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Numerous laboratory and field studies have found that female Pacific salmon have higher mortality than males during their once-in-a-lifetime upriver spawning migration. However, the proximate cause(s) of this increased mortality are poorly understood. This study exposed sockeye salmon (Oncorhynchus nerka) to a mild capture and tagging stressor and evaluated physiological recovery and movement behaviour at 1 and 4 h postrelease. Female sockeye salmon did not expend more anaerobic energy in response to the stressor but did have higher plasma lactate levels 4 h after the stressor, indicating that females took longer to physiologically recover compared with males. In addition, female salmon had lower plasma glucose but higher plasma cortisol, plasma [K.sup.+], and cardiac lactate levels compared with males. Male and female salmon had markedly different postrelease behaviours within the first hour of release; males were more likely to hold position within the staging area. Two potential mechanisms leading to increased mortality in female salmon were identified in this study: (a) prolonged recovery duration (possibly mediated by elevated plasma cortisol levels) and (b) insufficient oxygen delivery to the heart. De nombreuses etudes en laboratoire et de terrain ont releve que les saumons du Pacifique femelles presentent des taux de mortalite plus eleves que les males durant leur unique montaison de frai. Les causes immediates de cette mortalite accrue demeurent toutefois mal comprises. Des saumons sockeyes (Oncorhynchus nerka) ont ete exposes a un faible facteur de stress associe a la capture et au marquage, et leur retablissement physiologique et leur comportement de deplacement ont ete evalues 1 et 4 h apres qu'ils aient ete relaches. Les sockeyes femelles ne depensaient pas plus d'energie anaerobie en reaction au facteur de stress, mais presentaient des concentrations de lactate plasmatique plus elevees apres 4 h, indiquant que leur retablissement physiologique etait plus long que celui des males. En outre, les saumons femelles avaient de plus faibles teneurs en glucose plasmatique, mais de plus fortes teneurs en cortisol plasmatique, en [K.sup.+] plasmatique et en lactate cardiaque que les males. Les saumons males et femelles presentaient des comportements nettement differents durant la premiere heure apres leur lacher; les males etaient plus susceptibles de rester en place dans l'aire de repos. Deux mecanismes possibles menant a une plus importante mortalite des femelles ont ete cernes dans l'etude, a savoir : (a) une duree prolongee du retablissement (possiblement modulee par des concentrations elevees de cortisol plasmatique) et (b) un apport insuffisant d'oxygene au coeur., Introduction Sex-biased adult mortality can strongly influence population dynamics and extinction risk (Boukal et al. 2008; Melbourne and Hastings 2008), yet sex is often not considered as a factor in [...]

Cellular stress can result in dysfunction and disease, and mechanisms exist to combat this. Previously unknown steps have been uncovered in a pathway that signals when mitochondrial organelles are dysfunctional. Cleaved protein is a signal that activates the integrated stress response., Author(s): Bradford P. Tremblay, Cole M. Haynes Author Affiliations: Mitochondrial distress call moves to the cytosol to trigger a response to stress Organelles called mitochondria are responsible for storing energy [...]

Diapause is a widespread adaptation to seasonality across invertebrate taxa. It is critical for persistence in seasonal environments, synchronizing life histories with favorable, resource-rich conditions and mitigating exposure to harsh environments. Despite some promising recent progress, however, we still know very little about the molecular modifications underlying diapause. We used transcriptional profiling to identify key groups of genes and pathways differentially regulated during pupal diapause, dynamically regulated across diapause development, and differentially regulated after diapause was pharmacologically terminated in the flesh fly Sarcophaga crassipalpis. We describe major shifts in stress axes, endocrine signaling, and metabolism that accompany diapause, several of which appear to be common features of dormancy in other taxa. To assess whether invertebrates with different diapause strategies have converged toward similar transcriptional profiles, we use archived expression data to compare the pupal diapause of S. crassipalpis with the adult reproductive diapause of Drosophila melanogaster and the larval dauer of Caenorhabditis elegans. Although dormant invertebrates converge on a few similar physiological phenotypes including metabolic depression and stress resistance, we find little transcriptional similarity among dormancies across species, suggesting that there may be many transcriptional strategies for producing physiologically similar dormancy responses. dormancy | insulin signaling | metabolic depression | Sarcophaga crassipalpis | stress tolerance doi/ 10.1073/pnas.1007075107

Stress affects the hippocampus, a brain region crucial for memory. In rodents, acute stress may reduce density of dendritic spines, the location of postsynaptic elements of excitatory synapses, and impair long-term potentiation and memory. Steroid stress hormones and neurotransmitters have been implicated in the underlying mechanisms, but the role of corticotropin-releasing hormone (CRH), a hypothalamic hormone also released during stress within hippocampus, has not been elucidated. In addition, the causal relationship of spine loss and memory defects after acute stress is unclear. We used transgenic mice that expressed YFP in hippocampal neurons and found that a 5-h stress resulted in profound loss of learning and memory. This deficit was associated with selective disruption of long-term potentiation and of dendritic spine integrity in commissural/associational pathways of hippocampal area CA3. The degree of memory deficit in individual mice correlated significantly with the reduced density of area CA3 apical dendritic spines in the same mice. Moreover, administration of the CRH receptor type 1 ([CRFR.sub.1]) blocker NB130775 directly into the brain prevented the stress-induced spine loss and restored the stress-impaired cognitive functions. We conclude that acute, hours-long stress impairs learning and memory via mechanisms that disrupt the integrity of hippocampal dendritic spines. In addition, establishing the contribution of hippocampal CRH--[CRFR.sub.1] signaling to these processes highlights the complexity of the orchestrated mechanisms by which stress impacts hippocampal structure and function. corticotropin-releasing factorl long-term potentiation | memory | synaptic plasticity | hippocampus doi/ 10.1073/pnas.1003825107

We determined the effects of voluntary wheel running on the hypothalamic-pituitary-adrenal (HPA) axis, and the peripheral determinants of glucocorticoids action, in male Zucker diabetic fatty (ZDF) rats. Six-week-old euglycemic ZDF rats were divided into Basal, Sedentary, and Exercise groups (n = 8-9 per group). Basal animals were immediately killed, whereas Sedentary and Exercising rats were monitored for 10 wk. Basal (i.e., ~0900 AM in the resting state) glucocorticoid levels increased 2.3-fold by week 3 in Sedentary rats where they remained elevated for the duration of the study. After an initial elevation in basal glucocorticoid levels at week 1, Exercise rats maintained low glucocorticoid levels from week 3 through week 10. Hyperglycemia was evident in Sedentary animals by week 7, whereas Exercising animals maintained euglycemia throughout. At the time of death, the Sedentary group had ~40% lower glucocorticoid receptor (GR) content in the hippocampus, compared with the Basal and Exercise groups (P < 0.05), suggesting that the former group had impaired negative feedback regulation of the HPA axis. Both Sedentary and Exercise groups had elevated ACTH compared with Basal rats, indicating that central drive of the axis was similar between groups. However, Sedentary, but not Exercise, animals had elevated adrenal ACTH receptor and steroidogenic acute regulatory protein content compared with the Basal animals, suggesting that regular exercise protects against elevations in glucocorticoids by a downregulation of adrenal sensitivity to ACTH. GR and 11[beta]-hydroxysteroid dehydrogenase type 1 content in skeletal muscle and liver were similar between groups, however, GR content in adipose tissue was elevated in the Sedentary groups compared with the Basal and Exercise (P < 0.05) groups. Thus, the gradual elevations in glucocorticoid levels associated with the development of insulin resistance in male ZDF rats can be prevented with regular exercise, likely because of adaptations that occur primarily in the adrenal glands. glucocorticoid; stress; hippocampus; type 2 diabetes; wheel running doi: 10.1152/ajpregu.00155.2010.

Cellular adaptations to stress often involve changes in RNA metabolism. One RNA-binding protein that has been implicated in RNA handling during environmental stress in both animal cells and prokaryotes is the Ro autoantigen. However, the function of Ro in stress conditions has been unknown. We report that a Ro protein in the radiation-resistant eubacterium Deinococcus radiodurans participates in ribosomal RNA (tRNA) degradation during growth in stationary phase, a form of starvation. Levels of the Ro ortholog Rsf increase dramatically during growth in stationary phase and the presence of Rsr confers a growth advantage. Examination of rRNA profiles reveals that Rsr, the 3' to 5' exoribonuclease polynucleotide phosphorylase (PNP) and additional nucleases are ali involved in the extensive rRNA decay that occurs during starvation of this bacterium. We show that Rsr, PNP, and an Rsr-PNP complex exhibit increased sedimentation with ribosomal subunits during stationary phase. As the fractionation of PNP with ribosomal subunits is strongly enhanced in the presence of Rsr, we propose that Ro proteins function as cofactors to increase the association of exonucleases with certain substrates during stress. environmental stress | exonucleases | RNA-binding protein doi/10.1073/pnas.1000307107

Recent advances in the noninvasive analyses of plant metabolism include stress imaging techniques, mainly developed for vegetative tissues. We explored if infrared thermography can be used to predict whether a quiescent seed will germinate or die upon water uptake. Thermal profiles of viable, aged, and dead Pisum sativum seeds were recorded, and image analysis of 22,000 images per individual seed showed that infrared thermography can detect imbibilion- and germination-associated biophysical and biochemical changes. These 'thermal fingerprints' vary with viability in this species and in Triticum aestivum and Brassica napus seeds. Thermo-genesis of the small individual B. napus seeds was at the limit of the technology. We developed a computer model of 'virtual pea seeds,' that uses Monte Carlo simulation, based on the heat production of major seed storage compounds to unravel physico-chemical processes of thermogenesis. The simulation suggests that the cooling that dominates the early thermal profiles results from the dissolution of low molecular-weight carbohydrates. Moreover, the kinetics of the production of such 'cooling' compounds over the following 100 h is dependent on seed viability. We also developed a deterministic tool that predicts in the first 3 hours of water uptake, when seeds can be redried and stored again, whether or not a pea seed will germinate. We believe that the early separation of individual, ungerminated seeds (live, aged, or dead) before destructive germination assessment creates unique opportunities for integrative studies on cell death, differentiation, and development. aging | crop | germination | imaging | stress doi/10.1073/pnas.0914197107

Mechanical stress is an important modulator of lung morphogenesis, postnatal lung development, and compensatory lung regrowth. The effect of mechanical stress on stem or progenitor cells is unclear. We examined whether proliferative responses of epithelial progenitor cells, including dually immunoreactive (CCSP and proSP-C) progenitor cells (CCSP+/SP-C+) and type II alveolar epithelial cells (ATII), are affected by physical factors found in the lung of emphysematics, including loss of elastic recoil, reduced elastin content, and alveolar destruction. Mice underwent single lung pneumonectomy (PNY) to modulate transpulmonary pressure (mechanical stress) and to stimulate lung regeneration. Control mice underwent sham thoracotomy. Plombage of different levels was employed to partially or completely abolish this mechanical stress. Responses to graded changes in transpulmonary pressure were assessed in elastin-insufficient mice (elastin +/-, ELN+/-) and elastase-treated mice with elastase-induced emphysema. Physiological regrowth, morphometry (linear mean intercept; Lmi), and the proliferative responses of CCSP+/SP-C+, Clara cells, and ATII were evaluated. Plombage following PNY significantly reduced transpulmonary pressure, regrowth, and CCSP+/SP-C+, Clara cell, and ATII proliferation following PNY. In the ELN+/- group, CCSP+/SP-C+ and ATII proliferation responses were completely abolished, although compensatory lung regrowth was not significantly altered. In contrast, in elastase-injured mice, compensatory lung regrowth was significantly reduced, and ATII but not CCSP+/SP-C+ proliferation responses were impaired. Elastase injury also reduced the baseline abundance of CCSP+/SP-C+, and CCSP+/SP-C+ were found to be displaced from the bronchioalveolar duct junction. These data suggest that qualities of the extracellular matrix including elastin content, mechanical stress, and alveolar integrity strongly influence the regenerative capacity of the lung, and the patterns of cell proliferation in the lungs of adult mice. pneumocyte; bronchioalveolar stem cell; elastase; elastin; pneumonectomy; lung regeneration doi: 10.1152/ajplung.90594.2008.

The hippocampal formation is a brain region noted for its plasticity in response to stressful events and adrenal steroid hormones. Recent work has shown that chromatin remodeling in various brain regions, including the hippocampus, is associated with the effects of stress in a variety of models. We chose to examine the effects of stress, stress duration, corticosterone administration, and fluoxetine treatment on the levels of hippocampal histone H3 methylation at lysines 4, 9, and 27, marks associated, respectively, with active transcription, heterochromatin formation, and transcriptional repression. We found that acute stress increased the levels of H3K9 tri-methylation (H3K9me3) in the dentate gyrus (DG) and CA1, while it reduced levels of H3K9 mono-methylation (H3K9me1) and H3K27 tri-methylation (H3K27me3) in the same regions, and had no effect on levels of H3K4 tri-methylation (H3K4me3). Seven days of restraint stress reduced levels of H3K4me3 in the CA1 and H3K27me3 in the DG and CA1, while increasing basal levels of H3K9me3. Chronic restraint stress (CRS) for 21 days mildly increased levels of H3K4me3 and reduced H3K9me3 levels in the DG. Treatment with fluoxetine during CRS reversed the decrease in DG H3K9me3, but had no effect on the other marks. These results show a complex, surprisingly rapid, and regionally specific pattern of chromatin remodeling within hippocampus produced by stress and anti-depressant treatment that may open an avenue of understanding the interplay of stress and hippocampal gene expression, and reveal the outlines of a potential chromatin stress response that may be diminished or degraded by chronic stress. brain | chromatin | corticosteroids | fluoxetine doi/10.1073/pnas.0911143106

The nuclear factor [kappa]B (NF-[kappa]B) transcription factor regulates cellular stress responses and the immune response to infection. NF-[kappa]B activation results in oscillations in nuclear NF-[kappa]B abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor-[alpha] at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-[kappa]B nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical, models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-[kappa]B--dependent gene expression, which supports the idea that oscillation frequency has a functional role.

Here we investigated the effects of mineralocorticoid in the regulation of catecholamine biosynthesis using rat pheochromocytoma PC12 cells. Expression of mineralocorticoid receptor (MR) was confirmed in undifferentiated PC12 cells. Aldosterone stimulated dopamine production by PC12 cells without any increase in cAMP activity. Aldosterone-induced dopamine accumulation was enhanced in accordance with the increase in the rate-limiting enzyme tyrosine hydroxylase (TH). Blocking MR with eplerenone suppressed aldosterone-induced increases of TH mRNA and dopamine production. A glucocorticoid receptor (GR) antagonist, RU-486, attenuated dexamethasone- but not aldosterone-induced TH expression. Cycloheximide reduced both aldosterone- and dexamethasone-induced TH mRNA. A SAPK/JNK inhibitor, SP600125, suppressed aldosterone-induced TH mRNA expression; however, the aldosterone-induced TH expression was not affected by inhibition of ERK1/2, p38-MAPK, Rho-kinase, PI 3-kinase, and PKC. It was of note that cotreatment with eplerenone and SP600125 restored aldosterone-induced TH mRNA expression to basal levels. To investigate the involvement of bone morphogenetic protein (BMP) actions in aldosterone-induced catecholamine production, we examined the effects of BMP-4 and BMP-7, which are expressed in the adrenal medulla, on catecholamine biosynthesis. BMP-4 preferentially enhanced aldosterone-induced TH mRNA and dopamine production, although BMP-4 alone did not affect TH expression. The BMP-4 enhancement of aldosterone-induced TH expression was not observed in cells treated with eplerenone. BMP-4 did not affect MR expression of PC12 cells; however, it did enhance aldosterone-induced SAPK/JNK phosphorylation. Inhibition of SAPK/JNK or Rho suppressed BMP-4 enhancement of aldosterone-induced TH expression. Collectively, our findings demonstrate that aldosterone stimulates catecholamine biosynthesis in adrenomedullar cells via MR through genomic action and partly through nongenomic action by Rho-SAPK/JNK signaling, the latter of which is facilitated by BMP-4. A functional link between MR actions and endogenous BMP may be involved in the catecholamine production. stress-activated protein kinase; c-Jun N[H.sub.2]-terminal kinase; aldosterone; eplerenone; mineralocorticoid receptor; pheochromocytoma

Cigarette smoking decreases body weight, whereas molecular mechanisms underlying this phenomenon have not been elucidated. In this report, we investigated regulation of adipogenesis by cigarette smoke and involvement of aryl hydrocarbon receptor (AhR) and endoplasmic reticulum (ER) stress. We found that cigarette smoke extract (CSE) inhibited differentiation of preadipocytes into adipocytes dose dependently. It was associated with a decrease in lipid accumulation, blunted expression of adipocyte markers (adiponectin, PPAR-[gamma] and C/EBP[alpha]), and sustained expression of a preadipocyte marker MCP-1. CSE markedly induced activation of AhR, and AhR agonists (2,3,7,8tetrachlorodibenzo-p-dioxin, benzo[a]pyrene and 3-methylcholanthrene) reproduced the inhibitory effect of CSE on adipocyte differentiation. Furthermore, knockout of the AhR gene or blockade of AhR by a dominant-negative mutant attenuated the suppressive effects of CSE on adipocyte differentiation. We also found that CSE induced ER stress in preadipocytes, and ER stress inducers (thapsigargin, tunicamycin, and A23187) reproduced the suppressive effect of CSE on the differentiation of preadipocytes. Interestingly, AhR agonists did not cause ER stress, and ER stress inducers did not activate AhR. These results suggested that cigarette smoke has the potential to inhibit adipocyte differentiation via dual, independent mechanisms, i.e., through activation of the AhR pathway and induction of the unfolded protein response. adipocyte; adiponectin; peroxisome proliferator-activated receptor-[gamma]; CCAAT/enhancer-binding protein-[alpha]; monocyte chemoattractant protein-1

The pathophysiology of aging is accompanied by a decline in tolerance to environmental stress. While mitochondria are primary suspects in the etiology of aging, little is known about their ability to tolerate perturbations to homeostasis in older organisms. To investigate the role of mitochondria in the increased susceptibility to heat stress that accompanies aging, young and old Fischer 344 rats underwent a heat stress protocol known to elicit exaggerated cellular damage with aging. At either 2 or 24 h after heat stress, livers were removed from animals, and hepatic mitochondria were isolated. Electron microscopy revealed extensive morphological damage to mitochondria from young and, to a greater extent, old rats after heat stress. There was also a significant loss of cytochrome c from old, but not young, mitochondria and a persistent increase in 4-hydroxynonenal-modified proteins in old vs. young mitochondria exposed to heat stress. Electron paramagnetic resonance measurements of superoxide indicate greater superoxide production from mitochondria of old compared with young animals and suggest that mitochondrial integrity was altered during heat stress. The mitochondrial stress response, which functions to correct stress-induced damage to mitochondrial proteins, was also blunted in old rats. Delayed and reduced levels of heat shock protein 60 (Hsp60), the main inducible mitochondrial stress protein, were observed in old compared with young mitochondria after heat stress. Additionally, the amount of Hspl0 protein increased in young, but not old, rat liver mitochondria after hyperthermic challenge. Taken together, these data suggest that mitochondria in old animals are more vulnerable to incurring and less able to repair oxidative damage that occurs in response to a physiologically relevant heat stress. mitochondria; liver; stress response; ROS; cytochrome c

Exercise can activate the hypothalamo-pituitary-adrenocortical (HPA) axis, and regular exercise training can impact how the HPA axis responds to stress. The mechanism by which acute exercise induces HPA activity is unclear. Therefore, the purpose of this study was to test the hypothesis that nitric oxide modulates the neuroendocrine component of the HPA axis during exercise. Female Yucatan miniature swine were treated with N-nitro-L-arginine methyl ester (L-NAME) to test the effect of chronic nitric oxide synthase (NOS) inhibition on the ACTH response to exercise. In addition, we tested the effect of NOS inhibition on blood flow to tissues of the HPA axis and report the effects of handling and treadmill exercise on the plasma concentrations of ACTH and cortisol. Chronic NOS inhibition decreased plasma [NO.sub.x] levels by 44%, increased mean arterial blood pressure by 46%, and increased expression of neuronal NOS in carotid arteries. Vascular conductance was decreased in the frontal cortex, the hypothalamus, and the adrenal gland. Chronic NOS inhibition exaggerated the ACTH response to exercise. In contrast, chronic NOS inhibition decreased the ACTH response to restraint, suggesting that the role of NO in modulating HPA activity is stressor dependent. These results demonstrate that NOS activity modulates the response of the neuroendocrine component of the HPA axis during exercise stress. neuroendocrine; restraint; pigs; N-nitro-L-arginine methyl ester; stress

Stress is a risk factor for cardiovascular disease, such as atherosclerosis. Stress during pregnancy (maternal stress) may have long-term consequences for the health of the offspring. However, it is not known whether maternal stress affects the offspring's predisposition to develop atherosclerosis. Atherosclerosis is often related to vascular endothelial dysfunction. We hypothesized that maternal stress affects vascular endothelial function and accelerates development of atherosclerosis in offspring of apolipoprotein E-deficient mice, a model commonly used for atherosclerosis research. Stress was induced by restraining dams in small cylinders for five consecutive days (2 h/day) beginning on gestational day 8 [+ or -] 0.5. Vascular function and development of atherosclerosis in the aorta were determined in male and female offspring at 11-15 wk of age (with early lesions) and at 22-26 wk of age (with established lesions). Endotheliumdependent vasorelaxation was determined using methacholine (0.0001-10 [micro]mol/1) in the absence or presence of the nitric oxide synthase inhibitor [N.sup.[omega]]-nitro-L-arginine methyl ester hydrochloride (L-NAME; 100 [micro]mol/l). Male offspring (11-15 wk old) from stressed dams were less dependent on nitric oxide for relaxation compared with controls (L-NAME inhibition: 38 [+ or -] 10 vs. 69 [+ or -] 6%, P < 0.05). Atherosclerotic lesion area was larger in male and female 25- to 26-wk-old offspring from stressed dams compared with corresponding controls [median (interquartile range): males: 6.8 (5.4-7.7) vs. 5.1 (4.4-5.5), P < 0.05, females: 10.0 (8.9-10.9) vs. 7.0 (4.7-8.7), P < 0.05]. In conclusion, maternal stress renders the apolipoprotein E-deficient offspring more susceptible to develop atherosclerosis. fetal origins of adult disease; endothelial function; aorta

Animal reports suggest that reflex activation of cardiac sympathetic nerves can evoke coronary vasoconstriction. Conversely, physiological stress may induce coronary vasodilation to meet an increased metabolic demand. Whether the sympathetic nervous system can modulate coronary vasomotor tone in response to stress in humans is unclear. Coronary blood velocity (CBV), an index of coronary blood flow, can be measured in humans by noninvasive duplex ultrasound. We studied 11 healthy volunteers and measured beat-by-beat changes in CBV, blood pressure, and heart rate during 1) static handgrip for 20 s at 10% and 70% of maximal voluntary contraction; 2) lower body negative pressure at -10 and -30 mmHg for 3 min each; 3) cold pressor test for 90 s; and 4) hypoxia (10% [O.sub.2]), hyperoxia (100% [O.sub.2]), and hypercapnia (5% C[O.sub.2]) for 5 min each. At the higher level of handgrip, mean blood pressure increased (P < 0.001), whereas CBV did not change [P = not significant (NS)]. In addition, during lower body negative pressure, CBV decreased (P < 0.02; and P < 0.01, for -10 and -30 mmHg, respectively), whereas blood pressure did not change (P = NS). The dissociation between the responses of CBV and blood pressure to handgrip and lower body negative pressure is consistent with coronary vasoconstriction. During hypoxia, CBV increased (P < 0.02) and decreased during hyperoxia (P < 0.01), although blood pressure did not change (P = NS), suggesting coronary vasodilation during hypoxia and vasoconstriction during hyperoxia. In contrast, concordant increases in CBV and blood pressure were noted during the cold pressor test, and hypercapnia had no effects on either parameter. Thus the physiological stress known to be associated with sympathetic activation can produce coronary vasoconstriction in humans. Contrasting responses were noted during systemic hypoxia and hyperoxia where mechanisms independent of autonomic influences appear to dominate the vascular end-organ effects. exercise; hypoxia; oxygen; autonomic nervous system

We have previously demonstrated that the Bc1-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bc1-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57B1/6 mice and from mice cannulated and subjected to 1-4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings. redox stress; nitric oxide; hypoxia; cell death; inflammation

Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation. We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase. cytoskeleton; hypertonicty; cell volume; small GTPases

Secondary succession is impacted by both biotic and abiotic forces, but their relative importance varies due to environmental drivers. Across estuarine salinity gradients, physical stress increases with salinity, and biotic stresses are greater at lower salinities. In southern New England tidal marshes spanning a landscape-scale salinity gradient, we experimentally examined the effects of physical stress and consumer pressure by mammalian herbivores on secondary succession in artificially created bare patches. Recovery was slower in marshes exposed to full-strength seawater, where physical stress is high. Compared to full-strength salt marshes, recovery in low-salinity marshes was much faster and was influenced by small-mammal consumers. At lower salinities, small mammals selectively ate and prevented the establishment of several native and two invasive, nuisance species (Typha angustifolia and Phragmites australis) but were unable to control the expansion of established P. australis stands. By controlling the establishment of competitively dominant species and the trajectory of secondary succession in low-salinity marshes, small mammals may play a cryptic keystone role in estuarine plant communities and are a critical, overlooked consideration in the conservation and management of estuarine marshes. Key words: abiotic and biotic factors; consumer control; environmental stress models; herbivory; invasive species; New England tidal marshes; physical stress; salinity; secondary succession; small-mammal herbivores; stress gradients.

A dual childbirth injury model, including vaginal distension (VD) and pudendal nerve crush (PNC), may best represent the injuries seen clinically. The objective of this study was to investigate urethral function, anatomy, and neurotrophin expression after several simulated childbirth injuries. Groups of 140 rats underwent PNC, VD, PNC+VD, or neither (C). Four days after injury, all injury groups had significantly decreased leak-point pressure (LPP) compared with C rats. Ten days after injury, LPP in PNC and PNC+VD rats remained significantly lower than C rats. Three weeks after injury, LPP in all injury groups had recovered to C values. Histological evidence of injury was still evident in the external urethral sphincter (EUS) after VD and PNC+VD 10 days after injury. Three weeks after injury, the EUS of PNC+VD rats remained disrupted. One day after VD, brain-derived neurotrophic factor (BDNF) expression in the EUS was reduced, while neurotrophin-4 (NT-4) and nerve growth factor (NGF) expression was unchanged. BDNF, NT-4, and NGF expression was dramatically upregulated in the EUS after PNC. After PNC+VD, NGF expression was upregulated, and BDNF and NT-4 expression was upregulated somewhat but not to the same extent as after PNC. Ten days after injury, PNC+VD had the least number of normal nerve fascicles near the EUS, followed by PNC and VD. Twenty-one days after injury, all injury groups had fewer normal nerve fascicles, but without significant differences compared with C rats. PNC+VD therefore provides a more severe injury than PNC or VD alone. stress urinary incontinence; vaginal childbirth; pudendal nerve crush; anatomic recovery; rat

Visceral hypersensitivity and stress have been implicated in the pathophysiology of functional gastrointestinal disorders. We used a selective vasopressin 3 ([V.sub.3]) receptor antagonist SSR 149415 to investigate the involvement of the vasopressin (AVP)/[V.sub.3] signaling system in the development of stress-induced visceral hyperalgesia in rats. Rats were exposed to a daily 1-h session of water avoidance stress (WAS) or sham WAS for 10 consecutive days. The visceromotor response to phasic colorectal distension (CRD, 10-60 mmHg) was assessed before and after stress. Animals were treated daily with SSR149415 (0.3, 1, or 3 mg/kg ip 30 min before each WAS or sham WAS session), with a single dose of SSR149415 (1 mg/kg ip), or the selective corticotropin-releasing factor 1 (CR[F.sub.1]) antagonist DMP-696 (30 mg/kg po) before CRD at day 11. Effects of a single dose of SSR 149415 (10 mg/kg iv) on acute mechanical sensitization during repetitive CRD (12 distensions at 80 mmHg) were also assessed. In vehicle-treated rats, repeated WAS increased the response to CRD, indicating visceral hypersensitivity. Repeated administration of SSR149415 at 1 or 3 mg/kg completely prevented stress-induced visceral hyperalgesia. Similarly, a single dose of DMP-696 or SSR149415 completely blocked hyperalgesic responses during CRD. In contrast, a single dose of SSR149415 did not affect the acute hyperalgesic responses induced by repeated, noxious distension. These data support a major role for [V.sub.3] receptors in repeated psychological stress-induced visceral hyperalgesia and suggest that pharmacological manipulation of the AVP/[V.sub.3] pathway might represent an attractive alternative to the CRF/CR[F.sub.1] pathway for the treatment of chronic stress-related gastrointestinal disorders. corticotropin-releasing factor; DMP-696; functional gastrointestinal disorders; irritable bowel syndrome; SSR149415

This study compares the effects of rest intervals on isokinetic muscle torque recovery between sets of a knee extensor and flexor exercise protocol in physically active younger and older women. Twenty young (22.4 [+ or -] 1.7 years) and 16 older (70.7 [+ or -] 4.3 years) women performed three sets of eight maximum repetitions of knee extension/flexion at 60[degrees] x [s.sup.-1]. The rest interval between sets was 15, 30, and 60 seconds and was randomly assigned across three testing days. No significant interaction of rest by set by age group was observed. There was a significant decline in mean knee extensor torque when 15- and 30-second rest intervals were used between sets, but not when a 60-second rest interval was applied for both the young and the old women. No significant decline for mean knee flexor torque was observed in the older women when a 30-second rest interval was used, whereas a longer 60-second rest interval was required in younger women. Active younger and older women require similar rest intervals between sets of a knee extensor exercise (60 seconds) for complete recovery. However, older women recovered faster (30 seconds) than younger women (60 seconds) between sets of a knee flexor exercise. The exercise-to-rest ratio for knee extensors was similar for young and old women (1:2). Old women required only a 1:1 exercise-to-rest ratio for knee flexor recovery, whereas younger women required a longer 1:2 exercise-to-rest ratio. The results of the present study are specific to isokinetic testing and training and are more applicable in rehabilitation and research settings. Practitioners should consider age and gender when prescribing rest intervals between sets. KEY WORDS muscle fatigue, exercise-to-rest ratio, aging, resistance exercise, isokinetic

Head-up tilt (HUT) induces a reduction in preload, which is thought to be restored through sympathetic venoconstriction, reducing unstressed volume ([V.sub.u]) and venous compliance (VeC). In this study, we assessed venous inflow and outflow responses and their reproducibility and determined the relation with autonomic function during HUT. Eight healthy non-pregnant women were subjected to 20[degrees] head-down tilt to 60[degrees] HUT at 20[degrees] intervals. At each rotational step, we randomly assessed forearm pressure-volume (P-V) curves (venous occlusion plethysmography) during inflow (Ve[C.sub.IN]) and outflow [venous emptying rate ([VER.sub OUT])]. Ve[C.sub.IN] was defined as the ratio of the slope of the volume-time curve and pressure-time curve, with direct intravenous pressure measurement. [VER.sub.OUT] was determined using the derivate of a quadratic regression model using cuff pressure. We defined [V.sub.u] as the y-intercept of the P-V curve. We calculated, for both methods, the coefficients of reproducibility (CR) and variation (CV). Vascular sympathetic activity was determined by spectral analysis. Ve[C.sub.IN] decreased at each rotational step compared with the supine position (P < 0.05), whereas [VER.sub.OUT] increased. CR of Ve[C.sub.IN] was higher in the supine position than [VER.sub.OUT] but lower during HUT. CV varied between 19% and 25% (Ve[C.sub.IN]) and between 12% and 21% ([VER.sub.OUT]). HUT decreased [V.sub.u]. The change in Ve[C.sub.IN] and [VER.sub.OUT] correlated with the change in vascular sympathetic activity (r = -0.36, P < 0.01, and r = 0.48, P < 0.01). This is the first study in which a reproducible reduction in Ve[C.sub.IN] and [V.sub.u] and a rise in [VER.sub.OUT] during HUT are documented. The alterations in venous characteristics relate to changes in vascular sympathetic activity. head-up tilt; venous compliance; venous emptying rate; venous capacity; autonomic system

Intermittent restraint stress delays hyperglycemia in ZDF rats better than pair feeding. We hypothesized that intermittent stress would preserve [beta]-cell mass through distinct mechanisms from food restriction. We studied temporal effects of intermittent stress on [beta]-cell compensation during pre-, early, and late diabetes. Six-week-old obese male ZDF rats were restraint-stressed 1 h/day, 5 days/wk for 0, 3, 6, or 13 wk and compared with age-matched obese ZDF rats that had been food restricted for 13 wk, and 19-wk-old lean ZDF rats. Thirteen weeks of stress and food restriction lowered cumulative food intake 10-15%. Obese islets were fibrotic and disorganized and not improved by stress or food restriction. Obese pancreata had islet hyperplasia and showed evidence of neogenesis, but by 19 wk old [beta]-cell mass was not increased, and islets had fewer [beta]-cells that were hypertrophic. Both stress and food restriction partially preserved [beta]-cell mass at 19 wk old via islet hypertrophy, whereas stress additionally lowered [alpha]-cell mass. Concomitant with maintenance of insulin responses to glucose, stress delayed the sixfold decline in [beta]-cell proliferation and reduced [beta]-cell hypertrophy, translating into 30% more [beta]-cells per islet after 13 wk. In contrast, food restriction did not improve insulin responses or [beta]-cell hyperplasia, exacerbated [beta]-cell hypertrophy, and resulted in fewer [beta]-cells and greater [alpha]-cell mass than with stress. Thus, preservation of [beta]-cell mass with adaptation to intermittent stress is related to [beta]-cell hyperplasia, maintenance of insulin responses to glucose, and reductions in [alpha]-cell mass that do not occur with food restriction. restraint stress; Zucker diabetic fatty rat; islet dynamics; [alpha]-cell mass

The alternative sigma factor SigF of Mycobacterium tuberculosis has been characterized in detail as a general-stress, stationary-phase sigma factor involved in the virulence of the bacterium. While a homologous gene has been annotated in the genome of the fast-growing Mycobacterium smegmatis, little experimental evidence is available on the function of this gene. Here, we demonstrate that SigF of M. smegmatis is required for resistance to hydrogen peroxide, heat shock and acidic pH, but not for survival in human neutrophils. No difference in sensitivity to isoniazid was observed between the wild-type strain and the [DELTA]sigF mutant, suggesting that SigF-mediated resistance to hydrogen peroxide was via a pathway independent of KatG or AhpC. RT-PCR and 5'-RACE (rapid amplification of cDNA ends) analyses showed that sigF of M. smegmatis was co-transcribed with rsbW (thought to encode an anti-sigma factor for SigF) and MSMEG_1802 (unknown function) and was expressed from two promoters, one upstream of MSMEG_1802 and the second upstream of rsbW. Analysis of transcriptional lacZ fusion constructs in the sigF-deletion background revealed that the MSMEG_1802 promoter was dependent on SigF for expression. Moreover, MSMEG_1802-lacZ was induced twofold upon entry into stationary phase, while exposure of exponentially growing cultures to various stress conditions (e.g. heat, cold, ethanol, hydrogen peroxide or different pH values) did not lead to induction of MSMEG_1802-lacZ. Expression of rsbW-lacZ was independent of SigF and remained constant throughout the growth cycle and under various stress conditions unless the bacteria were challenged with D-cycloserine.

Although exercise training-induced changes in left ventricular (LV) structure are well characterized, adaptive functional changes are incompletely understood. Detailed echocardiographic assessment of LV systolic function was performed on 20 competitive rowers (10 males and 10 females) before and after endurance exercise training (EET; 90 days, 10.7 [+ or -] 1.1 h/wk). Structural changes included LV dilation (end-diastolic volume = 128 [+ or -] 25 vs. 144 [+ or -] 28 ml, P < 0.001), right ventricular (RV) dilation (end-diastolic area = 2,850 [+ or -] 550 vs. 3,260 [+ or -] 530 [mm.sup.2], P < 0.001), and LV hypertrophy (mass = 227 [+ or -] 51 vs. 256 [+ or -] 56 g, P < 0.001). Although LV ejection fraction was unchanged (62 [+ or -] 3% vs. 60 [+ or -] 3%, P = not significant), all direct measures of LV systolic function were altered. Peak systolic tissue velocities increased significantly (basal lateral S'[DELTA] = 0.9 [+ or -] 0.6 cm/s, P = 0.004; and basal septal S'[DELTA] = 0.8 [+ or -] 0.4 cm/s, P = 0.008). Radial strain increased similarly in all segments, whereas longitudinal strain increased with a base-to-apex gradient. In contrast, circumferential strain (CS) increased in the LV free wall but decreased in regions adjacent to the RV. Reductions in septal CS correlated strongly with changes in RV structure ([DELTA]RV end-diastolic area vs. [DELTA]LV septal CS; [r.sup.2] = 0.898, P < 0.001) and function ([DELTA]peak RV systolic velocity vs. [DELTA]LV septal CS, [r.sup.2] = 0.697, P < 0.001). EET leads to significant changes in LV systolic function with regional heterogeneity that may be secondary to concomitant RV adaptation. These changes are not detected by conventional measurements such as ejection fraction. athlete's heart; left ventricle; strain; ventricular interdependence

Chronic contractile activation, or tone, in asthma coupled with continuous stretching due to breathing may be involved in altering the contractile function of airway smooth muscle (ASM). Previously, we (11) showed that cytoskeletal remodeling and stiffening responses to acute (2 h) localized stresses were modulated by the level of contractile activation of ASM. Here, we investigated if altered contractility in response to chronic mechanical strain was dependent on repeated modulation of contractile tone. Cultured human ASM cells received 5% cyclic (0.3 Hz), predominantly uniaxial strain for 5 days, with once-daily dosing of either sham, forskolin, carbachol, or histamine to alter tone. Stiffness, contractility (KCl), and 'relaxability' (forskolin) were then measured as was cell alignment, myosin light-chain phosphorylation (pMLC), and myosin light-chain kinase (MLCK) content. Cells became aligned and baseline stiffness increased with strain, but repeated lowering of tone inhibited both effects (P < 0.05). Strain also reversed a negative tone-modulation dependence of MLCK, observed in static conditions in agreement with previous reports, with strain and tone together increasing both MLCK and pMLC. Furthermore, contractility increased 176% (SE 59) with repeated tone elevation. These findings indicate that with strain, and not without, repeated tone elevation promoted contractile function through changes in cytoskeletal organization and increased contractile protein. The ability of repeated contractile activation to increase contractility, but only with mechanical stretching, suggests a novel mechanism for increased ASM contractility in asthma and for the role of continuous bronchodilator and corticosteroid therapy in reversing airway hyperresponsiveness. asthma; mechanical stress; cytoskeletal remodeling; myosin light-chain kinase; optical magnetic twisting cytometry

The purpose of the present study was 1) to develop a stable model for measuring contraction-induced elevations in mRNA in single skeletal muscle fibers and 2) to utilize this model to investigate the response of heat shock protein 72 (HSP72) mRNA following an acute bout of fatiguing contractions. Living, intact skeletal muscle fibers were microdissected from lumbrical muscle of Xenopus laevis and either electrically stimulated for 15 min of tetanic contractions (EX; n = 26) or not stimulated to contract (REST; n = 14). The relative mean developed tension of EX fibers decreased to 29 [+ or -] 7% of initial peak tension at the stimulation end point. Following treatment, individual fibers were allowed to recover for 1 (n = 9), 2 (n = 8), or 4 h (n = 9) prior to isolation of total cellular mRNA. HSP72, HSP60, and cardiac [alpha]-actin mRNA content were then assessed in individual fibers using quantitative PCR detection. Relative HSP72 mRNA content was significantly (P < 0.05) elevated at the 2-h postcontraction time point relative to REST fibers when normalized to either HSP60 (18.5 [+ or -] 7.5-fold) or cardiac [alpha]-actin (14.7 [+ or -] 4.3-fold), although not at the 1or 4-h time points. These data indicate that 1) extraction of RNA followed by relative quantification of mRNA of select genes in isolated single skeletal muscle fibers can be reliably performed, 2) HSP60 and cardiac [alpha]-actin are suitable endogenous normalizing genes in skeletal muscle following contractions, and 3) a significantly elevated content of HSP72 mRNA is detectable in skeletal muscle 2 h after a single bout of fatiguing contractions, despite minimal temperature changes and without influence from extracellular sources. exercise; heat shock; stress

This study examined endotoxin-mediated cytokinemia during exertional heat stress (EHS). Subjects were divided into trained [TR; n = 12, peak aerobic power ([Vo.sub.2peak]) = 70 [+ or -] 2 ml*kg lean body [mass.sup.-1]*[min.sup.-1]] and untrained (UT; n = 11, [Vo.sub.2peak] = 50 [+ or -] 1 ml*kg lean body [mass.sup.-1*[min.sup.-1]) groups before walking at 4.5 km/h with 2% elevation in a climatic chamber (40[degrees]C, 30% relative humidity) wearing protective clothing until exhaustion (Exh). Venous blood samples at baseline and 0.5[degrees]C rectal temperature increments (38.0, 38.5, 39.0, 39.5, and 40.0[degrees]C/Exh) were analyzed for endotoxin, lipopolysaccharide binding protein, circulating cytoldnes, and intranuclear NF-[kappa]B translocation. Baseline and Exh samples were also stimulated with LPS (100 ng/ml) and cultured in vitro in a 37[degrees]C water bath for 30 min. Phenotypic determination of natural killer cell frequency was also determined. Enhanced blood (104 [+ or -] 6 vs. 84 [+ or -] 3 ml/kg) and plasma volumes (64 [+ or -] 4 vs. 51 [+ or -] 2 ml/kg) were observed in TR compared with UT subjects. EHS produced an increased concentration of circulating endotoxin in both TR (8 [+ or -] 2 pg/ml) and UT subjects (15 [+ or -] 3 pg/ml) (range: not detected to 32 pg/ml), corresponding with NF-[kappa]B translocation and cytokine increases in both groups. In addition, circulating levels of tumor necrosis factor-[alpha] and IL-6 were also elevated combined with concomitant increases in IL-1 receptor antagonist in both groups and IL-10 in TR subjects only. Findings suggest that the threshold for endotoxin leakage and inflammatory activation during EHS occurs at a lower temperature in U T compared with TR subjects and support the endotoxin translocation hypothesis of exertional heat stroke, linking endotoxin tolerance and heat tolerance. splanchnic permeability; immune function; blood volume; cardiovascular/thermoregulatory strain; flow cytometry

Exercise stress is associated with increased risk for upper respiratory tract infection. We have shown that exercise stress can increase susceptibility to infection. Quercetin, a flavonoid present in a wide variety of fruits and vegetables, has been reported to inhibit infectivity and replication of a broad spectrum of viruses and may offset the increase in susceptibility to infection associated with stressful exercise. This study examined the effects of quercetin feedings on susceptibility to the influenza virus A/Puerto Rico/8/34 (H1N1) following stressful exercise. Mice were randomly assigned to one of four treatment groups: exercise-placebo, exercise-quercetin, control-placebo, or control-quercetin. Exercise consisted of a run to fatigue (~140 min) on a treadmill for 3 consecutive days. Quercetin (12.5 mg/kg) was administered via gavage for 7 days before viral challenge. At 30 min after the last bout of exercise or rest, mice (n = 23-30) were intranasally inoculated with a standardized dose of influenza virus (0.04 hemagglutinating units). Mice were monitored daily for morbidity (time to sickness), symptom severity, and mortality (time to death) for 21 days. Exercise stress was associated with an increased susceptibility to infection [morbidity, mortality, and symptom severity on days 5-7 (P < 0.05)]; quercetin offset the increase in susceptibility to infection [morbidity, mortality, and symptom severity on days 5-7 (P < 0.05)] that was associated with stressful exercise. These data suggest that short-term quercetin feedings may prove to be an effective strategy to lessen the impact of stressful exercise on susceptibility to respiratory infection. nutrition; mice; infection; stress; virus

Repeated restraint stress (RRS; 3 h of restraint on 3 consecutive days) in rodents produces temporary hypophagia, but a long-term downregulation of body weight. The mild stress (MS) of an intraperitoneal injection of saline and housing in a novel room for 2 h also inhibits food intake and weight gain, but the effects are smaller than for RRS. Previous exposure to RRS exaggerates hypophagia, glucocorticoid release, and anxiety-type behavior caused by MS. Here we tested the involvement of brain stem corticotrophin-releasing factor receptors (CRFR) in mediating energetic and glucocorticoid responses to RRS or MS and in promoting stress hyperresponsiveness in RRS rats. Administration of 1.3 nmol [alpha]hCRF(9-41), a nonspecific CRFR antagonist, exaggerated hypophagia and weight loss in both RRS and MS rats, whereas 0.26 nmol had no effect in RRS or MS rats. In contrast, 2 nmol of the nonspecific antagonist astressin had no effect on weight loss or hypersensitivity to subsequent MS in RRS rats, but blocked weight loss and inhibition of food intake caused by MS alone. MS rats infused with 3 nmol antisauvagine-30, a CRFR2 antagonist, did not lose weight in the 48 h after MS, but 0.3 nmol did not prevent weight loss in MS rats. These data suggest that inhibition of food intake and weight loss induced by RRS or by MS involve different pathways, with hindbrain CRFR mediating the effect of MS on body weight and food intake. Hindbrain CRFR do not appear to influence stressinduced corticosterone release in RRS rats. brain stem; [alpha]hCRF(9-41); astressin; antisauvagine-30; rats

Heat stress (HS)-induced cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) to determine whether myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. After HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively with an adenoviral vector. Cell signaling was analyzed with Western blot analysis, while oncosis/apoptosis was assayed by measuring Trypan blue exclusion and/or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM before CI. HS increased association between FAK and phosphatidylinositol 3-kinase as well as causing a significant increase in AKT activity. Increased expression of wild-type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time 0 and after 10 min of CI compared with myocytes expressing empty virus. We conclude that myocardial stress activates cytoskeleton-hased signaling pathways that are associated with protection from lethal cell injury. cell signaling; protection; ischemia

We studied centrifugation-mediated mechanical stress-induced tumor necrosis factor-[alpha] (TNF-[alpha]) production in the monocyte-like cell line THP-1. The induction of TNF-[alpha] by mechanical stress was dependent on the centrifugation speed and produced the highest level of TNF-[alpha] after 1 h of stimulation. TNF-[alpha] production returned to normal levels after 24 h of stimulation. Mechanical stress also induced Toll-like receptor-2 (TLR2) mRNA in proportion to the expression of TNF-[alpha]. The inhibition of TLR2 signaling by dominant negative myeloid differentiation factor 88 (MyD88) blocked TNF-[alpha] expression response to mechanical stress. After transient overexpression of TLR2 in HEK-293 cells, mechanical stress induced TNF-[alpha] mRNA production. Interestingly, mechanical stress activated the c-Src-dependent TLR2 phosphorylation, which is necessary to induce [Ca.sup.2+] fluxes. When THP-1 cells were pretreated with BAPTA-AM, thapsigargin, and Ni[Cl.sub.2]*6[H.sub.2]O, followed by mechanical stimulation, both TLR2 and TNF-[alpha] production were inhibited, indicating that centrifugation-mediated mechanical stress induces both TLR2 and TNF-[alpha] production through [Ca.sup.2+] releases from intracellular [Ca.sup.2+] stores following TLR2 phosphorylation. In addition, TNF-[alpha] treatment in THP-1 cells induced TLR2 production in response to mechanical stress, whereas the preincubation of anti-TNF-[alpha] antibody scarcely induced the mechanical stress-mediated production of TLR2, indicating that TNF-[alpha] produced by mechanically stimulated THP-1 cells affected TLR2 production. We concluded that TNF-[alpha] production induced by centrifugation-mediated mechanical stress is dependent on MyD88-dependent TLR2 signaling that is associated with [Ca.sup.2+] release and that TNF-[alpha] production induced by mechanical stress affects TLR2 production. centrifugation; Toll-like receptor; THP-1 cells

Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection. lung; surfactant protein C; usual interstitial pneumonia; unfolded protein response

Rats of the JCR: LA-cp strain, which are homozygous for the cp gene (cp/cp), are obese, insulin-resistant, and hyperinsulinemic. They exhibit associated micro- and macrovascular disease and end-stage ischemic myocardial lesions and are highly stress sensitive. We subjected male cp/cp rats to pair feeding (providing the rats each day with the amount of food eaten by matched freely fed animals), a procedure that alters the diurnal feeding pattern, leading to a state of intermittent caloric restriction. Effects on insulin, glucose, and lipid metabolism, response to restraint stress, aortic contractile/relaxant response, and myocardial lesion frequency were investigated. Pair-fed young (12-wk-old) cp/cp rats had lower insulin and glucose levels (basal and following restraint), consistent with increased insulin sensitivity, but a greater increase in plasma nonesterified fatty acids in response to restraint. These effects were unrelated to lipolytic rates in adipose tissue but may be related to reduced fatty acid oxidation in skeletal muscle. Older (24-wk-old) pair-fed cp/cp rats had significantly reduced plasma triglyceride levels, improved micro- and macrovascular function, and reduced severity of ischemic myocardial lesions. These changes indicate a significant amelioration of end-stage disease processes in this animal model and the complexity of metabolic/physiological responses in studies involving alterations in food intake. The effects illustrate the sensitivity of the JCR:LA-cp rat, an animal model for the metabolic syndrome and associated cardiovascular disease, to the environmental and experimental milieu. Similar stress-related mechanisms may play a role in metabolically induced cardiovascular disease in susceptible human beings. metabolic syndrome; stress; fatty acids; vascular function; myocardial lesions doi: 10.1152/ajpendo.90257.2008.

Fluid shear stress modulates vascular production of endothelial superoxide anion ([[O.sub.2].sup.-]) and nitric oxide (NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress ([[tau].sub.ave]) of 23 dyn/[cm.sup.2] upregulated Mn-SOD mRNA expression at a higher level than did OSS at [[tau].sub.ave] = 0.02 dyn/[cm.sup.2] [+ or -] 3.0 dyn x [cm.sup.-2] x [s.sup.-1] and at 1 Hz (PSS by 11.3 [+ or -] 0.4-fold vs. OSS by 5.0 [+ or -] 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration. superoxide dismutase; superoxide anion; nitric oxide; nitrotyrosine; low-density lipoprotein

Mechanical forces are known to affect the biomechanical proper ties of native and engineered cardiovascular tissue. In particular, shear stress that results from the relative motion of heart valve leaflets with respect to the blood flow is one important component of their mechanical environment in vivo. Although different types of bioreactors have been designed to subject cells to shear stress, devices to expose biological tissue are few. In an effort to address this issue, the aim of this study was to design an ex vivo tissue culture system to characterize the biological response of heart valve leaflets subjected to a well-defined steady or time-varying shear stress environment. The novel apparatus was designed based on a cone-and-plate viscometer. The device characteristics were defined to limit the secondary flow effects inherent to this particular geometry. The determination of the operating conditions producing the desired shear stress profile was streamlined using a computational fluid dynamic (CFD) model validated with laser Doppler velocimetry. The novel ex vivo tissue culture system was validated in terms of its capability to reproduce a desired cone rotation and to maintain sterile conditions. The CFD results demonstrated that a cone angle of 0.5 deg, a cone radius of 40 mm, and a gap of 0.2 mm between the cone apex and the plate could limit radial secondary flow effects. The novel cone-and-plate permits to expose nine tissue specimens to an identical shear stress waveform. The whole setup is capable of accommodating four cone-and-plate systems, thus concomitantly subjecting 36 tissue samples to desired shear stress condition. The innovative design enables the tissue specimens to be flush mounted in the plate in order to limit flow perturbations caused by the tissue thickness. The device is capable of producing shear stress rates of up to 650 dyn [cm.sup.-2] [s.sup.-1] (i.e., maximum shear stress rate experienced by the ventricular surface of an aortic valve leaflet) and was shown to maintain tissue under sterile conditions for 120 h. The novel ex vivo tissue culture system constitutes a valuable tool toward elucidating heart valve mechanobiology. Ultimately, this knowledge will permit the production of functional tissue engineered heart valves, and a better understanding of heart valve biology and disease progression. Keywords: cone-and-plate, viscometer, bioreactor, shear stress, heart valve, tissue engineering, mechanotransduction

Responses to extracellular stress directly confer survival fitness by means of complex regulatory networks. Despite their complexity, the networks must be evolvable because of changing ecological and environmental pressures. Although the regulatory networks underlying stress responses are characterized extensively, their mechanism of evolution remains poorly understood. Here, we examine the evolution of three candidate stress response networks (chemotaxis, competence for DNA uptake, and endospore formation) by analyzing their phylogenetic distribution across several hundred diverse bacterial and archaeal lineages. We report that genes in the chemotaxis and sporulation networks group into well defined evolutionary modules with distinct functions, phenotypes, and substitution rates as compared with control sets of randomly chosen genes. The evolutionary modules vary in both number and cohesiveness among the three pathways. Chemotaxis has five coherent modules whose distribution among species shows a clear pattern of interdependence and rewiring. Sporulation, by contrast, is nearly monolithic and seems to be inherited vertically, with three weak modules constituting early and late stages of the pathway. Competence does not seem to exhibit well defined modules either at or below the pathway level. Many of the detected modules are better understood in engineering terms than in protein functional terms, as we demonstrate using a control-based ontology that classifies gene function according to roles such as 'sensor,' 'regulator,' and 'actuator.' Moreover, we show that combinations of the modules predict phenotype, yet surprisingly do not necessarily correlate with phylogenetic inheritance. The architectures of these three pathways are therefore emblematic of different modes and constraints on evolution. chemotaxis | competence | module | regulatory | sporulation

Sleep deprivation (SD) can suppress cell proliferation in the hippocampal dentate gyrus of adult male rodents, suggesting that sleep may contribute to hippocampal functions by promoting neurogenesis. However, suppression of cell proliferation in rats by the platform-over-water SD method has been attributed to elevated corticosterone (Cort), a potent inhibitor of cell proliferation and nonspecific correlate of this procedure. We report here results that do not support this conclusion. Intact and adrenalectomized (ADX) male rats were subjected to a 96-h SD using multiple- and single-platform methods. New cells were identified by immunoreactivity for 5-bromo-2'-deoxyuridine (BrdU) or Ki67 and new neurons by immunoreactivity for BrdU and doublecortin. EEG recordings confirmed a 95% deprivation of rapid eye movement (REM) sleep and a 40% decrease of non-REM sleep. Cell proliferation in the dentate gyrus was suppressed by up to 50% in sleep-deprived rats relative to apparatus control or home cage control rats. This effect was also observed in ADX rats receiving continuous low-dose Cort replacement via subcutaneous minipumps but not in ADX rats receiving Cort replacement via drinking water. In these latter rats, Cort intake via water was reduced by 60% during SD; upregulation of cell proliferation by reduced Cort intake may obscure inhibitory effects of sleep loss on cell proliferation. SD had no effect on the percentage of new cells expressing a neuronal phenotype. These results demonstrate that the Cort replacement method is critical for detecting an effect of SD on cell proliferation and support a significant role for sleep in adult neurogenesis. cell proliferation; hippocampus; rapid eye movement sleep; stress; corticosterone

Approximately one-third of patients with stress urinary incontinence (SUI) also suffer from urgency incontinence, which is one of the major symptoms of overactive bladder (OAB) syndrome. Pudendal nerve injury has been recognized as a possible cause for both SUI and OAB. Therefore, we investigated the effects of pudendal nerve ligation (PNL) on bladder function and urinary continence in female Sprague-Dawley rats. Conscious cystometry with or without capsaicin pretreatment (125 mg/kg sc), leak point pressures (LPPs), contractile responses of bladder muscle strips to carbachol or phenylephrine, and levels of nerve growth factor (NGF) protein and mRNA in the bladder were compared in sham and PNL rats 4 wk after the injury. Urinary frequency detected by a reduction in intercontraction intervals and voided volume was observed in PNL rats compared with sham rats, but it was not seen in PNL rats with capsaicin pretreatment that desensitizes C-fiber-afferent pathways. LPPs in PNL rats were significantly decreased compared with sham rats. The contractile responses of detrusor muscle strips to phenylephrine, but not to carbachol, were significantly increased in PNL rats. The levels of NGF protein and mRNA in the bladder of PNL rats were significantly increased compared with sham rats. These results suggest that pudendal nerve neuropathy induced by PNL may be one of the potential risk factors for OAB, as well as SUI. Somato-visceral cross sensitization between somatic (pudendal) and visceral (bladder) sensory pathways that increases NGF expression and [[alpha].sub.1]-adrenoceptor-mediated contractility in the bladder may be involved in this pathophysiological mechanism. nerve growth factor; cross-talk sensitization; [[alpha].sub.1]-adrenoceptor; capsaicin

Previous studies report greater postexercise heat loss responses during active recovery relative to inactive recovery despite similar core temperatures between conditions. Differences have been ascribed to nonthermal factors influencing heat loss response control since elevations in metabolism during active recovery are assumed to be insufficient to change core temperature and modify heat loss responses. However, from a heat balance perspective, different rates of total heat loss with corresponding rates of metabolism are possible at any core temperature. Seven male volunteers cycled at 75% of V[O.sub.2peak] in the Snellen whole body air calorimeter regulated at 25.0[degrees]C, 30% relative humidity (RH), for 15 min followed by 30 min of active (AR) or inactive (IR) recovery. Relative to IR, a greater rate of metabolic heat production (M - W) during AR was paralleled by a greater rate of total heat loss (HE) and a greater local sweat rate, despite similar esophageal temperatures between conditions. At end-recovery, rate of body heat storage, that is, [(M - W) - [H.sub.L]] approached zero similarly in both conditions, with M - W and [H.sub.L] elevated during AR by 91 [+ or -] 26 W and 93 [+ or -] 25 W, respectively. Despite a higher M - W during AR, change in body heat content from calorimetry was similar between conditions due to a slower relative decrease in IlL during AR, suggesting an influence of nonthermal factors. In conclusion, different levels of heat loss are possible at similar core temperatures during recovery modes of different metabolic rates. Evidence for nonthermal influences upon heat loss responses must therefore be sought after accounting for differences in heat production. body heat storage; calorimetry; heat production: heat stress: nontherreal factors; thermoregulation

Endothelial function is modulated by wall shear stress acting on the vessel wall, which is determined by fluid velocity and the local viscosity near the vessel wall. Red blood cell (RBC) aggregation may affect the local viscosity by favoring axial migration. The aim of this study was to investigate the role of RBC aggregation, with or without altered plasma viscosity, in the mechanically induced nitric oxide (NO)-related mechanisms of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured on the inner surface of cylindrical glass capillaries that were perfused with RBC suspensions having normal and increased aggregation at a nominal shear stress of 15 dyn/[cm.sup.2]. RBC aggregation was enhanced by two different approaches: 1) poloxamer-coated RBC suspended in normal, autologous plasma, resulting in enhanced aggregation but unchanged plasma viscosity and 2) normal RBC suspended in autologous plasma containing 0.5% dextran (tool mass 500 kDa), with a similar level of RBC aggregation but higher plasma viscosity. Compared with normal cells in unmodified plasma, perfusion with suspensions of poloxamer-coated RBC in normal plasma resulted in decreased levels of NO metabolites and serine 1177 phosphorylation of endothelial nitric oxide synthase (eNOS). Perfusion with normal RBC in plasma containing dextran resulted in a NO level that remained elevated, whereas only a modest decrease of phosphorylated eNOS level was observed. The results of this study suggest that increases of RBC aggregation tendency affect endothelial cell functions by altering local blood composition, especially if the alterations of RBC aggregation are due to modified cellular properties and not to plasma composition changes. wall shear stress; human umbilical vein endothelial cells; nitric oxide; endothelial nitric oxide synthase; plasma viscosity

Shear stress is a pathophysiologically relevant mechanical signal in cartilage biology and tissue engineering. Cyclooxygenase-2 (COX-2) is a pivotal proinflammatory enzyme, which is induced by mechanical loading-derived shear stress in chondrocytes. In the present study, we investigated the transcriptional machinery and signaling pathway regulating shear-induced COX-2 expression in human chondrocytic cells. Deletion and mutation analyses of the human cox-2 promoter reveal that the CCAAT/enhancer-binding protein (C/EBP) and activator protein-1 (AP-1) predominantly contribute to the shear-induced cox-2 promoter activity. Supershift assays disclose that C/EBP[beta], but not C/EBP[alpha] or C/EBP[delta], binds to the C/EBP site, whereas c-Jun binds to AP-1. Individual gene knockdown experiments demonstrate the direct regulation of C/EBP[beta] expression by c-Jun, and the critical roles of both c-Jun and C/EBP[beta] in shear-induced COX-2 synthesis. Our studies also indicate that Rac and, to a lesser extent, Cdc42 transactivate MEKK1, which is, in turn, responsible for activation of mitogen-activated protein kinase kinase 7 (MKK7). MKK7 regulates c-Jun N[H.sub.2]-terminal kinase 2 activation, which, in turn, triggers the phosphorylation of c-Jun that controls shear-mediated COX-2 upregulation in chondrocytes. Reconstructing the signaling network regulating shear-induced COX-2 expression and inflammation may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic interventions for arthritic disorders. mechanobiology; signal transduction; shear stress; cyclooxygenase; enhancer-binding protein; mitogen-activated protein kinase kinase; c-Jun N[H.sub.2]-terminal kinase

Restraint stress induces permeability changes in the small intestine, but little is known about the role of tumor necrosis factor (TNF)-[alpha] in the defects of the TJ function. In the present study, we used tumor necrosis factor-R1 knockout mice (TNF-[alpha]-R1KO) to understand the roles of TNF-[alpha] on ileum altered permeability function in models of immobilization stress. The genetic TNF-[alpha] inhibition significantly reduced the degree of 1) TNF-[alpha] production in ileum tissues; 2) the alteration of zonula occludens-I (ZO-1), claudin-2, claudin-4, claudin-5, and [beta]-catenin (immunohistochemistry); and 3) apoptosis (TUNEL staining, Bax, Bc1-2 expression). Taken together, our results demonstrate that inhibition of TNF-[alpha] reduces the tight junction permeability in the ileum tissues associated with immobilization stress, suggesting a possible role of TNF-[alpha] on ileum barrier dysfunction. apoptosis; TNF-[alpha]-deficient mice; zonula occludens-1; claudin-2; [beta]-catenin; tight junction

Endothelial hyperpermeability is regulated by a myosin light chain-2 (MLC2) phosphorylation-dependent contractile mechanism. Thrombin is a potent inducer of hyperpermeability of cultured monolayers of endothelial cells (ECs) via Rho kinase-mediated MLC2-phosphorylation. The aim of the present study was to investigate the effects of thrombin on in situ endothelial morphology and barrier integrity. Cytoskeletal dynamics, regions of paracellular flux, and MLC2-phosphorylation of ECs were visualized by digital three-dimensional imaging microscopy of pressurized rat kidney arterioles. Myosin phosphatase targeting subunit (MYPT 1)-phosphorylation was used as a surrogate marker for Rho kinase activity. Thrombin induced the formation of F-actin filaments in ECs in situ and rounding of the ECs in the absence of obvious formation of gaps between ECs. These changes were accompanied by an increase in MLC2 phosphorylation and a decrease in barrier integrity. In vitro analysis revealed that Rho kinase activity on F-actin filaments was associated with a contractile response that enhanced opening of the barrier. Rho kinase activity was not detectable on F-actin filaments induced by histamine, an inducer of a more transient hyperpermeability response. Inhibition of the myosin phosphatase mimicked the effects of thrombin on barrier function. The thrombin-induced changes in in situ MLC2 phosphorylation and barrier function were Rho kinase dependent. These data demonstrate a direct effect of thrombin on EC morphology and barrier integrity in intact microvessels. Furthermore, they establish an important contribution of enhanced Rho kinase activity to the development of prolonged but not transient types of endothelial barrier dysfunction. vascular biology; three-dimensional digital imaging; cytoskeleton; stress fibers

Linear enamel hypoplasia (LEH) is an enamel defect that records the effects of physiological stress on tooth formation. Estimating the age at which LEH defects form is integral to the reconstruction of population health in bioarcheological studies. Two principal methods for aging LEH defects have been introduced in the literature. The conventional approach employs regression equations based on a linear model of tooth growth. The newer, Reid and Dean [Am J Phys Anthropol 113 (2000) 135-139] approach, is based upon a histologically derived curvilinear model of enamel development and therefore likely provides more accurate age estimates. However, the extent to which the Reid and Dean method produces estimated ages at defect formation differing from those of the regression equations has not, until now, been determined. This study quantifies the differences between these two methods. Evaluating the degree to which these methods differ is essential for interpreting the accuracy of LEH age estimates given in previous bioarcheological studies. Age estimates of LEH defects on 338 anterior teeth from the Hamann--Todd osteological sample were calculated using both methods. The resulting estimated ages were compared through a randomized block ANOVA. However, the mean differences between the estimated ages yielded by both methods range from 4 months or less depending on the tooth type with an overall average of 2.63 months. The discussion focuses on the degree to which this difference affects answers to bioarcheological questions. KEY WORDS enamel defects; developmental stress; crown height; age estimation