Investigation of emission-transmission misalignment artifacts on rubidium-82 cardiac PET with adenosine pharmacologic stress.

PURPOSE: This study was undertaken to determine if artifacts from misalignment of cardiac emission to transmission data is present in adenosine stress studies and if the artifact could be reproduced by intentional misalignment in normal exams. PROCEDURES: Seventy consecutive 82Rb myocardial perfusion studies were reviewed. Utilizing a quality control program, misalignment was assessed. The study was reprocessed after manual realignment to determine if the defect extent changed. Emission and transmission acquisitions in six normal studies also were intentionally misaligned. RESULTS: Twenty of 69 rest studies (29.0%) and 17 of 69 (24.6%) stress studies demonstrated misalignment. In four patients with stress misalignment, there was a significant change in clinical interpretation. Upon intentionally misaligning six normal studies, a lateral wall defect was reproduced. CONCLUSIONS: Emission-transmission misalignment occurs in 29.0% and 24.6% of 82Rb rest and adenosine stress studies, respectively. While there is a positive correlation of artifactual defects with misalignment, the presence and size of artifacts is variable and unpredictable at seemingly lesser degrees of misalignment.

Preventive effect of a traditional herbal medicine, Hochu-ekki-to, on immunosuppression induced by surgical stress.

PURPOSE: To examine the effect of preoperative administering of a Japanese traditional herbal medicine, Hochu-ekki-to (TJ-41), on immunosuppression induced by surgical stress in patients with gastrointestinal malignancies. METHODS: To monitor the immune functions, the mitochondrial membrane potential (MMP) and natural killer (NK) cell activity prior to and following operation were measured in peripheral blood lymphocytes (PBL) in patients with (n = 20) or without (n = 27) the preoperative administering of TJ-41 for 7 days. The plasma catecholamine and interleukin (IL)-6 levels were also analyzed prior to and following the operation. RESULTS: The numbers of MMP-high CD56-positive cells (NK cells) and NK cell activities in the TJ-41-treated group were significantly higher than those in the control group (P = 0.026 and P = 0.037, respectively). An elevation of plasma noradrenaline and IL-6 following surgery was also inhibited by the preoperative administering of TJ-41 (P = 0.023 and P = 0.039, respectively). A positive correlation between MMP-high CD56-positive cell numbers and NK cell activity in PBL treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) in vitro suggested that MMP measurement in CD56-positive cells can serve as a convenient alternative to evaluate the NK cell activity. CONCLUSION: Our findings suggest that the preoperative administering of TJ-41 prevents surgical stress-induced immunosuppression by maintaining the NK cell activity and inhibiting the elevation of stress mediators.

Mitochondria are involved in stress response of A549 alveolar cells to halothane toxicity.

During inhalation anaesthesia lung epithelial cells are directly exposed to halogenated hydrocarbons such as halothane. Information about the effects of volatile anaesthetics on lung cells is rather limited although their noxious effect on the A549 alveolar cells has been shown recently. The present study indicated that halothane decreases cell viability, impairs DNA integrity and provokes stress-induced apoptosis in A549 cells when applied at clinically relevant concentrations. Data obtained clearly demonstrated intensive expression of anti-apoptotic Bcl-2 protein during treatment with all tested concentrations. In post-treatment periods the increased DNA injury was accompanied by reduction of Bcl-2 expression. We concluded that the in vitro effect of halothane on lung cells involved alteration in the expression of proteins of the mitochondrial apoptotic pathway.

Decreased epithelial barrier function evoked by exposure to metabolic stress and nonpathogenic E. coli is enhanced by TNF-alpha.

A defect in mitochondrial activity contributes to many diseases. We have shown that monolayers of the human colonic T84 epithelial cell line exposed to dinitrophenol (DNP, uncouples oxidative phosphorylation) and nonpathogenic Escherichia coli (E. coli) (strain HB101) display decreased barrier function. Here the impact of DNP on macrophage activity and the effect of TNF-alpha, DNP, and E. coli on epithelial permeability were assessed. DNP treatment of the human THP-1 macrophage cell line resulted in reduced ATP synthesis, and, although hyporesponsive to LPS, the metabolically stressed macrophages produced IL-1beta, IL-6, and TNF-alpha. Given the role of TNF-alpha in inflammatory bowel disease (IBD) and the association between increased permeability and IBD, recombinant TNF-alpha (10 ng/ml) was added to the DNP (0.1 mM) + E. coli (10(6) colony-forming units), and this resulted in a significantly greater loss of T84 epithelial barrier function than that elicited by DNP + E. coli. This increased epithelial permeability was not due to epithelial death, and the enhanced E. coli translocation was reduced by pharmacological inhibitors of NF-kappabeta signaling (pyrrolidine dithiocarbamate, NF-kappabeta essential modifier-binding peptide, BAY 11-7082, and the proteosome inhibitor, MG132). In contrast, the drop in transepithelial electrical resistance was unaffected by the inhibitors of NF-kappabeta. Thus, as an integrative model system, our findings support the induction of a positive feedback loop that can severely impair epithelial barrier function and, as such, could contribute to existing inflammation or trigger relapses in IBD. Thus metabolically stressed epithelia display increased permeability in the presence of viable nonpathogenic E. coli that is exaggerated by TNF-alpha released by activated immune cells, such as macrophages, that retain this ability even if they themselves are experiencing a degree of metabolic stress.

Involvement of mitochondria in endoplasmic reticulum stress-induced apoptotic cell death pathway triggered by the prion peptide PrP(106-126).

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrP(Sc). Recently, we have shown that PrP(106-126) induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress-mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP(106-126) on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 rho0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 rho+ cells. In this study, we show that PrP(106-126) induces ER stress in both cell lines, given that ER Ca2+ content is low, glucose-regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 rho+ cells, PrP(106-126)-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 rho+ from PrP(106-126)-induced cell death, demonstrating the involvement of Ca2+ release through ER ryanodine receptors. However, in PrP(106-126)-treated NT2 rho0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders.

Abeta 1-42 induces mild endoplasmic reticulum stress in an aggregation state-dependent manner.

Alzheimer's disease (AD) is characterized by the aggregation of misfolded proteins. Previously we reported activation of the unfolded protein response (UPR) in AD neurons. A potential source for UPR activation in AD neurons may be the increased levels of beta-amyloid (Abeta). In this study, we used preparations enriched in oligomeric or fibrillar Abeta (1-42) to investigate the role of the conformational state of Abeta in UPR activation in differentiated neuroblastoma cells. Both oligomeric and fibrillar Abeta (1-42) do not induce BiP expression to the extent that it can be detected in a pool of cells. However, using a fluorescent UPR reporter cell line that allows analysis of individual cells, we demonstrated mild activation of the UPR by oligomeric but not fibrillar Abeta (1-42). We showed that oligomeric Abeta (1-42) is significantly more toxic to cells primed for UPR than is fibrillar Abeta (1-42), indicating that activation of the UPR contributes to oligomer-specific Abeta (1-42) toxicity. Because UPR activation is observed in AD brain at a stage that precedes the massive fibrillar Abeta deposition and tangle formation, this may indicate a role for nonfibrillar Abeta in the induction of the UPR in AD neurons.

Prenatal exposure to a pro-inflammatory stimulus causes delays in the development of the innate immune response to LPS in the offspring.

Growing evidence suggests that maternal health during the prenatal period is a critical determinant of adult immuno-competence. This study aimed to characterise the innate immune response to bacterial exposure in rat offspring following maternal exposure to a pro-inflammatory stimulus. The offspring's innate immune responses were investigated at four developmental timepoints in the rat by determination of immune cell subtypes and TNF-alpha and IL-1beta response to in-vivo LPS exposure. The pre-weaned offspring of exposed dams demonstrated no immune response to the LPS challenge, whereas control offspring responded with a typical elevation in cytokine levels. In pubescence no differences were observed between the responses of the control and exposed offspring. In adulthood and senescence, offspring of endotoxin treated dams had significantly less monocytes in circulation than control offspring and differential sex effects were only evident in these older animals. The developmental profile of immune functioning following prenatal immune activation has not previously been demonstrated. This study highlights the prenatal period as one of importance in determining later immune function.

Grape polyphenols affect mRNA expression of PGHS-2, TIS11b and FOXO3 in endometrium of heifers under ACTH-induced stress.

Stress activates the hypothalamo-pituitary-adrenal axis leading to enhanced glucocorticoid secretion and concurrently disrupts ovarian cycle. Plant polyphenols are known to posses antioxidant and anti-inflammatory proprieties. This could be of interest for ovarian cycle when stressing conditions lead to progesterone enhancement and hamper normal reproduction activity. The present study examined whether ovarian follicular development and progesterone secretory pattern are affected by exogenous ACTH administration in heifers. Moreover, the effect of grape polyphenols in endometrium of heifers, under adrenocorticotropic hormone challenge, is evaluated in terms of transcriptional patterns of genes related to inflammation, oxidative stress and endometrial functions. At day 14 of synchronized estrous cycle, Holstein Friesian heifers received injections of either saline (CTR group) or adrenocorticotropic hormone (ACT group) agonist every 12 h for 7 days. Another group (POL group) of animals received the same treatment plus an oral supplementation of 15 g/day of grape skin extract. Cortisol and progesterone were analysed in the blood samples collected at days 0, 3, 6, 9, 12, 14, 17, 21, 24 of the estrous cycle. Endometrial biopsies were collected at diestrus (day 18) and at estrus and a panel of gene expressions were quantified by real-time PCR. ACTH administration increased both cortisol (P<0.001) and progesterone concentrations (P<0.01) compared to CTR group. PGHS-2 was significantly (P<0.01) up-regulated in the POL group compared to ACT and CTR groups at diestrus and at estrus. FOXO3 and TIS11b were down-regulated in the CTR group compared to ACT and POL groups. The PGHS-2, SOD2 (P<0.05), FOXO3 and TIS11b (P<0.10) genes were down-regulated at estrus in all groups compared to diestrus. An interesting role of polyphenols in modulating the expression levels of PGHS-2 in endometrial tissue and on the activation of TIS11b and SOD2 through c-AMP-dependent signalling was suggested.

Disruption of type 5 adenylyl cyclase enhances desensitization of cyclic adenosine monophosphate signal and increases Akt signal with chronic catecholamine stress.

BACKGROUND: Desensitization of the cyclic adenosine monophosphate signal protects cardiac myocytes against catecholamine stress, thus preventing the development of apoptosis. Molecular mechanisms of desensitization have been well studied at the level of adrenergic receptors but less so at the level of the effector enzyme, adenylyl cyclase (AC). METHODS AND RESULTS: When the effects of long-term (1 to 2 weeks) isoproterenol infusion were compared between type 5 AC-null mice (AC5KO) and wild-type controls, we found that the subsequent responses of left ventricular ejection fraction to sudden intravenous isoproterenol challenge were reduced in AC5KO compared with wild-type mice (ie, physiological desensitization was more effective in AC5KO), consistent with enhanced downregulation of AC catalytic activity in AC5KO. One mechanism for the less effective desensitization in wild-type mice was paradoxical upregulation of type 5 AC protein expression. The number of apoptotic myocytes was similar at baseline but was significantly less in AC5KO after infusion. This was accompanied by a 4-fold greater increase in Bcl-2 and a 3-fold greater increase in phospho-Akt in AC5KO. The latter is most likely mediated by increased membrane localization of phosphoinositide-dependent protein kinase 1, which is known to be inhibited by the cyclic adenosine monophosphate signal. CONCLUSIONS: The absence of type 5 AC results in more effective desensitization after long-term catecholamine stress and protects against the development of myocyte apoptosis and deterioration of cardiac function, potentially elucidating a novel approach to the therapy of heart failure.

111Indium-trastuzumab visualises myocardial human epidermal growth factor receptor 2 expression shortly after anthracycline treatment but not during heart failure: a clue to uncover the mechanisms of trastuzumab-related cardiotoxicity.

AIM: Trastuzumab can induce cardiotoxicity, particularly when combined with anthracyclines. Myocardial human epidermal growth factor receptor 2 (HER2) expression may be transiently upregulated by a compensatory mechanism following cardiac stress. 111In-DTPA-trastuzumab, scintigraphy can detect HER2 positive tumour lesions, however previously, we found myocardial uptake in only 1 of the 15 anthracycline-pre-treated patients with a median of 11 months after the last anthracycline administration. To evaluate whether myocardial HER2 expression is upregulated by anthracycline-induced cardiac stress or in case of heart failure by chronic pressure or volume overload, we performed 111In-DTPA-trastuzumab scans in patients shortly after anthracyclines and with non-anthracycline-related heart failure. METHODS: Patients within 3 weeks after undergoing 4-6 cycles first-line anthracycline-based chemotherapy and patients with heart failure due to cardiac disease underwent gammacamera imaging 48 and 96 h after 111In-DTPA-trastuzumab intravenously. RESULTS: Myocardial 111In-DTPA-trastuzumab uptake was observed in 5 out of 10 anthracycline-treated patients, who all were without symptomatic cardiac dysfunction. None of the 10 heart failure patients showed myocardial uptake. CONCLUSION: Shortly after completion of anthracycline treatment, myocardial HER2 over-expression was detectable in 50% of the patients. 111In-DTPA-trastuzumab scintigraphy after anthracyclines prior to adjuvant trastuzumab potentially identifies patients susceptible for trastuzumab-related cardiotoxicity and thus may facilitate the optimal timing of trastuzumab therapy.

[High-resolution MRI for the quantitative evaluation of subendocardial and subepicardial perfusion under pharmacological stress and at rest]. / Hochaufgelöste quantitative MR-tomografische Bestimmung der subendo- und subepimyokardialen Perfusion unter Stress und in Ruhe.

PURPOSE: MR stress perfusion imaging of the heart allows the quantification of myocardial perfusion and the evaluation of myocardial perfusion reserve (MPR) and the ratio of subendocardial to subepicardial perfusion at rest and under adenosine stress. The aim of this study was to evaluate a high-resolution GRAPPA sequence for quantitative MR first pass perfusion imaging in healthy volunteers. MATERIALS AND METHODS: First pass stress and rest perfusion studies were performed on 10 healthy volunteers using a 1.5 T MR scanner with a multislice SR-TrueFISP first pass perfusion sequence with a GRAPPA algorithm (acceleration factor 3) in prebolus technique and an image resolution of 1.8 x 1.8 mm. For the comparison group, we examined 12 different healthy volunteers with a standard first pass perfusion SR-TrueFISP sequence using a resolution of 2.7 x 3.3 mm. Myocardial contours were manually delineated followed by an automatic division of the myocardium into two rings with an equal thickness for the subendo- and subepicardial layer. Eight sectors per slice were evaluated using contamination and baseline correction. RESULTS: Using the GRAPPA sequence, the ratio of subendo- to subepimyocardial perfusion was 1.18 +/- 0.32 for the examination at rest. Under pharmacologically induced stress, the ratio was 1.08 +/- 0.27. For the standard sequence the ratio was 1.15 +/- 0.28 at rest and 1.11 +/- 0.33 under stress. For the high resolution sequence higher mean values for the subendo- to subepimyocardial ratio were obtained with comparable standard deviations. The difference between the sequences was not significant. CONCLUSION: The evaluation of subendomyocardial and subepimyocardial perfusion is feasible with a high-resolution first pass perfusion sequence. The use of a higher resolution to avoid systematic error leads to increased image noise. However, no relevant reduction in the quantitative perfusion values under stress and at rest was able to be depicted.

Carbon monoxide produced by heme oxygenase-1 in response to nitrosative stress induces expression of glutamate-cysteine ligase in PC12 cells via activation of phosphatidylinositol 3-kinase and Nrf2 signaling.

Induction of heme oxygenase-1 (HO-1) expression has been associated with adaptive cytoprotection against a wide array of toxic insults, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigated the potential role of carbon monoxide (CO), one of the by-products of the HO-1 reaction, in the adaptive survival response to peroxynitrite-induced PC12 cell death. Upon treatment of rat pheochromocytoma (PC12) cells with the peroxynitrite generator 3-morpholinosydnonimine hydrochloride (SIN-1), the cellular GSH level decreased initially, but was gradually restored to the basal level. This was accompanied by increased expression of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in GSH biosynthesis. The SIN-1-induced GCLC up-regulation was preceded by induction of HO-1 and subsequent CO production. Inhibition of HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression by small interfering RNA abrogated the up-regulation of GCLC expression and the subsequent GSH restoration induced by SIN-1. In contrast, additional exposure to the CO-releasing molecule (CO-RM) restored the GSH level previously reduced by inhibition of CO production using zinc protoporphyrin IX. Furthermore, CO-RM treatment up-regulated GCLC expression through activation of Nrf2. The CO-RM-induced activation of Nrf2 was under the control of the phosphatidylinositol 3-kinase/Akt signaling pathway. In conclusion, CO produced by HO-1 rescues PC12 cells from nitrosative stress through induction of GCLC, which is mediated by activation of phosphatidylinositol 3-kinase/Akt and subsequently Nrf2 signaling.

Establishment of cholinergic neuron-like cell lines with differential vulnerability to nitrosative stress.

Cholinergic cell lines were established by fusion of embryonic day 17 wild-type neurons from rat basal forebrain (BF) and upper brainstem (BS) with N18tg neuroblastoma cells. Isolated clones expressed choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) activities that were increased upon differentiation with retinoic acid. Clones from the BF expressed high levels of the tyrosine kinase type A (TrkA) receptor expression and activation of the mitogen-activated kinase ERK2 upon treatment with nerve growth factor. Like wild-type cholinergic populations, the six clones studied were variably resistant to nitric oxide (NO) excess from addition of S-nitroso-N-acetyl-D, L-penicillamine (SNAP). Of these, the BS2 clone exhibited resistance like in vivo BS cholinergic neurons, while the MS10 clone mimicked in vivo BF vulnerability. Apoptosis in response to NO excess was preceded by increases in mitochondrial responses bax/bcl-2 ratios, but cytochrome C was not released. Mitochondrial levels of apoptosis initiating factor (AIF) were either unchanged or increased, and only in MS clones was endonuclease G (EndoG) released. Microarray data indicated the existence of endoplasmic reticular (ER) stress and caspase-4 and caspase-12 were involved in the pathway to DNA fragmentation. The array data also indicated a survival role for mdm2, and its blockade rendered vulnerable the brainstem survivor clone BS2. Akt and ERK1/2 pathways were activated in response to NO and their blockade increased DNA fragmentation. Blockade of GSK-3 alpha/beta, a downstream target of Akt, reduced SNAP toxicity and this was more prominent in basal forebrain clones. We have identified two cholinergic cell lines useful for molecular studies of cholinergic vulnerability. We hypothesize that, in cholinergic neurons, control of ER stress signaling may be a major factor in differential vulnerability.

[Effects of noopept and cortexin on the behavior of matured rats treated with corticoliberin or 70-kDa heat shock proteins in early ontogeny].

Young Wistar rats aged 4 days were injected intraperitoneally with corticotropin releasing hormone (CRH), which is an agent activating the stress system, or 70-kDa heat shock proteins (HSP-70)--intracellular shaperons, possessing antistress properties. In grown adult rats aged 90-100 days, the effects of nootropic drugs noopept and cortexin (1 mg/kg, i.p.) were assessed. The activation of stress or antistress systems with CRH or HSP-70 significantly altered the drug action. The effects were different in males and females and depended on animal gender. The spectrum of pharmacological activity of noopept and cortexin changed: noopept demonstrated preferable psychoactivating and antiaggressive effects, whereas cortexin showed mild anxiolytic and antidepressant activity. It is suggested that the behavioral effects of nootropes depend on the conditions of the stress system formation in early ontogeny.

Increased expression of specific thioredoxin family proteins; a pilot immunohistochemical study on human hepatocellular carcinoma.

Hepatocellular Carcinoma (HCC) is one of the most frequent cancers worldwide, however, prognosis remains poor following its discovery. We investigate the Thioredoxin superfamily of proteins as diagnostic markers for HCC. Furthermore, we delineate possible roles of the endoplasmic reticulum member of the superfamily, ERdj5, in carcinogenesis. Using antibodies against Thioredoxin 1, Thioredoxin Reductase 1 and ERdj5, we performed immunohistochemistry on paraffin embedded liver biopsy sections from HCC patients. All three redox proteins exhibited elevated expression levels in tumor tissue compared to internal control, with ERdj5 showing a remarkable 3-fold increase. In vitro cell viability experiments using Hepatocellular Carcinoma HuH7 cells treated with ERdj5 small interfering RNA showed that ERdj5 knockdown cells exhibited less resistance to Doxorubicin (chemotherapy drug), but more resistance to Tunicamycin (Endoplasmic Stress inducer), compared to control cells. In conclusion, we introduce members of the Thioredoxin superfamily as possible immunohistochemical markers in the diagnostics of hepatocellular carcinoma and indicate a potential defensive role for ERdj5 in chemotherapeutic drug resistance.

The effect of the choice of irrigation fluid on cardiac stress during transurethral resection of the prostate: a comparison between 1.5% glycine and 5% glucose.

PURPOSE: Variable amounts of irrigation fluid are absorbed during transurethral prostate resection. Previous studies suggest that cardiac stress occurs as a result of transurethral prostate resection, possibly due to glycine absorption. We performed a prospective, blinded, randomized trial comparing 1.5% glycine with 5% glucose irrigating solution. We assessed whether glycine or glucose irrigation for transurethral prostate resection is associated with cardiotoxicity, as measured by troponin I and echocardiogram changes. MATERIALS AND METHODS: Between December 2001 and March 2003, 250 patients were recruited. Changes in immediate postoperative vs preoperative echocardiogram and serum cardiac troponin I indicated perioperative myocardial stress. Intraoperative irrigating fluid absorption was measured with 1% ethanol as a marker. Operative details recorded were anesthesia type, resection time, resected tissue weight and temperature change. Blood loss was measured with transfusions considered. Postoperatively blood assessments included serum glycine assay. RESULTS: Five patients (4%) in the glycine group and 3 (2%) in the glucose group had significantly increased troponin I after surgery. Of these men 1 per group had myocardial infarction and the remainder had transient ischemia. Logistic regression was used to identify factors associated with an unfavorable outcome, which was recorded as a significant increase in troponin I or ischemic changes on echocardiography. Increasing patient age and blood loss were associated with an unfavorable outcome (OR 1.84 and 1.24, respectively). We noted no significant differences in the 1.5% glycine and 5% glucose groups with regard to troponin I/echocardiogram. However, when the glycine assay was compared with adverse outcomes, an increased glycine assay was found to be associated with echocardiogram changes (p = 0.001) and with increased troponin I levels (relative risk 10.71). CONCLUSIONS: Transurethral prostate resection has an effect on the myocardium perioperatively. Glycine absorption causes echocardiogram changes and it is associated with increased troponin I. Increasing patient age and blood loss are associated with myocardial insult. The risk of increased blood loss was accumulative with each unit lost. Unrecognized blood loss or glycine absorption may explain the increase in morbidity and mortality previously reported in patients who undergo transurethral prostate resection.

Kainic acid induces early and transient autophagic stress in mouse hippocampus.

Kainic acid (KA) treatment is a well-established model of hippocampal neuron death mediated in large part by KA receptor-induced excitotoxicity. KA-induced, delayed neuron death has been shown previously to follow the induction of seizures and exhibit characteristics of both apoptosis and necrosis. Growing evidence supports a role of autophagic stress-induced death of neurons in several in vitro and in vivo models of neuron death and neurodegeneration. However, whether autophagic stress also plays a role in KA-induced excitotoxicity has not been previously investigated. To examine whether KA alters the levels of proteins associated with or known to regulate the formation of autophagic vacuoles, we isolated hippocampal extracts from control mice and in mice following 2-16 h KA injection. KA induced a significant increase in the amount of LC3-II, a specific marker of autophagic vacuoles, at 4-6h following KA, which indicates a transient induction of autophagic stress. Levels of autophagy-associated proteins including ATG5 (conjugated to ATG12), ATG6 and ATG7 did not change significantly after treatment with KA. However, ratios of phospho-mTOR/mTOR were elevated from 6 to 16 h, and ratios of phospho-Akt/Akt were elevated at 16 h following KA treatment, suggesting a potential negative feedback loop to inhibit further stimulation of autophagic stress. Together these data indicate the transient induction of autophagic stress by KA which may serve to regulate excitotoxic death in mouse hippocampus.

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

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

Mice lacking the transcription factor Mist1 exhibit an altered stress response and increased sensitivity to caerulein-induced pancreatitis.

Several animal models have been developed to investigate the pathobiology of pancreatitis, but few studies have examined the effects that altered pancreatic gene expression have in these models. In this study, the sensitivity to secretagogue-induced pancreatitis was examined in a mouse line that has an altered acinar cell environment due to the targeted deletion of Mist1. Mist1 is an exocrine specific transcription factor important for the complete differentiation and function of pancreatic acinar cells. Mice lacking the Mist1 gene [Mist1 knockout (KO) mice] exhibit cellular disorganization and functional defects in the exocrine pancreas but no gross morphological defects. Following the induction of pancreatitis with caerulein, a CCK analog, we observed elevated serum amylase levels, necrosis, and tissue damage in Mist1 KO mice, indicating increased pancreatic damage. There was also a delay in the regeneration of acinar tissue in Mist1 KO animals. Molecular profiling revealed an altered activation of stress response genes in Mist1 KO pancreatic tissue compared with wild-type (WT) tissue following the induction of pancreatitis. In particular, Western blot analysis for activating transcription factor 3 and phosphorylated eukaryotic initiation factor 2alpha (eIF2alpha), mediators of endoplasmic reticulum (ER) stress, indicated limited activation of this pathway in Mist1 KO animals compared with WT controls. Conversely, Mist1 KO pancreatic tissue exhibits increased expression of growth arrest and DNA damage inducible 34 protein, an inhibitor of eIF2alpha phosphorylation, before and after the induction of pancreatitis. These finding suggest that activation of the ER stress pathway is a protective event in the progression of pancreatitis and highlight the Mist1 KO mouse line as an important new model for studying the molecular events that contribute to the sensitivity to pancreatic injury.

Bax and the mitochondrial permeability transition cooperate in the release of cytochrome c during endoplasmic reticulum-stress-induced apoptosis.

Endoplasmic reticulum (ER) stress induces apoptosis by mechanisms that are not fully clear. Here we show that ER stress induced by the Ca(2+)-ATPase inhibitor thapsigargin (THG) activates cytochrome c-dependent apoptosis through cooperation between Bax and the mitochondrial permeability transition (MPT) in human leukemic CEM cells. Pharmacological inhibition of the MPT as well as small interfering RNA (siRNA) knockdown of the MPT core component cyclophilin D blocked cytochrome c release and caspase-dependent apoptosis but did not prevent Bax activation, translocation or N-terminal exposure in mitochondria. siRNA knockdown of Bax also blocked THG-mediated cytochrome c release and apoptosis, but did not prevent MPT activation and resulted in caspase-independent cell death. Our results show that ER-stress-induced cell death involves a caspase and Bax-dependent pathway as well as a caspase-independent MPT-directed pathway.