3A_01_S

An Integrated View of the Stress Response and Stress-related Behavioral and /or Somatic Disorders

George P. Chrousos

Athens University Medical School, Athens, Greece

Stress activates the central and peripheral components of the stress system, i.e., the hypothalamic-pituitary-adrenal (HPA) axis and the arousal/sympathetic system. The principal effectors of the stress system are corticotropin-releasing hormone (CRH), arginine vasopressin, the proopiomelanocortin-derived peptides alpha -melanocyte- stimulating hormone and beta-endorphin, the glucocorticoids, and the catecholamines norepinephrine and epinephrine. The developing brain undergoes rapid growth and is characterized by high turnover of neuronal connections during the prenatal and early extrauterine life. These processes and, hence, brain plasticity, slow down during childhood and puberty and plateau in young adulthood. Hormonal actions in early life, and to a much lesser extent later, can be organizational, i.e., can have effects that last for long periods of time, frequently for the entire life of the individual. Hormones of the stress system and sex steroids have such effects, which influence the behavior and certain physiologic functions of individuals for life. Exposure of the developing brain to severe and/or prolonged stress may result in hyperactivity/hyperreactivity of the stress system, with resultant amygdala hyperfunction (fear reaction), decreased activity of the hippocampus (defective glucocorticoid negative feedback, cognition) and the mesocorticolimbic dopaminergic system (dysthymia, novelty seeking, addictive behaviors), hyperactivation of the HPA axis (hypercortisolism), suppression of reproductive, growth, thyroid and immune functions, and changes in pain and fatigue perception. These changes may be accompanied by disturbed childhood, adolescent and adult behaviors, including excessive fear (“inhibited child syndrome”) and addictive behaviors, dysthymia and/or depression and gradual development of components of the metabolic syndrome X, including visceral obesity, diabetes mellitus type 2 and essential hypertension. Prenatal stress exerted during the period of sexual differentiation may be accompanied by impairment of this process, with behavioral and/or somatic sequelae. The vulnerability of individuals to develop varying degrees and/or components of the above life-long syndrome is defined by as yet unidentified genetic factors, which account for up to 60% of the variance. CRH has marked kindling and glucocorticoids have strong consolidating properties, hence both of these hormones are crucial in the development of, and can each alone produce, the above syndrome. CRH and glucocorticoids may act in synergy, as in acoustic startle, while glucocorticoids may suppress or stimulate CRH, as in the hypothalamus and amygdala respectively. A CRH receptor type 1 antagonist antalarmin inhibits both the development and expression of conditioned fear in rats and has anxiolytic properties in monkeys. Profound stressors, such as those from sexual abuse, rape, etc., may elicit components of post-stress the syndrome in older children, adolescents and adults as well. Most frequently, chronic dysthymia and/or depression may ensue, associated with gastrointestinal complaints and/or the premenstrual tension syndrome. A lesser proportion of individuals may develop the classic posttraumatic stress disorder characterized by hypocortisolism and intrusive and avoidance symptoms; in younger individuals it may present as dissociative personality disorder.

3A_02_S

STRESS AND ADAPTATION: CHANGES IN THE SYMPATHOADRENAL SYSTEM ACTIVITY

Kvetnansky, R.

Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia

According to Hans Selye stress is an unspecific response of the organism to any demand made upon it. However, the specific activation of two components of the sympathoadrenal system (adrenomedullary and sympathoneural) by various stressors has been shown.

The aim of the present work was to investigate changes in enzymes involved in norepinephrine and epinephrine (EPI) synthesis - tyrosine hydroxylase (TH), dopamine-ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) – their gene expression, immunoprotein levels and activities in the adrenal medulla (AM), sympathetic ganglia, hearts and brains of rats and mice after a single or repeated exposure to various stressors. Immobilization for 2h (IMO), cold 4oC (COLD), administration of insulin 5IU (INS) or 2-deoxyglucose 500 mg/kg (2DG) were used. A single exposure to IMO, COLD, INS or 2DG was found to induce increases in TH, DBH and PNMT mRNA levels in the studied organs. Increased transcription rate is responsible for stress-induced TH and PNMT gene expression. Repeated exposure to these stressors elevated besides mRNA also activity and immunoprotein levels of the enzymes. Various stressors regulate TH, DBH and PNMT gene expression by different transcriptional mechanisms. PNMT gene expression is mainly regulated by HPA axis and in corticoliberine gene knock-out mice is basically reduced especially after stress exposure. One day cold exposure elevated TH mRNA levels in AM, however, 28 day cold exposure did not show any changes. Cold-adapted rats, however, responded to heterotypic novel stressors (IMO, INS, or 2DG) by exaggerated responses.

Thus, our data suggest an adaptation of TH and PNMT gene expression during long-term exposure to stressors. An exposure of adapted rats to novel stressors induces exaggerated responses. It is the readiness of the long-term stressed organism to overrespond, already at the level of expression of genes, to the changed quality of stressor, that we consider as an important adaptive phenomenon of the sympathoadrenal system.

Supported by Slovak APVV Grant 0148-06 and VEGA Grant 2-5125.

3A_03_S

CHOLINESTERASE MODULATIONS OF MAMMALIAN STRESS AND ANXIETY REACTIONS

Hermona Soreq, PhD

The Hebrew University of Jerusalem; Safra Campus-Givat Ram; Jerusalem 91904; Israel

Stress and anxiety disorders present a major mental health problem, but their putative involvement in the initiation and/or progression of neurodegenerative diseases is being debated. Recent research in the lab focuses on the molecular mechanism(s) underlying anxiety-induced changes in cholinergic neurotransmission. These mechanisms modulate the motor control over movement, regulate working memory, and activate brain-to-body communication through the neuron-immune interface modifying blood cells composition and platelet production. Importantly, stress-associated changes were found in the expression pattern of the acetylcholinesterase ACHE gene, which encodes the acetylcholine hydrolyzing enzyme AChE. AChE is not one, but a combinatorial series of proteins having indistinguishable enzymatic activity yet with variant N- and C-termini due to alternate promoter usage and 3'-alternative splicing. Differentially induced under stress, they show distinct non-hydrolytic properties, interact with variant-specific protein partners and induce inverse signaling cascades. Surprisingly, transcriptional and post-transcriptional regulation of AChE pre-mRNA not only protects blood and nerve cells from acute dangers, but may also entail long-term advantages. Specifically, causal involvement of both AChE and its closely related enzyme butyrylcholinesterase (BChE) in the progression of Alzheimer's and Parkinson's diseases, anticipates future therapeutic needs for drugs targeting specific cholinesterases or the corresponding RNA transcripts.

3A_04_S

Keeping All Together in a Fragmented World: The evidence base of the salutogenic research

Monica Eriksson and Bengt Lindström

Folkhälsan Research Center, Health Promotion Research Program, Paasikivigatan 4, FIN-00250 Helsinki, Finland

This conference focuses on linking knowledge from different areas of stress research particularly exploring the system level. Thus trying to make sense of our fragmented world. Social trends point to a major upset of the traditional social structures such as the rupture of local and intimate networks because of migration into urban areas, changed function and structure of family networks, changes in the patterns of working life and communication and information technology. At the same time there are concerns regarding increasing violence, alienation, a decreasing physical and mental health. All this makes it difficult to find and run a coherent life. Besides the research of Selye there are other theories and frameworks contributing to stress research. One of them is the salutogenic theory developed by Aaron Antonovsky in the late 1970s. This presentation focuses on the salutogenic approach such as Aaron Antonovsky formulated his salutogenic theory “Sense of Coherence” (SOC) as a global life orientation to view the world as comprehensible, manageable and meaningful. He claimed that the way people view their life has a positive influence on their health.[] The salutogenesis has become an established concept and considered as a paradigm shift from the pathogenic focus on risk factors for disease to the salutogenic focus on the strengths and determinants for health. How do people manage the lack of control of the life? The answer was formulated in terms of SOC and General Resistance Resources. The SOC is a resource that enables people to manage tension, to reflect about their external and internal resources (GRRs), to identify and mobilize them, to promote effective coping by finding solutions and resolve tension in a health promoting manner.

The findings from an ongoing review of the salutogenic research 1992-2003 by the authors is presented.[] The review consists of 458 scientific articles and 13 doctoral theses. To date the SOC questionnaire has been used in at least 33 languages in 32 countries all over the World. The SOC scale seems to be a reliable, valid, and cross culturally applicable instrument measuring health. SOC tends to increase with age. SOC is strongly related to perceived good health, especially mental health, and QoL. The stronger the SOC the better the perceived health in general. SOC seems to have a main, moderating or mediating role in the explanation of health. It seems to be able to predict health and quality of life. The applicability of the SOC concept in practice on both an individual, a group and on a societal level is discussed. With the evidence base of the salutogenic research on hands the conclusion is that the Salutogenesis is a valuable approach for individuals and groups in order to build capacity and coherence and healthy societies.

3A_05_S

Glycobiology of Stress

Olga Gornik and Gordan Lauc

1University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia. glauc@pharma.hr

Psychological stress is associated with numerous diseases, but molecular mechanisms linking stress to the development of disease are only starting to be understood. Stress alert is conveyed by hormonal signals throughout the body, yet a particular cell response to a hormonal signal is not determined by the signal itself, but by the molecular composition, energy content, and by the physiological role and current status of the target cell. Stress induced changes in glycoconjugate structures and expression of their receptors lectins appear to be an important molecular consequences of stress experience. At the moment only several fragments of the glycobiological mechanisms involved in the physiological response to psychological stress are known, but the complete picture is slowly emerging. Corticosteroids affect activity of at least one glycosyltransferase both in vitro and in vivo. Altered activity of glycosyltransferases results in different carbohydrate structures attached to glycoproteins, and these changes have been demonstrated both in humans and in experimental animals. A change in the carbohydrate structures attached to a glycoprotein is a well-established way to change its structural and functional properties, and recently this was shown to be one of the mechanisms that control activity of membrane receptors. Although this type of glycosylation-mediated receptor modulation in stress still has to be proven, it is a very interesting hypothesis. On the other hand, new glycoconjugate structures could also represent novel signals on the cell surface that could alter interaction of the cell with neighboring cells. Stress is also known to be associated with the appearance of novel lectins that could be receptors for either novel, or also “normal” glycoconjugate structures, translating their structures into molecular functions. Although most of this is still largely speculative, hopefully more will be known soon about the molecular role of glycoconjugates, their lectin receptors, and glycosyltransferases in the physiological response to psychological stress.

Module 3 – Poster lectures:

SEVERE STRESS AND ITS IMPLICATIONS: THE NEED TO RECOGNISE CONTRA-VITAL PATHOPHYSIOLOGICAL RESPONSES

C.P. Arun

Kellogg College, University of Oxford. Email: arunpeter@yahoo.com

INTRODUCTION For animals in the wild, predation, not old age is the most common mode of death. Nature appears to provide for the almost inevitable event of death from some form of trauma by allowing a quick final exit. REVIEW OF LITERATURE Our previous work (Arun, 2004) presented a contrarian view of pathophysiology, asserting that the catecholamine response (that lasts for only 24 hours) in acute stress plays a deciding role in the survival of the animal exposed to severe stress and could actually hasten death if the situation is hopeless. HYPOTHESIS We propose the use of the term ‘contra-vital’(= deleterious to life) to qualify actions taken by an organism’s organ systems to accelerate its own death. Our view is at variance with contemporary scientific thinking that reflexes exist only to promote the organism’s survival. From the point of view of the individual animal, initiation of the contra-vital response is beneficial when death is inevitable since it would diminish suffering. From a sociobiology standpoint, a quick death of a severely injured member is advantageous to the herd since rather than a futile wait for an irrecoverably injured animal to recover, the herd can move on, accepting the animal’s death as a fact. DISCUSSION The systemic response to sepsis appears to incorporate a contra-vital reflex and this will be elaborated in this paper. The recent worldwide initiative ‘Surviving Sepsis’ campaign is a tacit recognition of the existence of contra-vital responses. CONCLUSIONS Physiological systems appear to have a self-destruct mechanism available to accept defeat in the face of insuperable stress. There is a need for the scientific community to mature in its thinking, fight the taboo of studying death and recognize that contra-vital reflexes are a reality in severe stress.

These decreased responses would also appear to have a major effect on longevity.

3B_01_P

Psychophysiological effects of Neurexan^® on stress-induced electropsychograms. A double blind, randomized, placebo-controlled study in human volunteers

Wilfried Dimpfel

Justus Liebig University Giessen c/o NeuroCode AG, D 35578 Wetzlar, Sportparkstr. 9, Germany, w.dimpfel@neurocode-ag.com

Objective. There is evidence for the effectiveness of natural drugs in treatment of anxiety. However, there is a need for further prospective, double blind, randomized, placebo controlled clinical studies which demonstrate efficacy.

Methods. A preparation containing (amongst others) low dose extracts of passiflora incarnata and avena sativa (Neurexan^®) was tested in 30 healthy human volunteers in a randomised, placebo controlled cross-over trial. Eligible subjects aged 30 – 60 years were assessed under conditions of relaxation and under experimental stress-induction (calculation of mathematical tasks with financial reward or punishment according to performance). After recording of the electropsychogram (electrical activity in the presence of emotional provocations) frequency analysis using Fast Fourier Transformation and analysis of source density was performed 1 to 4 hours after administration of 4 tablets verum or placebo. Task dependent increase of beta 2 power was used as a surrogate parameter of a stress induced anxiety. The same experimental procedure was repeated for each subject on the ‘crossed-over’ medication after an interval of at least one week.

Results. In the presence of verum, circadian enhancement of alpha 2 waves was blunted. Beta 2 power was statistically significantly lower during the second and third hour after administration in comparison to placebo. Values returned to baseline values 5 hours after administration. The preparation was very well tolerated.

Conclusion. Recording of electrical activity during a stress-related emotional situation revealed a smaller increase of beta 2 power under verum compared to placebo. This is indicative of better coping with task related stress whilst on Neurexan^®.

3B_03_P

Second-Hand Stress: Neurobiological Evidence for a Human Alarm Pheromone

LR Mujica-Parodi, Helmut H Strey, Blaise Frederick, Robert Savoy, David Cox, Bosky Ravindranath, Yevgeny Botanov

Laboratory for the Study of Emotion and Cognition, Departments of Biomedical Engineering and Psychiatry,Stony Brook University School of Medicine, Health Sciences Center T18, Stony Brook, NY 11794-8181 USA

Pheromones function as airborne chemical signals released by an individual into the environment that, even in the absence of conscious perception, affect the physiology or behavior of other members of the same species. While almost all clinical research in the area has concentrated on human reproductive pheromones, non-human mammals are known to also possess alarm pheromones, which rapidly transmit warning of danger to others of the same species via olfaction. The existence of alarm pheromones is well-established in mammals, with animals exposed to odors secreted by acutely stressed conspecifics expressing physiological and behavioral changes that are indistinguishable from their reactions to predators, including increased neural activity in the amygdala, analgesia, increased hiding and avoidance, freezing and rearing, hyperthermia, and air-sampling. Using functional MRI, we demonstrate here the first direct evidence for a human alarm pheromone, with humans showing brain activation of the amygdala and hypothalamus—the primary brain regions responsible for the fear response—during inhalation of sweat taken from a sample of first-time skydivers, with exercise sweat as a control. The fMRI subjects were unable to identify the samples, and rated the odors for both conditions as mild and non-aversive. The results were found for breathing the fear sweat, but not sniffing it, most likely reflecting the fact that the neural circuitry associated with sniffing is explicitly tied to conscious odor detection, while pheromones are hypothesized to operate unconsciously. Both males and females showed equivalent neural activation, thereby ruling out the influence of sexual pheromones in accounting for our results. Our findings indicate that there may be a hidden biological component to human social dynamics, in which emotional stress is, quite literally, “contagious.”

3B_04_P

Over-expression of the Estrogen Receptor Beta Associated Protein HSP27 is Atheroprotective

Recently, we discovered that HSP27 is an estrogen receptor beta associated protein that represses estrogen mediated transcription (Miller et al, ATVB 2005). Moreover, in human coronary arteries the expression of HSP27 decreases with the progression of atherosclerotic disease stage and serum levels are >3-fold higher in normal controls compared to patients with angiographic evidence of coronary artery disease. Purpose: To determine if HSP27 is protective against the development of atherosclerosis. Methods: Mice over-expressing human HSP27 (hHSP27) were crossbred with atherosclerosis-prone apoE null mice (apoE) to yield hHSP27apoE mice (n=9 female, 5 male) and compared to apoE mice (n=7 female, 6 male) for aortic lesion development. All mice were fed a cholesterol supplemented diet for 4 weeks and euthanized at age 10 weeks. Results: All mice were viable, of similar body weight and had equally elevated cholesterol levels (approximately 1100 mg/dL). Quantitative histomorphological studies of en face aortic specimens revealed that the percentage aortic area with overt atherosclerosis was similar in both groups of male mice. However, amongst female mice there was a 41% reduction in atherosclerotic lesion area in the hHSP27apoE vs. apoE mice (p<0.001). Moreover, there was a decrease in the degree of vessel wall lipid deposition and inflammation in the hHSP27apoE vs. apoE mice. Conclusions: Over-expression of HSP27 is associated with attenuated atherogenesis but appears to occur only in female mice – thereby suggesting that ovarian hormones may be instrumental in mediating this effect. Studies focusing on the precise mechanisms of the HSP27 atheroprotective effect including the interplay with estrogen receptor modulation are ongoing.

3B_05_P

Characterization of autonomic nervous system (ANS) stability and variations by the ANS spot

Markku Paloheimo, MD, PhD

Eye Hospital, Helsinki University Hospital, Box 220, 00029 HUS, Finland, and GE Medical, Research, Helsinki, Finland, markku.paloheimo@hus.fi

The autonomic nervous system enables all of our body systems to operate in an external environment that is both physically and emotionally challenging. A Google search using words “stress autonomic nervous system” yielded 43,500 hits. Among the tests of parasympathetic cardiovagal regulation include heart rate analysis, heart rate variation with deep breathing, variation of beat-to-beat blood pressure and heart rate (HR), and the Valsalva ratio. Tests of sympathetic adrenergic vascular regulation include blood pressure analysis in various situations, the Valsalva manoeuvre, sustained handgrip, mental stress, and cold water immersion. Tests of sympathetic cholinergic sudomotor function include the sympathetic skin response, quantitative sudomotor axon reflex test, sweat box testing, and quantification of sweat imprints.

We have developed a method to display neural or hormonal ß-adrenergic responses of heart rate and peripheral cholinergic neural vascular responses of finger pulse amplitude to characterize the autonomic state and variations during general anaesthesia and sedation. This was done by displaying pulse-to-pulse intervals (in ms) on the X-axis and subsequent plethysmographic pulse wave amplitudes on the Y-axis (see Figures). The data is obtained from Datex-Ohmeda monitors via serial interface and the source of the plethysmographic signal is a common pulse oximetry sensor on the subject’s finger. The number of traced observations can be set (Number of points). Six most recent data is shown as red dots and the older ones in white, which forms a trend. Low sympathetic activity is characterized by high pulse amplitudes and low heart rates (long intervals). Changes in autonomic activity is shown as a movement of the spot; vagal activation is seen as pulse rate slowing and increase in sympathetic activity is heralded by a decrease in pulse plethysmographic amplitude and increase in heart rate. During anticholinergic and ß-blocking medication, heart rate variability is decreased, but these drugs do not affect pulse amplitude variability.

This noninvasive method has been used to follow autonomic state and responses during general anaesthesia, conscious sedation, intensive care and psychological testing. A Surgical Stress Index (see Google) displayed on a scale of 0 -100 is under development.

3B_08_P

O-GlcNAc, A New Paradigm for Regulating Stress-Signaling Networks

Natasha E. Zachara¹, Henrik Molina^1,2, Akilesh Pandey^1,2, Gerald W. Hart¹.

1. Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21218.

2. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21287.

nzachara@jhmi.edu

In response to numerous forms of cellular stress, levels of the O-GlcNAc protein modification are elevated rapidly and dynamically on myriad nucleocytoplasmic proteins. Increased O-GlcNAc levels are linked to stress tolerance in both cell culture and animal models, suggesting that this phenomenon is a survival response of cells. Elevated levels of O-GlcNAc have been linked to changes in Hsp70 and Hsp40 protein levels, as well as altered capacitive calcium entry, suggesting at least two mechanisms by which O-GlcNAc may protect cells. These, and other data, suggest a new paradigm in the regulation of stress mediated signal transduction pathways, further investigation of which will elucidate new roles for O-GlcNAc in diverse clinical and cellular settings such as Ischemia-reperfusion injury, neurodegenerative diseases and aging. In order to better understand the mechanism(s) by which O-GlcNAc modulates the cellular response to stress, we have determined which proteins are dynamically O-GlcNAc modified in response to cellular stress using Stable Isotope Labeling with Amino Acids in Cell Culture. We have identified 27 O-GlcNAc modified proteins, of which 19 exhibited an increase in O-GlcNAc protein modification in response to heat shock. These proteins are predominantly involved in signal transduction, transcription and vesicle transport. Currently, we are examining the role of glycosylation on these proteins, and determining how this contributes to O-GlcNAc mediated stress tolerance. Under a licensing agreement between Covance and Johns Hopkins University, GWH receives a share of royalties on sales of CTD110.6.

3B_09_P

Chromosome aberrations, DNA reparation, micronuclei, proliferation and apoptosis in lymphocytes’ cultures of human under emotional stress

F.I.Ingel

A.N.Sysin State Research Institute for Human Ecology and Environmental Hygiene of Russian Academy of Medical Sciences, Moscow, Russia, faina_ingel@uk2.net

Our experiments on mice (Ingel et al, 1993 - 1999) demonstrated that emotional stress (immobilization) induced chromosome aberrations in bone marrow and thymus in common dynamics during 25 days, and, additionally, increased susceptibility of genome of these cells to standard mutagen cyclophosphamide. So we supposed that the same effects may be induced with human emotional stress in stage of disadaptation. Then 303 adults and 248 children from different industrial towns of Russia and settlements in Kazakhstan were tested for emotional stress expression (Cholms-Ray’, Taylor’, Akkles’ scales – for adults and Luscher’ test – for children) in parallel with medical and biochemical (blood, urine) studies, PCR analysis (GSTM1, GSTT1, CYP1A1, CYP1A2, PON 54) and cytogenetical investigations of peripheral blood cell in cultures (without and with additional load of mutagen in vitro). Results demonstrated that level of stress expression positively correlated with: a) industrial contacts with toxic and mutagenic compounds or residing in zone of ecological disaster (Aral Sea Basin) as well as blood contamination (dioxin, r = 0,75-0,87; p?0,001); b) frequencies of chromosome aberrations and micronuclei in cultivated blood lymphocytes (r = 0,74-0,78; p?0,001); c) coefficient of UV-induced DNA reparation (r = 0,86; p?0,00001); d) radiosensitivity of cells in culture (r=0,081; p=0,001); e) proliferation  of cells in cultures of peripheral blood  (r = 0,75; p=0,000001); f) speed and symmetry of cellular division (r=0,56 – 0,62; p= 0,0001); g) apoptosis of cells in culture of adults (r = 0,45; p=0,002). Negative correlation between stress expression and apoptosis of lymphocytes in culture was detected for children (r=-0,42; p=0,003). Additionally, disadaptative stress expression was significantly associated with mutation in tested genes of detoxification of xenobiotics. Conclusions: the most of results for human and animals were identical - emotional stress induced mutations and increased susceptibility of genome to environmental mutagens. So, chronic emotional disadaptation shows the same cytogenetic properties as full carcinogen.  

3C_01_P

The impact of the yeast RAC-Ssb co-translational system of chaperones on the cellular activity of mutated proteins

Katarzyna Tomala, Ryszard Korona

Jagiellonian University, Institute of Environmental Sciences, address: Gronostajowa 7, 30-387 Kraków, Poland; email: tomala@eko.uj.edu.pl

A large fraction of spontaneous mutations lead to destabilization of protein structure. This results in exposure of hydrophobic patches on the protein surface. It is thus very likely that mutant proteins are more frequently bound by the molecular chaperones. The subsequent processing of excessively unstable proteins is poorly known. The more frequent binding by the chaperones may help to overcome the effect of mutation and secure functioning of the protein in the cell. Alternatively, the excessive activity of the chaperones concentrated on a defective polypeptide may constitute a signal that it has to be degraded. Our research was focused on the RAC-Ssb system of co-translational chaperones in yeast. We asked whether its role is to uphold folding efficiency of mutated proteins or rather sort them out and exclude from the cellular metabolism. We prepared a library of mutagenized ADE2 gene whose inactivity or malfunctioning results in red pigmentation of the yeast colonies. We then screened for mutations producing red color in strains in which RAC-Ssb was inactivated by gene deletion. No such synthetic phenotypes were found indicating that mutational destabilization of Ade2p is not aggravated in the absence of the chaperone activity. In a second screen, we first identified an array of mutationally destabilized variants of Ade2p proteins. This was marked by their thermosensitivity. In case of all mutants, their growth on media lacking adenine was better when the RAC-Ssb was inactivated. This indicates that the activity of chaperones leads to a decline of a cellular pool of active Ade2(ts) molecules. Current experiments show that the defective protein is present in the cell in form of aggregates. We suggest that the RAC-Ssb can have a role in sequestering defective proteins from the cytosol and restricting their uncontrolled interactions with other elements of the cellular metabolism.

3C_02_P

Molecular study of acid shock proteins in a diarrheagenic Enteroaggregative Escherichia coli (EAEC)

Puneet Badesha

EAEC is an emerging pathotype for persistent pediatric diarrhea characterized by an aggregative adherence (AA) pattern to HEp-2 cells. Its pathogenesis is poorly understood. Like other enterics, it faces acidic conditions in stomach and small intestine. Therefore, the adaptive strategies like acid tolerance responses (ATRs) are induced to ensure its survival during the disease progression. Outer membrane proteins (OMPs) are the key molecules that interface cell with the environment and are important in adhesion, invasion and intracellular survival of pathogens in the host. Most notable pH-response regulators like Fur, PhoP, OmpR and RpoS are reported and the rpoS, an alternative sigma factor is well known for the induction of ATRs in S. typhimurium and EHEC.

ATR in EAEC was studied by growing cells at variable pH range (2.0-7.4). Cell survival was observed till pH 4.0 but the preadapted cells (adapted at mild acidic pH 5.0 for 2 hours), survived two fold better than that of the non-adapted cells (pH 4.0). Phenotypic characterization under acid stress conditions was checked in vitro. At pH 4.0, clump formation was totally abolished. The biofilm score was <+1.0 (partial honeycomb formation) even at 12h of growth at pH 4.0 whereas in control it was +3 (three dimensional mounds with visible substratum). Diffusely adherent (DA) pattern with HEp-2 cells was observed at pH 4.0 in contrast to typical aggregative adherence (AA) in control. The overlapping of 2-D gels of OMPs (pH 7.4 vs. 4.0) and digitized PDQuest bioinformatic software analysis confirmed the six differentially expressed proteins at pH 4.0. The intensity of the main porins was lower at pH 4.0. The spots of interest (pH 4.0) were digested with trypsin and were analyzed by MALDI-TOF/MS, displayed a “peptide fingerprint” of the protein. Western blot analysis with stress specific antibodies further revealed ASPs in EAEC are differentially expressed, which might be the reason for variation in phenotypic characteristics. The expression of ASPs in rpoS mutant was absent (pH 4.0). Therefore, the putative ASPs associated with an acid-tolerant phenotype under rpoS, provides a new information regarding EAEC survival at low pH. Further, characterization of rpoS may reveal more specific targets aimed for the disease treatment.

3C_03_P

Systemic Analysis of Heat Shock and Proteasome Inhibition Using Proteomics and Functional Genomics

Hee-Jung Kim, Yung Hee Kim, Jaeho Jeong, Kong-Joo Lee

kjl@ewha.ac.kr

Heat shock response is well conserved adaptation phenomenon characterizing drastic repression of total cellular protein synthesis and induction of heat shock proteins (Hsps). Various extracellular stresses as well as heat shock induce heat shock response and render cells cross resistance to lethal stresses. Recently, denatured proteins have been suggested as the sensor of extracellular physical stresses. Cells have two clearance systems of the misfolded proteins, proteasomal and autophagic degradation. For the proteasomal degradation, misfolded protein are tagged by polyubiquitin and guided by proteasome shuttle chaperone CDC48/p97. MG132 is the inhibitor of proteasomal function, resulting in accumulation of the denatured proteins in cells. In accordance with the suggested possibility above, MG132 activates JNK2 mediated transcriptional activity of heat shock factor 1. In this study, we compared differentially expressed mRNAs and proteins between heat shock and MG132 treatment using DNA chip, fluorescence 2-D difference gel electrophoresis (DIGE), and ³⁵S labeling proteomics. These systemic analyses provide the information on the common and differential signaling pathways between proteasome inhibition and heat shock response. [Supported by KOSEF NCRC for CCS & DDR and FPR05A2-480. HJ Kim is supported by BK21]

3C_04_P

Local and systemic changes in protein expression following biotic stress in European beech (Fagus sylvatica)

Cristina-Maria Valcu and Katja Schlink

Technical University of Munich, Section of Forest Genetics, Am Hochanger 13, D-85354 Freising, email: valcu@wzw.tum.de

Pathogen and herbivore attack induced changes in protein expression patterns were investigated at proteome level in European beech (Fagus sylvatica) saplings. Two model systems were employed: (1) infection with the root pathogen Phytophthora citricola which induces root necrosis and plant wilting and (2) wounding as elicitor of the response to herbivore attack. The plant defence response was investigated at both local and systemic level. Protein expression patterns were characterised for root and leaf samples by means of two-dimensional electrophoresis at three hours after wounding and at different stages of root infection. Infection experiments were performed in two experimental setups involving in vitro infection of saplings as well as infections in soil. Within the first system we investigated both local and systemic response to pathogen infection while the latter experiments were used for the study of systemic response in conditions close to the natural ones. Wounding experiments were carried out in similar conditions in order to enable the direct comparison of plant reaction to the two types of stress. The local response to wounding was characterised both for roots and leaves of plants grown in liquid system, while the systemic response was studied in leaf samples from plants grown in liquid culture or in soil. Protein spots up-regulated or down-regulated in response to infection or wounding were further identified by means of mass spectrometry. We were able to identify several proteins specifically regulated in response to pathogen or herbivore attack. Additionally, a significant overlap of the two defence pathways known to exist in plants was confirmed.

A physiological and homology based proteomics characterization of salinity-induced glycolate metabolism in nitrogen-fixing Anabaena doliolum

Ashish Kumar Srivastava^1,2 and Lal Chand Rai²

2Molecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India

This study provides first hand physiological and proteomic information on the occurrence of glycolate metabolism in fresh water nitrogen fixing cyanobacterium Anabaena doliolum LCR1 under salinity. A reduction in growth, O₂-evolution, photosystem II, carbon fixation, chlorophyll and NADPH content and an increase in intracellular Na⁺, photosystem I, respiration and ATP content was observed after 150 mM NaCl treatment for 1 and 24h. In view of the alteration in the above mentioned physiological parameters, the proteomic study (using two dimensional gel electrophoresis and MALDI-TOF MS/MS) was done. Of the 201 protein spots in control only 90 sustained the high dose of salinity. Of these protein spots, six showed significant and reproducible alterations, which showed homology with Fe-SOD, SOD, phycocyanin alpha chain, EF-Tu, RuBisCO and phosphoribulokinase of Nostoc PCC7120. Thus the physiological and proteomics data jointly suggests that the organism is deriving carbon from a different source; one such source could be the glycolate metabolism. To examine the alternate route of carbon, phosphoglyceric acid and the intermediates of the glycolate pathway were measured. Evidence on the operation of this pathway came form the: (i) enhanced accumulation of phosphoglyceric acid and inhibited phosphoenolpyruvate carboxykinase activity, (ii) the increased activity of enzyme glycolate oxidase, and (iii) increase in the intermediates of the C2 glycolate cycle (photorespiration) i.e. glycine, serine and ammonia. In view of the production of ammonia, the possible generation of serine from PGA through non-photorespiratory pathway is ruled out. However, this pathway generates peroxide whose scavenging enzymes are sensitive to salt stress; therefore the organism fails to acclimatize to salt. Taking recourse to the above, occurrence of salt-induced glycolate metabolism (photorespiration) in A. doliolum is proposed.

Keywords: Anabaena doliolum, glycolate metabolism, MALDI-TOF MS/MS, NaCl, Nostoc PCC7120, two dimensional gel electrophoresis

Proteome analysis of human macrophages reveals up-regulation of apoptosis and cell stress associated proteins in response to influenza A virus infection

Tuula A. Nyman¹, Tiina Öhman¹, Johanna Rintahaka², Nisse Kalkkinen¹, and Sampsa Matikainen²

¹Protein Chemistry Research Group, Institute of Biotechnology, University of Helsinki and ²Unit of Excellence in Immunotoxicology, Finnish Institute of Occupational Health, Helsinki, Finland

Alveolar macrophages of the lung are the primary targets for respiratory viruses. Virus-encoded single-stranded and double-stranded RNA are recognized by host pattern recognition receptors. This ssRNA and dsRNA recognition leads to the changes in the host cell protein expression. Here we have studied the regulation of influenza A induced protein expression in human macrophages. Macrophages were infected with influenza A virus after which the cytosolic and mitochondrial cell fractions were prepared and analyzed by using two-dimensional electrophoresis for protein separation and mass spectrometry for protein identification. In mitochondrial proteomes expression of actin was highly up-regulated upon Influenza A virus infection. Actin is known to associate with mitochondria at early phases of apoptosis and accordingly caspase-3, which is the effector caspase in apoptosis, was activated in infected macrophages. In cytosolic proteomes expression of several heat shock proteins (Hsp60, 70, and 90) and small calcium binding proteins (S-100A and S-100 Ca+ binding protein) was clearly up-regulated. In addition, expression of Rho GDP dissociation inhibitor as well as cathepsins B and D were clearly down-regulated upon Influenza A infection. In conclusion, our results show that the expression of several key proteins involved in apoptosis and stress response are regulated during influenza A virus infection of human primary macrophages.