and 45 minutes after the epidural block. Salivary alpha-amylase was simultaneously measured using a portable analyzer. The relationship between the VAS and salivary alpha-amylase in chronic pain patients was assessed. RESULTS: After the epidural block both heart rate and systolic blood pressure decreased by approximately 8%. In the pain (+) group, the epidural block markedly decreased the VAS pain scale and salivary alpha-amylase from 56 +/- 22 to 19 +/- 16 mm (P < .01) and from 82 +/- 48 to 45 +/- 28 U/mL (P < .01), respectively, with a significant correlation between the 2 measures (r = 0.561, P < .01). In contrast, salivary alpha-amylase did not change significantly in the pain (-) group. CONCLUSIONS: Because there was a significant correlation between VAS pain scale and salivary alpha-amylase, we suggest that this biomarker may be a good index for the objective assessment of pain intensity. In addition, a simple to use portable analyzer may be useful for such assessment.

Terminating the stress: peripheral peptidolysis of proopiomelanocortin-derived regulatory hormones by the dermal microvascular endothelial cell extracellular peptidases neprilysin and angiotensin-converting enzyme.

The skin including the microvascular endothelium is an established peripheral source and target of the immunomodulatory proopiomelanocortin (POMC) peptides ACTH and alpha-MSH. Whereas intracellular POMC peptide generation is well characterized, less is known on their extracellular processing in peripheral tissues by the neuropeptide-specific zinc metalloproteases neprilysin (NEP) and angiotensin-converting enzyme (ACE). This may locally control POMC peptide bioavailability and activation of ACTH/alpha-MSH-specific melanocortin receptors (MCs). In a cell-free system, endothelial cell (EC) membranes prepared from ACE(high)/NEP(low)-expressing primary human dermal microvascular ECs and the ACE(low)/NEP(high) expressing EC line HMEC-1 degraded ACTH(1-39) over time, resulting in temporary increased alpha-MSH immunoreactivity. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy peptide mapping and electrospray ionization-mass spectroscopy sequencing identified several stable fragments generated from ACTH(1-39), ACTH(1-24), and alpha-MSH by EC membranes or recombinant NEP and ACE. Whereas some fragments could be assigned to a cell-specific NEP or ACE activity, other degradation products require additional enzyme activity. Pharmacological NEP inhibition enhanced the ACTH and alpha-MSH-mediated activation of EC ectopically expressing MC(1). Likewise, selected peptides such as alpha-MSH(2-12) generated from ACTH(1-39) and alpha-MSH by recombinant NEP displayed equipotent MC(1)-activating properties in vitro and antiinflammatory activity in murine allergic contact dermatitis in vivo as compared with the parental peptides. Thus, NEP and ACE significantly contribute to the EC processing of stress hormones (ACTH) and antiinflammatory peptides (alpha-MSH), which modulates MC(1) activation but does not completely inactivate the peptide ligand. Because NEP and ACE are regulated by inflammatory mediators and UV light, this may be important for ACTH/MSH-modulated skin inflammation.

Exercise, MAPK, and NF-kappaB signaling in skeletal muscle.

Mitogen-activated protein kinases (MAPKs) and NF-kappaB are two major regulators of gene transcription and metabolism in response to oxidative, energetic, and mechanical stress in skeletal muscle. Chronic activation of these signaling pathways has been implicated in the development and perpetuation of various pathologies, such as diabetes and cachexia. However, both MAPK and NF-kappaB are also stimulated by exercise, which promotes improvements in fuel homeostasis and can prevent skeletal muscle atrophy. This review will first discuss the major MAPK signaling modules in skeletal muscle, their differential activation by exercise, and speculated functions on acute substrate metabolism and exercise-induced gene expression. Focus will then shift to examination of the NF-kappaB pathway, including its mechanism of activation by cellular stress and its putative mediation of exercise-stimulated adaptations in antioxidant status, tissue regeneration, and metabolism. Although limited, there is additional evidence to suggest cross talk between MAPK and NF-kappaB signals with exercise. The objectives herein are twofold: 1) to determine how and why exercise activates MAPK and NF-kappaB; and 2) to resolve their paradoxical activation during diseased and healthy conditions.

SIRT2, a tubulin deacetylase, acts to block the entry to chromosome condensation in response to mitotic stress.

We previously identified SIRT2, an nicotinamide adenine dinucleotide (NAD)-dependent tubulin deacetylase, as a protein downregulated in gliomas and glioma cell lines, which are characterized by aneuploidy. Other studies reported SIRT2 to be involved in mitotic progression in the normal cell cycle. We herein investigated whether SIRT2 functions in the mitotic checkpoint in response to mitotic stress caused by microtubule poisons. By monitoring chromosome condensation, the exogenously expressed SIRT2 was found to block the entry to chromosome condensation and subsequent hyperploid cell formation in glioma cell lines with a persistence of the cyclin B/cdc2 activity in response to mitotic stress. SIRT2 is thus a novel mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability (CIN), characteristics previously reported for the CHFR protein. We further found that histone deacetylation, but not the aberrant DNA methylation of SIRT2 5'untranslated region is involved in the downregulation of SIRT2. Although SIRT2 is normally exclusively located in the cytoplasm, the rapid accumulation of SIRT2 in the nucleus was observed after treatment with a nuclear export inhibitor, leptomycin B and ionizing radiation in normal human fibroblasts, suggesting that nucleo-cytoplasmic shuttling regulates the SIRT2 function. Collectively, our results suggest that the further study of SIRT2 may thus provide new insights into the relationships among CIN, epigenetic regulation and tumorigenesis.

Stress-induced inactivation of the c-Myb transcription factor through conjugation of SUMO-2/3 proteins.

Post-translational modifications, such as phosphorylation, acetylation, ubiquitination, and SUMOylation, play an important role in regulation of the stability and the transcriptional activity of c-Myb. Conjugation of small ubiquitin-like modifier type 1 (SUMO-1) to lysines in the negative regulatory domain strongly suppresses its transcriptional activity. Here we report conjugation of two other members of the SUMO protein family, SUMO-2 and SUMO-3, and provide evidence that this post-translational modification negatively affects transcriptional activity of c-Myb. Conjugation of SUMO-2/3 proteins is strongly enhanced by several different cellular stresses and occurs primarily on two lysines, Lys(523) and Lys(499). These lysines are in the negative regulatory domain of c-Myb and also serve as acceptor sites for SUMO-1. Stress-induced SUMO-2/3 conjugation is very rapid and independent of activation of stress-activated protein kinases of the SAPK and JNK families. PIAS-3 protein was identified as a new c-Myb-specific SUMO-E3 ligase that both catalyzes conjugation of SUMO-2/3 proteins to c-Myb and exerts a negative effect on c-Myb-induced reporter gene activation. Interestingly, co-expression of a SPRING finger mutant of PIAS-3 significantly suppresses SUMOylation of c-Myb under stress. These results argue that PIAS-3 SUMO-E3 ligase plays a critical role in stress-induced conjugation of SUMO-2/3 to c-Myb. We also detected stress-induced conjugation of SUMO-2/3 to c-Myb in hematopoietic cells at the levels of endogenously expressed proteins. Furthermore, according to the negative role of SUMO conjugation on c-Myb capacity, we have observed rapid stress-induced down-regulation of the targets genes c-myc and bcl-2 of c-Myb. Our findings demonstrate that SUMO-2/3 proteins conjugate to c-Myb and negatively regulate its activity in cells under stress.

Autophagy is activated for cell survival after endoplasmic reticulum stress.

Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.

Perk-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress.

It has been well established that the tumor microenvironment can promote tumor cell adaptation and survival. However, the mechanisms that influence malignant progression have not been clearly elucidated. We have previously demonstrated that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR). Here, we show that tumors derived from K-Ras-transformed Perk(-/-) mouse embryonic fibroblasts (MEFs) are smaller and exhibit less angiogenesis than tumors with an intact ISR. Furthermore, Perk promotes a tumor microenvironment that favors the formation of functional microvessels. These observations were corroborated by a microarray analysis of polysome-bound RNA in aerobic and hypoxic Perk(+/+) and Perk(-/-) MEFs. This analysis revealed that a subset of proangiogenic transcripts is preferentially translated in a Perk-dependent manner; these transcripts include VCIP, an adhesion molecule that promotes cellular adhesion, integrin binding, and capillary morphogenesis. Taken with the concomitant Perk-dependent translational induction of additional proangiogenic genes identified by our microarray analysis, this study suggests that Perk plays a role in tumor cell adaptation to hypoxic stress by regulating the translation of angiogenic factors necessary for the development of functional microvessels and further supports the contention that the Perk pathway could be an attractive target for novel antitumor modalities.

Expression profiles of Na+,K+-ATPase during acute and chronic hypo-osmotic stress in the blue crab Callinectes sapidus.

During acclimation to dilute seawater, the specific activity of Na+,K+-ATPase increases substantially in the posterior gills of the blue crab Callinectes sapidus. To determine whether this increase occurs through regulation of pre-existing enzyme or synthesis of new enzyme, mRNA and protein levels were measured over short (<24 h) and long (18 days) time courses. Na+,K+-ATPase expression, both mRNA and protein, did not change during the initial 24-h exposure to dilute seawater (10 ppt salinity). Thus, osmoregulation in C. sapidus during acute exposure to low salinity likely involves either modulation of existing enzyme or mechanisms other than an increase in the amount of Na+,K+-ATPase enzyme. However, crabs exposed to dilute seawater over 18 days showed a 300% increase in Na+,K+-ATPase specific activity as well as a 200% increase in Na+,K+-ATPase protein levels. Thus, it appears that the increase in Na+,K+-ATPase activity during chronic exposure results from the synthesis of new enzyme. The relative amounts of mRNA for the alpha-subunit increased substantially (by 150%) during the acclimation process, but once the crabs had fully acclimated to low salinity, the mRNA levels had decreased and were not different from levels in crabs fully acclimated to high salinity. Thus, there is transient induction of the Na+,K+-ATPase mRNA levels during acclimation to dilute seawater.

Protective role of caffeic acid phenethyl ester in the liver of rats exposed to cold stress.

Cold exposure can induce a form of environmental stress. Cold stress (CS) alters homeostasis, results in the creation of reactive oxygen species and leads to alterations in the antioxidant defense system. The caffeic acid phenethyl ester (CAPE), an active component of propolis, has an antioxidant capacity. We investigated the effect of CS on oxidative stress and antioxidant defense system and the possible protective effect of CAPE in rat liver tissue. Twenty-four female Wistar Albino rats were divided into four groups: Control, CAPE-treated, CS, and CAPE-treated CS (CS + CAPE) group. Catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities and total glutathione (GSH) and malondialdehyde (MDA) levels were measured. In addition, histological changes in liver tissue were examined by light microscopy. SOD, CAT and GSH-Px activities and total GSH level were significantly declined in the CS group. In the CS + CAPE group, the activities of these three enzymes and GSH level significantly raised with regard to the CS group. MDA levels increased in the CS group and decreased in the CS + CAPE group. The tissues of the CS group showed some histopathological changes such as necrosis, hepatocyte degeneration, sinusoidal dilatation, hemorrhage and vascular congestion and dilatation. In the CS + CAPE group, the histopathological evidence of hepatic damage was markedly reduced. Histological parameters were consistent with biochemical parameters. In this study, CS increased oxidative stress in liver tissue. CAPE regulated antioxidant enzymes, inhibited lipid peroxidation and reduced hepatic damage.

Effects of dexamethasone treatment (to mimic stress) and Vitamin E oral supplementation on the spermiogram and on seminal plasma spontaneous lipid peroxidation and antioxidant enzyme activities in dogs.

The objective was to determine if treatment with dexamethasone (to mimic stress) has a deleterious effect on the spermiogram and on the composition of seminal plasma in the dog and whether adverse effects were reduced by oral supplementation with Vitamin E. Eighteen adult male Rottweiler dogs were randomly allocated in a 2 x 2 factorial treatment design (with or without dexamethasone treatment versus with or without Vitamin E supplementation). Dogs in the supplemented group received 500 mg of alpha-tocopherol (Vitamin E)/dog/day per os for 10 weeks. Dexamethasone (0.01 mg/kg/day i.m.) was given once daily for 7 days, starting 7 days after the onset of Vitamin E supplementation. Food intake, body condition score and body weight were assessed daily. Semen collections (digital manipulation) were performed twice weekly for 14 weeks and blood samples (for plasma concentrations of cortisol and testosterone) were collected once a week. Dexamethasone treatment significantly reduced ejaculate volume and increased thiobarbituric acid-reactive substances (TBARS) in the seminal plasma. In contrast, supplementation with Vitamin E increased sperm motility, vigor and concentration and decreased the percentage of major sperm defects. In conclusion, dexamethasone treatment (to mimic stress) had a deleterious effect on the spermiogram and on the seminal plasma lipid peroxidation in dogs; however, some of these effects were prevented by oral supplementation with Vitamin E.

Hydrolytic and nonenzymatic functions of acetylcholinesterase comodulate hemopoietic stress responses.

Glucocorticoid-initiated granulocytosis, excessive proliferation of granulocytes, persists after cortisol levels are lowered, suggesting the involvement of additional stress mediator(s). In this study, we report that the stress-induced acetylcholinesterase variant, AChE-R, and its cleavable, cell-penetrating C-terminal peptide, ARP, facilitate granulocytosis. In postdelivery patients, AChE-R-expressing granulocyte counts increased concomitantly with serum cortisol and AChE activity levels, yet persisted after cortisol had declined. Ex vivo, mononuclear cells of adult peripheral blood responded to synthetic ARP26 by overproduction of hemopoietically active proinflammatory cytokines (e.g., IL-6, IL-10, and TNF-alpha). Physiologically relevant ARP26)levels promoted AChE gene expression and induced the expansion of cultured CD34+ progenitors and granulocyte maturation more effectively than cortisol, suggesting autoregulatory prolongation of ARP effects. In vivo, transgenic mice overexpressing human AChE-R, unlike matched controls, showed enhanced expression of the myelopoietic transcription factor PU.1 and maintained a stable granulocytic state following bacterial LPS exposure. AChE-R accumulation and the consequent inflammatory consequences can thus modulate immune responses to stress stimuli.

Stress-activated signaling pathways mediate the stimulation of pregnancy-associated plasma protein-A expression in cultured human fibroblasts.

Pregnancy-associated plasma protein-A (PAPP-A) is an IGF binding protein protease that appears to function as a posttranslational modulator of IGF bioavailability in response to injury. A previous study indicated that the proinflammatory cytokines, TNFalpha and IL-1beta, were potent stimulators of PAPP-A expression in cultured human fibroblasts. In this study, we investigated the intracellular signaling pathways mediating cytokine-stimulated PAPP-A expression. Treatment of human fibroblasts with TNFalpha and IL-1beta (1 nm) had little or no effect on phosphatidylinositol 3-kinase and Erk1/2 activation, pathways commonly associated with proliferation. On the other hand, TNFalpha and IL-1beta induced p38, c-Jun N-terminal kinase (JNK), and nuclear factor (NF)kappaB activation, pathways more closely related to stress response. An inhibitor of p38 activation (SB203580) had no effect on TNFalpha- or IL-1beta-stimulated PAPP-A expression. The JNK inhibitor, SP600125, had no effect on IL-1beta- or TNFalpha-stimulated PAPP-A mRNA expression. However, SP600125 effectively inhibited IL-1beta-induced PAPP-A protein expression. MG-132, a proteasome inhibitor that blocked degradation of the intrinsic NFkappaB inhibitor, IkappaB, and thereby prevented NFkappaB activation, was a potent inhibitor of both TNFalpha- and IL-1beta-stimulated PAPP-A mRNA and protein expression and IGF binding protein-4 protease activity. MG-132 had no effect on JNK phosphorylation or p38 activation, and SB203580 and SP600125 had no effect on IkappaB degradation, documenting inhibitor specificity. BAY11-7082, another inhibitor of NFkappaB activation, also inhibited TNFalpha- and IL-1beta-stimulated PAPP-A expression and IGF binding protein-4 protease activity. These data indicate that NFkappaB activation is the primary mediator of cytokine-stimulated PAPP-A expression in human fibroblasts.

Stress-induced enhancement of activity of lymphocyte lysosomal enzymes in pigs of different stress-susceptibility.

To evaluate a possible mechanism of stress-induced lymphopenic effect we assessed the activity of lymphocyte lysosomal enzymes (LE) under immobilization. The effects of immobilization stress on LE (AP, acid phosphatase, cathepsin D and L, beta-N-acetyl-glucosamidase) activity in lymphocytes, number of lymphocytes and plasma cortisol (COR) level in the peripheral blood were examined in the cross-bred Pietrain pigs showing genotypic (presence or lack of RyR1 gene mutation) and phenotypic (reactivity to halothane) differences. It was found that immobilization stress evoked an increase in LE which was concomitant with lymphopenia and a rise of COR level. The most pronounced enhancement of LE, which may reflect a tendency to lymphocyte cytolysis, was found in the recessive homozygotes RyR1 (nn) phenotypically defined as stress/halothane susceptible as well as in the heterozygotes RyR1 (Nn) included in the group of stress/halothane resistant. Despite this individual variability the stress-induced increase in LE activity was present in all the animals. It seems that a possibility of destruction (lysis) of lymphocyte cells should not be excluded as one of the causes of stress lymphopenia.