Atlantic salmon possess three mitogen activated protein kinase kinase 6 paralogs responding differently to stress.

Mitogen activated protein kinase kinase (MKK) 3 and 6 are the main p38 mitogen-activated protein kinase activators in mammals. In the present study, three Atlantic salmon MKK6 orthologs were identified. The deduced amino acid sequences of the salmon MKK6 proteins were highly similar to mammalian MKK6 sequences, and they were ubiquitously expressed. All three were shown to be upstream activators of salmon p38. In cells exposed to sorbitol, sodium arsenite and UV radiation, the different salmon MKK6s were shown to be selectively activated. Thus, our results suggest a specific function of the three salmon MKK6s depending on which stress stimuli the cells are exposed to. Phylogenetic analysis of MKK6 and MKK3 sequences from different species indicate that salmon is unique in having three MKK6 gene copies, whereas other fish species possess one or two MKK6 genes. Interestingly, in contrast to mammals, fish do not have an MKK3 gene. We propose that two major duplication events have occurred for the ancestral MKK3/6 gene: one in tetrapods yielding MKK3 and MKK6, and another one in fish yielding two MKK6 paralogs. The third MKK6 copy found in salmon is probably the result of the salmonid-specific tetraploidization event. In conclusion, we report for the first time in any species the existence of three MKK6 genes displaying distinct expression and activation patterns. Furthermore, MKK3 is dispensable in some vertebrates because it is absent from fish genomes despite being present in chicken and all mammals sequenced so far.

[Influence repeated stress on immune responciveness and monooxigenase activity of normotensive and hypertensive rats].

Repeated stress led to antipodal directions in immune system and cytochrome P450 activities of normotensive and hypertensive rats. Enhancement of the Reaction of Delayed Hypersensitivity, suppression of cytochrome P450-mediated monooxigenase activities were observed in Wistar rats. On the contrary, in the NISAG decrease of the Reaction Delayed Hypersensitivity, elevation of cytochrome P450-mediated monooxigenase activities were observed, as comparison with Wistar rats.

Methodological considerations in the use of salivary alpha-amylase as a stress marker in field research.

Salivary alpha-amylase recently has been identified as a stress-related biomarker for autonomic nervous system activity. This study addresses sample collection and handling considerations for field researchers. Saliva was collected by unstimulated passive drool from 14 adults and pooled. Incubation of pooled saliva at 22 or 37 degrees C for 21 days did not diminish amylase activity. However, sodium azide added at concentrations

Transport and acclimation conditions for the use of an estuarine fish (Pomatoschistus microps) in ecotoxicity bioassays: effects on enzymatic biomarkers.

Acclimation of organisms for ecotoxicity testing is in general processed according to Organisation for Economic Co-operation and Development (OECD) and/or Environmental Protection Agency (EPA) guidelines, under controlled conditions. However, when organisms are collected in the field, their capture, transport and adaptation to laboratory conditions are factors of stress. In their natural habitat, estuarine fish are exposed to considerable fluctuations of environmental variables, while in laboratory they are acclimated to constant conditions and this can be per se a factor of stress that may influence biomarker responses. Therefore, it is important to investigate the effects of these procedures on estuarine fish performance before using them as test organisms in ecotoxicity bioassays. The goal of the present study was to investigate the effects of transporting the common goby, Pomatoschistus microps from the field (natural populations) to the laboratory and of its acclimation to laboratory conditions on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferases (GSTs). Fish were collected in a reference site of the Minho River estuary (NW of Portugal) and the activities of the biomarkers were monitored before and after the transport of organisms to the laboratory and during the acclimation period (at 5, 10 and 15 days). The activities of all the enzymes indicated that capture and transport conditions had no effects on enzymatic activities. Furthermore, AChE, LDH and GST presented higher activities at the end of the acclimation period than at beginning, suggesting a physiological adaptation to laboratory conditions. This adaptation should be taken into consideration in the experimental design to avoid bias in the interpretation of effects of xenobiotics on biomarkers.

Decreased expression of phospholipase C-beta 1 protein in endoplasmic reticulum stress-loaded neurons.

The endoplasmic reticulum (ER) plays a critical role in the maintenance of intracellular homeostasis and its dysfunction is thought to lead to neuronal death, which results in neurodegenerative disorders. Since phospholipase C (PLC) isozymes are involved in maintenance of the intracellular Ca2+ concentration by regulating Ca2+ release from the ER, their expression might be affected by ER stress. Of these isozymes, PLC-beta 1 and -gamma 1, in particular, are known to protect cells from oxidative stress and thus alteration of their expression profile under ER stress-loaded conditions is interesting. Using primary cultured rat cortical neurons, we here examined whether expression of PLC-beta 1 and -gamma 1 was altered in ER stress-loaded neurons induced by tunicamycin (Tm). In ER stress-loaded neurons treated with Tm in the range of 0.03-3 microg/ml for 20 h, the viability of the neurons was decreased dose-dependently, the decrease being significant with 0.3 or more microg/ml, and expression of the representative ER stress markers, GRP78/BiP, and cleaved caspase-3 and -12, was increased after 24 h postincubation, confirming the induction of ER stress in the neurons. In the ER stress-loaded neurons obtained on Tm treatment, the expression level of PLC-beta 1 decreased dose-dependently. On the other hand, there was no difference in the PLC-gamma 1 protein expression level between control and ER stress-loaded neurons. Overall, we demonstrated that ER stress decreases the expression of PLC-beta 1, but not -gamma 1, in neurons.

Effects of cold stress on the messenger ribonucleic acid levels of corticotrophin-releasing hormone and thyrotropin-releasing hormone in hypothalami of broilers.

In this study, seventy 1-d-old male broiler chicks were randomly allocated to 10 groups to investigate the effect of cold stress on the messenger RNA (mRNA) levels of corticotrophin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH) in hypothalami. The chicks were maintained at 30 +/- 2 degrees C temperature and given free access to standard chow and water. Until 15 d old, the 6 treatment groups were maintained at 12 +/- 1 degrees C. Hypothalami were collected for the assessment of the mRNA levels by semiquantitative reverse transcription-PCR after stress termination. Cold stress significantly decreased the mRNA levels of CRH in 6 and 12 h treatment groups and significantly increased the mRNA levels of TRH in 1, 6, and 12 h treatment groups during acute cold stress. There were no significant differences in the mRNA levels of CRH and TRH among different control groups during chronic cold stress. However, chronic cold stress resulted in a significant increase of the mRNA levels of CRH and a significant decrease of the mRNA levels of TRH compared with the corresponding control groups. The results indicate that the 2 genes show different response to cold stress at the mRNA levels, and on the other hand, the different degree of cold stress also produces different effects on the identical gene.

Long-term increase in GAD67 mRNA expression in the central amygdala of rats sensitized by drugs and stress.

Repeated administration of addictive drugs and prolonged exposure to stressful stimuli induce sensitization to their behavioural stimulant properties. In this study, male Sprague-Dawley rats were repeatedly exposed to morphine [twice a day for 3 days at increasing doses, 10, 20, 40 mg/kg subcutaneously (s.c)], amphetamine (1 mg/kg s.c., once a day for 10 days), nicotine (0.4 mg/kg s.c., once a day for 5 days) and stress (food restriction for 7 days). After an interval of 3-30 days, depending on the pretreatment, rats were challenged with vehicle, with the same drug received as pretreatment (5 mg/kg of morphine, 0.5 mg/kg of amphetamine or 0.4 mg/kg of nicotine, respectively) or, in the case of food-restricted rats, with 0.5 mg/kg of amphetamine. Thereafter, changes in the expression of glutamic acid decarboxylase (GAD)67 mRNA were estimated by in situ hybridization in the central nucleus of the amygdala (CeA), basolateral amygdala (BLA), dorsolateral striatum (dLStr), nucleus accumbens shell (AcS) and core (AcC). All sensitizing pretreatments increased GAD67 mRNA in the CeA. Drug challenge did not further affect GAD67 mRNA in the CeA of saline, drug and stress pre-exposed rats. As to the other areas, no differences were observed in drug pre-exposed compared with saline pre-exposed and fed ad libitum rats, except for amphetamine. Amphetamine pre-exposure decreased GAD67 mRNA levels in the dLStr and the AcC and AcS, and this effect was reversed by amphetamine challenge. The results show that different drugs and stress models of behavioural sensitization have in common an increase of GA67 in the CeA but not in the BLA, and suggest the changes of GAD67 in the CeA are a substrate of the sensitized response to drug challenge.

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.

Role of neuronal nitric oxide synthase in the regulation of the neuroendocrine stress response in rodents: insights from mutant mice.

Nitric oxide (NO) is a free radical gas synthesised from arginine and oxygen by enzymes of the family of the nitric oxide synthase. In particular, the neuronal nitric oxide synthase (nNOS) is highly expressed by cells of the hypothalamic paraventricular nucleus, where the sympatho-adrenal system, the hypothalamic-pituitary-adrenal axis and the hypothalamic-neurohypophyseal system originate. These structures are deputed to regulate the neuroendocrine stress response. In the past years, evidence has been accumulated to suggest that NO of nNOS origin plays a significant role in modulating the activity of the above mentioned systems under acute stressor exposure. The availability of nNOS knock-out mice allowed to investigate not only the physiological consequences of a constitutive lack of NO of nNOS origin at the hormonal and molecular level, but also to examine possible behavioural alterations. In this review, we shall discuss and confront the current trends of research in this area, especially focusing on the latest findings gained from genetically modified mice.

Transient stress and stress enzyme responses have practical impacts on parameters of embryo development, from IVF to directed differentiation of stem cells.

In this review, we discuss the expression, regulation, downstream mechanisms, and function of stress-induced stress enzymes in mammalian oocytes, peri-implantation embryos, and the stem cells derived from those embryos. Recent reports suggest that stress enzymes mediate developmental functions during early mammalian development, in addition to the homeostatic functions shared with somatic cells. Stress-induced enzymes appear to insure that necessary developmental events occur: many of these events may occur at a slower rate, although some may occur more rapidly. Developmental events induced by stress may be mediated by a single dominant enzyme, but there are examples of responses that require the integration of more than one stress enzyme. The discussion focuses on the consequences of stress as a function of duration and magnitude, and this includes an emerging understanding of the threshold levels of duration and magnitude that lead to pathology. Other topics discussed are the reversibility of the developmental as well as homeostatic consequences of stress, the further problems with readaptation after stress subsides, and the mechanisms and functions of stress enzymes during early mammalian development. The analyses are done with specific concern for their practical impact in assisted reproductive technology (ART) and stem cell technologies.

AMP-activated protein kinase subunit interactions: beta1:gamma1 association requires beta1 Thr-263 and Tyr-267.

AMP-activated protein kinase (AMPK) plays multiple roles in the body's overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK alpha1beta1gamma1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian alphabetagamma complex shows that beta binds alpha and gamma. Here we show that a beta1(186-270)gamma1 complex can form in the absence of detectable alpha subunit. Moreover, using alanine mutagenesis we show that beta1 Thr-263 and Tyr-267 are required for betagamma association but not alphabeta association.

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.

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response.

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2alpha, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.

Intracerebroventricular injection of L-arginine induces sedative and hypnotic effects under an acute stress in neonatal chicks.

L-arginine participates in many important and diverse biochemical reactions associated with the normal physiology of the organism. In the present study, we investigated the effect of central administration of L-arginine on the stress response and its mechanism in neonatal chicks. Intracerebroventricular (i.c.v.) injection of L-arginine clearly attenuated the stress response in a dose-dependent manner, and induced sleep-like behavior during 10 min. To clarify the mechanism by which L-arginine induces sedative and hypnotic effects in chicks, we investigated the effects of nitric oxide (NO) synthase (NOS) inhibitors on L-arginine-induced sedative and hypnotic effects, and as well as the effects of a NO donor. L-Arginine-induced (1.9 micromol) sedative and hypnotic effects were attenuated by i.c.v. co-injection with a non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester HCl (400 nmol). In addition, the effects of L-arginine were slightly attenuated by the inactive isomer of the NOS inhibitor N(G)-nitro-D-arginine methyl ester HCl (400 nmol). The i.c.v. injection of 3-morpholinosylnomine hydrochloride, a spontaneous NO donor, had little effect on postures. The i.c.v. injection of L-arginine had no effect on NOx concentration at various brain sites. These results suggested that the contribution of NO generation via NOS may be low in the sedative and hypnotic actions of L-arginine. Therefore, L-arginine and/or its metabolites, excluding NO, may be necessary for these actions.

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.

Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs.

OBJECTIVE: To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs. ANIMALS: 24 pigs (mean weight, 20 +/- 1 kg). PROCEDURES: Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA. RESULTS: No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.

Activation of caspase 3, 9, 12, and Bax in masseter muscle of mdx mice during necrosis.

The mdx mouse, a model of muscular dystrophy, lacks dystrophin, a cell membrane protein. It is known that the lack of dystrophin causes muscle fiber necrosis from 2 weeks after birth, and the majority of necrotic muscle fibers are replaced by regenerated muscle fibers by 4 weeks after birth. A recent study indicated the possibility that mitochondria-mediated intracellular stress, a phenomenon similar to apoptosis, may be produced during muscle fiber necrosis, but did not analyze endoplasmic reticulum-mediated intracellular stress. Therefore, we examined the expression of the caspase-12 gene involved in the endoplasmic reticulum stress pathway and the Bax, caspase-9, and caspase-3 genes involved in the mitochondrial stress pathway in the mdx masseter muscle. We found over-expression of caspase-12 in cells at 2-3 weeks after birth when muscle fiber necrosis was not prominent. This suggests that stress occurs in the endoplasmic reticulum to maintain cell morphology in the absence of dystrophin. In addition, Bax was abundantly expressed in the mdx masseter muscle at 3 weeks after birth, and the expression of caspase-9 and -3 was prominent at 3-4 weeks after birth when necrosis and regeneration were marked. These results indicate that endoplasmic reticulum and mitochondrial stresses are produced during necrosis of the mdx masseter muscle, and suggest that these events are a phenomenon similar to apoptosis.

Investigation into the signal transduction pathway via which heat stress impairs intestinal epithelial barrier function.

BACKGROUND AND AIMS: Intact protein absorption is thought to be a causative factor in several intestinal diseases, such as food allergy, celiac disease and inflammatory bowel disease. However, the mechanism remains unclear. The aim of this study was to characterize a novel signal transduction pathway via which heat stress compromises intestinal epithelial barrier function. METHODS: Heat stress was carried out by exposing confluent human intestinal epithelial cell line T84 cell monolayers to designated temperatures (37-43 degrees C) for 1 h. Transepithelial electric resistance (TER) and permeability to horseradish peroxidase (HRP, molecular weight = 44 000) were used as indicators to assess the intestinal epithelial barrier function. Phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and protein kinase C (PKC) protein of the T84 cells were evaluated in order to identify the signal transduction pathway in the course of heat stress-induced intestinal epithelial barrier dysfunctions. RESULTS: The results showed that exposure to heat stress significantly increased intact protein transport across the intestinal epithelial monolayer; the amount of phospho-PKC, phospho-MLCK and phospho-MLC proteins in T84 cells decreased significantly at 41 degrees C and 43 degrees C although they increased at 39 degrees C. The heat stress-induced T84 monolayer barrier dysfunction was inhibited by pretreatment with PKC inhibitor, MLCK inhibitor, or HSP70. CONCLUSION: Heat stress can induce intestinal epithelial barrier dysfunction via the PKC and MLC signal transduction pathway.

Visible light induces matrix metalloproteinase-9 expression in rat eye.

Up-regulation of matrix metalloproteinase-9 (MMP-9, gelatinase B) in the nervous system has been demonstrated when excitotoxicity-induced tissue remodeling and neuronal death occurs. Induction of MMP-9 by a natural stimulus has not been observed yet. Using RT-PCR and gelatin-zymography we demonstrated MMP-9 induction at transcriptional and protein levels in different structures of the rat eye following over-stimulation with white light. MMP-9 elevation occurred in the retina without reduction in photoreceptor number or major anatomical reorganization. A transient decrease in electroretinogram b-wave indicated the functional recovery. Retrobulbar injection of a broad-spectrum MMP-inhibitor GM6001, slowed the recovery rate of b-wave amplitude. Even room-light applied to dark-adapted awake animals induced MMP-9 increase in the retina, which suggests a role for MMP-9 in physiological functional plasticity of the nervous system, such as light adaptation. This is the first demonstration of MMP-9 induction by a sensory stimulus.

Adipose stress-sensing kinases: linking obesity to malfunction.

Obesity has been proposed to inflict a variety of stresses on adipose tissue, including inflammatory, metabolic, oxidative and endoplasmic reticulum stress. Through the activation of 'stress-sensing pathways', metabolic and endocrine alterations are produced, which probably contribute to the co-morbidities associated with obesity. Here, we review the evidence supporting the development of various obesity-related stresses and the activation of several stress-sensing pathways, specifically in adipocytes and/or adipose tissue, which manifest metabolic and endocrine dysfunction frequently in obesity. As the central role of adipose tissue in regulating whole-body metabolism is elucidated, understanding adipose tissue stress-sensing pathways might provide potential new therapeutic targets to attenuate obesity-related morbidity.