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Bypassing of DNA lesions by damage-tolerant DNA polymerases depends on the interaction of these enzymes with the monoubiquitylated form of the replicative clamp protein, PCNA. We have analyzed the contributions of ubiquitin and PCNA binding to damage bypass and damage-induced mutagenesis in Polymerase {eta} (encoded by RAD30) from the budding yeast Saccharomyces cerevisiae. We report here that a ubiquitin-binding domain provides enhanced affinity for the ubiquitylated form of PCNA and is essential for in vivo function of the polymerase, but only in conjunction with a basal affinity for the unmodified clamp, mediated by a conserved PCNA interaction motif. We show that enhancement of the interaction and function in damage tolerance does not depend on the ubiquitin attachment site within PCNA. Like its mammalian homolog, budding yeast Polymerase {eta} itself is ubiquitylated in a manner dependent on its ubiquitin-binding domain.
Background: West Nile virus (WNV) infection can cause severe meningitis and encephalitis in humans. Apoptosis was recently shown to contribute to the pathogenesis of WNV encephalitis. Here, we used WNV-infected glioma cells to study WNV-replication and WNV-induced apoptosis in human brain-derived cells. Results: T98G cells are highly permissive for lytic WNV-infection as demonstrated by the production of infectious virus titre and the development of a characteristic cytopathic effect. WNV replication decreased cell viability and induced apoptosis as indicated by the activation of the effector caspase-3, the initiator caspases-8 and -9, poly(ADP-ribose)polymerase (PARP) cleavage and the release of cytochrome c from the mitochondria. Truncation of BID indicated cross-talk between the extrinsic and intrinsic apoptotic pathways. Inhibition of the caspases-8 or -9 inhibited PARP cleavage, demonstrating that both caspases are involved in WNV-induced apoptosis. Pancaspase inhibition prevented WNV-induced apoptosis without affecting virus replication. Conclusions: We found that WNV infection induces cell death in the brain-derived tumour cell line T98G by apoptosis under involvement of constituents of the extrinsic as well as the intrinsic apoptotic pathways. Our results illuminate the molecular mechanism of WNV-induced neural cell death.
Imatinib (GleevecTM; GlivecTM; formerly STI571), a specific inhibitor of Abl tyrosine kinase, is efficacious in treating Philadelphiachromosomepositive (Ph+) leukaemias such as chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL) (Ottmann, Druker et al. 2002). Within a few years of its introduction to the clinic, Imatinib had dramatically altered the firstline therapy for CML, because it was found that most newly diagnosed CML patients in the chronic phase achieve durable responses when treated with Imatinib (Goldman and Melo 2003). However, a small percentage of these patients, as well as most advancedphase CML and Ph+ ALL patients, relapse on Imatinib therapy (Yokota, Kimura et al. 2006). Several mechanisms of refractoriness and relapse have been reported. These include point mutations within the Abl kinase domain, overexpression of BcrAbl mRNA (Hofmann, Jones et al. 2002), decreased intracellular drug levels mediated by Pglycoprotein (Pgp) (Hegedus, Orfi et al. 2002), and nonBcrAbl dependent mechanisms (activation of the SFKs) (Donato, Wu et al. 2003). In this research work, a possible means of overcoming resistance to Imatinib by the use of the specific dual Src/Abl kinase inhibitor AZD0530 has been investigated. The efficacy of AZD0530 in the treatment of Ph+ leukaemias, sensitive to or resistant to Imatinib, has been tested on cell lines, primary patient material and in vivo in transduction/transplantation mouse model of Imatinib sensitive or resistant BcrAbl dependent CML-like disease. Data with AZD0530 has been compared to cells treated with Imatinib. The potential of inhibiting both Src and Abl kinases while inducing growth arrest and apoptosis has been analysed. AZD0530 specifically inhibited the growth of CML and Ph+ ALL cells in a dosedependent manner, but has shown a marginal effect on Ph- ALL cells. Treatment of p185BcrAbl expressing Ba/F3 cells with AZD0530 has led to apoptosis induction and growth inhibition in these cells, while the untransformed Ba/F3 cells have remained unaffected. Resistance to Imatinib due to mutation in the Ba/F3MutY253F cells has been overcomed by this compound. The growth inhibitory effect of AZD0530 correlates with its induction of apoptosis. Combination of AZD0530 and Imatinib at low concentrations has shown an additive effect on the inhibition of proliferation of BV173 cells. The growth inhibition and apoptosis induction by AZD0530 have shown to be uncoupled to major changes in cell cycle. An exception is the CML blast crisis cell line BV173 which has shown a considerable G0/G1 arrest in the presence of AZD0530 and Imatinib as single agents. Immunoblotting of whole cell lysates from Imatinib or AZD0530 treated BV173, Ba/F3 expressing p185(BcrAbl) MutT253F cells and the WTSupB15 cells, for Src and BcrAbl clearly demonstrates that there is an ongoing transphosphorylation taking place between the SFKs and BcrAbl. This transphosphorylation synergizes and influences the aggressive nature of CML blast crisis and Ph+ ALL. Investigations have been carried out on downstream signaling events to determine how Src family members contribute to BcrAbl signaling. Specifically, Stat, Erk and PI3K/ Akt activation status have been characterised in Imatinib sensitive and resistant Ph+ cells. AZD0530 has significantly downregulated the activation of survival signaling pathways as shown by it’s inhibition of Stat5, Akt and Erk kinases in Ph+ cells, resistant or sensitive to Imatinib. The only exception to this has been the Imatinib resistant cell line RTSupB15, in which activated Akt kinase level has remained unaffected. AZD0530 has shown to be efficient in the treatment of cells isolated from three Ph+ leukaemic patients (resistant or sensitive to Imatinib), and has led to an induction of apoptosis. Equally, in the same patients, growth and survival pathways have been inhibited in vitro in the presence of AZD0530. An overall therapeutic effect of AZD0530 in vivo has been studied in mouse model of Imatinib sensitive and Imatinib resistant, BcrAbldependent desease. Mice with a BcrAbllike disease responded to Imatinib treatment but not to AZD0530. Using the CFU assay, an influence on the differentiation status of primary leukaemic blast stem cells have been tested. The in vivo studies as well as the CFU results have shown discrepancies to the effects of AZD0530 tested so far in this research work. These discrepancies have paralleled with the upregulation of BcrAbl in most AZD0530 treated cells. These are to be further analysed. These data elucidate the role of Src kinases in BcrAbl leukaemogenesis. Results gotten from this research work has shown that AZD0530 targets both Src and BcrAbl kinase activity and reduces the transforming potential of BcrAbl. It also shows that there is an ongoing transphosphorylation between SFKs and BcrAbl kinase. AZD0530 has proven effective in CML cell lines, Ph+ ALL cell lines and patient cells resistant to Imatinib. These have demonstrated that AZD0530 is a potential drug target which can be used to overcome Imatinib resistance.
Prostaglandin D2 (PGD2) is involved in a variety of physiological and pathophysiological processes, but its role in fever is poorly understood and the data obtained so far are rather controversial. Here we investigated the effects of central PGD2 delivery and of systemic prostaglandin D synthase (PGDS) or cyclooxygenase (COX) inhibition on core body temperature (TC) and on prostaglandin levels in the cerebrospinal fluid (CSF) of rats. Both PGE2 and PGD2 were detectable in CSF samples from control rats (6.2 ± 1.1 and 17.3 ± 3.1 pg/ml, respectively). Lipopolysaccharide (LPS) injection (50 μg i.p.) induced fever during the 5-hour observation period. Five hours after LPS injection, the levels of PGE2 and PGD2 were increased in the CSF about 90-fold (541.0 ± 47.5 pg/ml) and 5-fold (95.4 ± 23.1 pg/ml), respectively. Administration of PGD2 (50 - 500 ng) into the cisterna magna (i.c.m) evoked a delayed fever response in a dose-dependent manner that was accompanied by increased levels of PGE2 in the CSF. RT-PCR analyses revealed that the increased levels of PGE2 after PGD2 administration were not caused by up-regulation of COX-2 or microsomal prostaglandin E synthase 1 (mPGES-1) in the hypothalamus. Interestingly, i.c.m. pretreatment of animals with PGD2 considerably sustained the pyrogenic effects of i.c.m. administered PGE2. Pretreatment with a novel PGDS inhibitor, EDJ300520 (10 – 40 mg/kg p.o.), 1 h prior to the LPS injection impaired the LPS-induced increase of both PGD2 and PGE2 in the CSF and inhibited the fever response. In contrast, administration of EDJ300520 3 h after LPS injection did not ameliorate the LPS-induced fever. Accordingly, the concentration of PGE2 in the CSF was not decreased after EDJ300520 treatment. However, the CSF levels of PGD2 were reduced after administration of a high dose of EDJ300520 (40 mg/kg). We also investigated the effects of antipyretic drugs on the CSF levels of PGE2 and PGD2 during LPS-induced fever. Four antipyretic drugs with different mechanisms of action were used, including ibuprofen (5 - 20 mg/kg), celecoxib (10 - 50 mg/kg), SC560 5 - 20 mg/kg), and paracetamol (50 - 150 mg/kg). Each drug was used in three different doses and was orally administered 3 h after the LPS injection. All drugs were capable to attenuate the LPS-induced fever. The decrease of TC paralleled the reduction of PGE2 levels in the CSF. Of note, there was a tendency to reduced PGD2 levels in the CSF after treatment with the antipyretic drugs. However, only SC560 and the high dose of celecoxib (50 mg/kg) reduced the PGD2 levels significantly. In summary, our experiments underscore the pivotal role of PGE2 as the principal downstream mediator of fever. Moreover, we demonstrate that PGD2 is also involved in the mechanisms underlying fever. Our data suggest that PGD2 exerts an indirect pyrogenic effect by modulating the availability of PGE2 in the CSF. Additional studies are needed to explore the exact mechanism by
Molecular mechanism of intracellular signal transduction by the angiotensin-converting enzyme
(2007)
The angiotensin converting enzyme (ACE) is an important component of the renin-angiotensin system (RAS) and is crucially involved in the homeostasis of fluid and electrolyte balance and thus in the regulation of blood pressure. The zinc metallopeptidase is involved in the generation of angiotensin II, a potent vasoconstrictor and in the degradation of bradykinin, a potent vasodilator. It is worth noting that ACE more readily hydrolyzes bradykinin than it does angiotensin I thus culminating in the net physiological effect of the production of a vasoconstrictor and the decrease in the availability of a vasodilator. ACE inhibitors have become one of the most successful therapeutic approaches as a first line of therapy in hypertension, and are also widely used in treating heart failure, myocardial infarction, stroke, coronary artery disease and impaired left ventricular function. However, one unexpected clinically relevant finding related to ACE inhibitors is their ability to delay the onset of type II diabetes that was revealed by various large clinical trials. However, the mechanisms underlying these beneficial effects of ACE inhibitor therapy are currently unclear and cannot be explained by the prevention of angiotensin II formation or the attenuated degradation of bradykinin. Thus the potential beneficial effects attributed to ACE inhibitors may occur independent of reductions in blood pressure paving way for new and/or unknown mechanism. Our group has recently redefined ACE as a signal transduction molecule which upon binding to ACE inhibitor turns on a signalling cascade leading to phosphorylation of Ser1270 by CK2, activation of JNK and changes in gene expression in endothelial cells. However the mechanism by which ACE inhibitor initiates the signalling cascade was not clear. It was hypothesized that ACE, which is anchored to the membrane with a single transmembrane domain should dimerize prior to initiating further downstream signalling events in endothelial cells. Therefore, we sought to explore whether or not ACE forms dimers in endothelial cells and whether ACE dimerization is essential for the initiation of ACE signalling in endothelial cells. Using native gel electrophoresis, we found that ACE forms dimers in endothelial cells and that there is an increase in the dimer formation upon treatment of endothelial cells with ACE inhibitors. ACE homodimerization was also demonstrated using the split-ubiquitin system and chemical cross-linking experiments. ACE dimers are also formed in endothelial cells overexpressing the non-phosphorylatable ACE, wherein ACE signalling was abolished indicating that dimerization process is not influenced by the phosphorylation of the serine residue residing in the cytoplasmic tail. Monosaccharides like glucose, galactose and mannitol did not have any influence on ACE-inhibitor induced dimerization. Making use of different monoclonal antibodies directed to the epitopes of N-domain which harbours carbohydrate recognizing domain, also did not affect dimerization. However, inactivation of the C-domain active site by introducing mutation of the key histidine residues in HEMGH consensus sequences, which complexes the zinc ions, abolished enzyme dimerization both in the basal state and in response to ramiprilat. Mutation of the C-domain also resulted in the loss of ACE inhibitor-induced ACE signalling, that is we failed to observe ramiprilat-induced increase in the phosphorylation of the Ser1270 and the subsequent JNK activation. ACE-inhibitor induced dimerization precedes the phosphorylation of Ser1270 and activation of JNK. Thus the ACE-inhibitor induced dimerization via the C-domain of ACE represents the initial step in the ACE signalling pathway which involves the activation of JNK/c-Jun pathway and leading to the changes in the gene expression in endothelial cells. Our group previously identified ACE itself as well as cyclooxygenase-2 (COX-2) as two “ACE signalling-regulated” genes. To screen for additional genes regulated in a similar manner we used DNA microarray technology, to assess ramiprilat-induced changes in the endothelial cell gene expression. 21 genes were identified to be differentially regulated of which, 7 were upregulated and 14 were downregulated by ramiprilat. However, when screened at the protein level, we found no significant differences between the untreated control cells and those treated with ramiprilat. As several other cells and tissues possess a fully functional RAS we screened plasma samples from healthy volunteers as well as from patients with coronary artery disease for the proteins identified in the microarray. We observed that the cellular retinal binding protein-1 (CRBP-1) was detectable at low levels in plasma from patients and that ramipril markedly increased serum levels of this protein. Endothelial cells overexpressing CRBP-1 demonstrated increased RXRE and PPRE activity when stimulated with 9-cis retinoic acid and rosiglitazone respectively suggesting that CRBP-1 might affect gene expression via heterodimerization of PPAR elements with RXR elements by virtue of its function as a transport protein of retinoic acid. Studies aimed at determining the consequences of elevated CRBP-1 expression on endothelial cell homeostasis are ongoing. Although the RAS has been described in many other tissues apart from endothelial cells, ACE signalling has not yet been addressed in tissues such as monocytes/macrophages, which have an increased ACE expression in an atherosclerotic setting. We observed that upon stimulation of cultured ACE expressing monocytes with ramiprilat, JNK is activated suggesting the occurrence of ACE signalling in human monocytes. It is worth noting that ACE inhibitors delay the onset of type II diabetes in spite of moderate decrease in blood pressure. To further elucidate the mechanism underlying this effect, we found that ACE inhibitors increase the PPARgamma levels in the nuclear extracts of ACE expressing monocytes which were also reproduced in human endothelial cells overexpressing human somatic ACE. However, ramiprilat did not have any direct effect on the activity of a luciferase-coupled promoter containing several copies of the PPRE in human endothelial cells. These results contrasted with the actions of the PPARgamma agonist suggesting that ramiprilat enhances PPARgamma levels through an indirect mechanism. We next hypothesized that ramiprilat might increase the levels of 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2) which is a natural ligand for PPARgamma via COX enzymes in monocytes. We observed that ramiprilat was able to decrease the diminution of COX-2 levels upto 48 hours of treatment but the levels of 15dPGJ2 were too low to be detected by ELISA. However ramiprilat enhanced the plasma levels of adiponectin, a downstream target of PPARgamma, which is a anti-atherogenic and anti-inflammatory adipokine, in patients with coronary artery disease. Though adiponectin is a PPARgamma-regulated gene, the observed increase in adiponectin might be attributed to the increase in RXR rather than via PPARgamma. Taken together, the results of this investigation have revealed that ACE inhibitors initiate ACE signalling by eliciting the dimerization of the enzyme, more specifically via its C-domain active centers. The ACE signalling cascade when activated leads to the enhanced expression of ACE, COX-2 and CRBP-1 which in turn favours the heterodimerization of PPARgamma with RXR and thus results in the increased expression of “PPARgamma regulated” genes such as adiponectin. The latter results provide a molecular basis for the observation that ACE inhibitors can delay the onset of type 2 diabetes in as much as it was possible to link ramipril with CRBP-1, RXR activity and the expression of adiponectin, an adipokine associated with improved insulin sensitivity. Further work is however required to elucidate the consequences of ACE inhibitors in monocytes and adipocytes as well as in intact animals.
Background A procedure for including activity against enveloped viruses in the post-contamination treatment of hands has been recommended, but so far no European standard is available to implement it. In 2004, the German Robert Koch-Institute (RKI) and the German Association for the Control of Virus Disease (DVV) suggested that vaccinia virus and bovine viral diarrhea virus (BVDV) should be used as test viruses in a quantitative suspension test to determine the activity of a disinfectant against all enveloped viruses. Methods We have studied the activities of three commonly-used alcohol-based hand rubs (hand rub A, based on 45% propan-2-ol, 30% propan-1-ol and 0.2% mecetronium etilsulfate; hand rub B, based on 80% ethanol; hand rub C, based on 95% ethanol) against vaccinia virus and BVDV, and in addition against four other clinically relevant enveloped viruses: herpes simplex virus (HSV) types 1 and 2, and human and avian influenza A virus. The hand rubs were challenged with different organic loads at exposure time of 15, 30 and 60 s. According to the guidelines of both BGA/RKI and DVV, and EN 14476:2005, the reduction of infectivity of each test virus was measured on appropriate cell lines using a quantitative suspension test. Results All three alcohol-based hand rubs reduced the infectivity of vaccinia virus and BVDV by >= 4 log10-steps within 15 s, irrespective of the type of organic load. Similar reductions of infectivity were seen against the other four enveloped viruses within 15 s in the presence of different types of organic load. Conclusions Commonly used alcohol-based hand rubs with a total alcohol concentration >= 75% can be assumed to be active against clinically relevant enveloped viruses if they effectively reduce the infectivities of vaccinia virus and BVDV in a quantitative suspension test.
Why is it hard to divide attention between dissimilar activities, such as reading and listening to a conversation? We used functional magnetic resonance imaging (fMRI) to study interference between simple auditory and visual decisions, independently of motor competition. Overlapping activity for auditory and visual tasks performed in isolation was found in lateral prefrontal regions, middle temporal cortex and parietal cortex. When the visual stimulus occurred during the processing of the tone, its activation in prefrontal and middle temporal cortex was suppressed. Additionally, reduced activity was seen in modality-specific visual cortex. These results paralleled impaired awareness of the visual event. Even without competing motor responses, a simple auditory decision interferes with visual processing on different neural levels, including prefrontal cortex, middle temporal cortex and visual regions.
Aims: To analyze the relationship between exposure to chlorinated and aromatic organic solvents and malignant lymphoma in a multi-centre, population-based case-control study.
Methods: Male and female patients with malignant lymphoma (n=710) between 18 and 80 years of age were prospectively recruited in six study regions in Germany (Ludwigshafen /Upper Palatinate, Heidelberg/ Rhine-Neckar-County, Wurzburg/ Lower Frankonia, Hamburg, Bielefeld/ Gutersloh, and Munich). For each newly recruited lymphoma case, a gender, region and age-matched (+/- 1 year of birth) population control was drawn from the population registers. In a structured personal interview, we elicited a complete occupational history, including every occupational period that lasted at least one year. On the basis of job task-specific supplementary questionnaires, a trained occupational physician assessed the exposure to chlorinated hydrocarbons (trichloroethylene, tetrachloroethylene, dichloromethane, carbon tetrachloride) and aromatic hydrocarbons (benzene, toluene, xylene, styrene). Odds ratios (OR) and 95% confidence intervals (CI) were calculated using conditional logistic regression analysis, adjusted for smoking (in pack years) and alcohol consumption. To increase the statistical power, patients with specific lymphoma subentities were additionally compared with the entire control group using unconditional logistic regression analysis.
Results: We observed a statistically significant association between high exposure to chlorinated hydrocarbons and malignant lymphoma (Odds ratio = 2.1; 95% confidence interval 1.1-4.3). In the analysis of lymphoma subentities, a pronounced risk elevation was found for follicular lymphoma and marginal zone lymphoma. When specific substances were considered, the association between trichloroethylene and malignant lymphoma was of borderline statistical significance. Aromatic hydrocarbons were not significantly associated with the lymphoma diagnosis.
Conclusions: In accordance with the literature, this data point to a potential etiologic role of chlorinated hydrocarbons (particularly trichloroethylene) and malignant lymphoma. Chlorinated hydrocarbons might affect specific lymphoma subentities differentially. Our study does not support a strong association between aromatic hydrocarbons (benzene, toluene, xylene, or styrene) and the diagnosis of a malignant lymphoma.
Einleitung: Es wurden die Leistungen beim Verstehen im Störgeräusch von CI-Patienten mit unterschiedlichen Implantattypen verglichen. Der TEMPO+ Sprachprozessor (MED-EL, Implantat C40+) verwendet ein Mikrophon mit Kugelcharakteristik, während der ESPrit 3G Prozessor (COCHLEAR, Implantat CI24R(CA)) mit einem frontal ausgelegten Richtmikrophon ausgestattet ist.
Methode: Von den zwei untersuchten Patientengruppen (n=20) war eine mit einem C40+ Implantat (MED-EL, Innsbruck), die andere mit dem CI24RCA Implantat (Cochlear, Melbourne) versorgt. Es wurde die S0N180 Lautsprecheranordnung im Freifeld für den HSM-Test (Hochmair, Schulz und Moser, 1997) und die S0N0 Anordnung für den Oldenburger Satztest (Wagener, Kühnel und Kollmeier, 1999) verwendet. Der OLSA wurde mit festem Sprachpegel (65 dB SPL) und adaptivem Störgeräusch durchgeführt. Der HSM-Satztest wurde bei Signal-/ Rauschverhältnissen von 15 dB, 10 dB, 5 dB, 0dB sowie ohne Störgeräusch durchgeführt.
Ergebnisse: Im HSM-Satztest (S0N180) wurden signifikant bessere Leistungen beim Verstehen im Störgeräusch für die Gruppe mit dem Richtmikrophon nachgewiesen. Im Oldenburger Satztest zeigten sich keine signifikanten Unterschiede.
Schlussfolgerungen: Im Vergleich zu einem Mikrophon mit Kugelcharakteristik verbessert ein Richtmikrophon das Sprachverstehen in Situationen, in denen die Sprache frontal und der Störschall von hinten dargeboten werden.
Background The Deltaretrovirus genus comprises viruses that infect humans (HTLV), various simian species (STLV) and cattle (BLV). HTLV-I is the main causative agent in adult T-cell leukemia in endemic areas and some of the simian T-cell lymphotropic viruses have been implicated in the induction of malignant lymphomas in their hosts. BLV causes enzootic bovine leukosis in infected cattle or sheep. During the past few years several new Deltaretrovirus isolates have been described in various primate species. Two new HTLV-like viruses in humans have recently been identified and provisionally termed HTLV-III and HTLV-IV. In order to identify a broad spectrum of Deltaretroviruses by a single PCR approach we have established a novel consensus PCR based on nucleotide sequence data obtained from 42 complete virus isolates (HTLV-I/-II, STLV-I/-II/-III, BLV). The primer sequences were based on highly interspecies-conserved virus genome regions. We used this PCR to detect Deltaretroviruses in samples from adult patients with a variety of rare T-cell neoplasms in Germany. Results: The sensitivity of the consensus PCR was at least between 10-2 and 10-3 with 100% specificity as demonstrated by serial dilutions of cell lines infected with either HTLV-I, HTLV-II or BLV. Fifty acute T-cell lymphoblastic leukemia (T-ALL) samples and 33 samples from patients with various rare mature T-cell neoplasms (T-PLL, Sezary syndrome and other T-NHL) were subsequently investigated. There were no cases with HTLV-I, HTLV-II or any other Deltaretroviruses. Conclusions: The results rule out a significant involvement of HTLV-I or HTLV-II in these disease entities and show that other related Deltaretroviruses are not likely to be involved. The newly established Deltaretrovirus PCR may be a useful tool for identifying new Deltaretroviruses.
Transcranial magnetic stimulation (TMS) is a non-invasive technique which can be used to study different intracortical excitatory and inhibitory neuronal circuits in the intact human being. In the primary motor cortex, there are essentially three different TMS measures of inhibitory neuronal circuits as determined by paired-pulse TMS: short-interval intracortical inhibition (SICI), long-interval intracortical inhibition (LICI) and interhemispheric inhibition (IHI). It was hypothesized that SICI is a GABAA receptor mediated inhibition (Ilic et al., 2002) whereas LICI and IHI are mediated by GABAB receptors (Daskalakis et al., 2002; McDonnell et al., 2006). Additionally, it was shown that these inhibitory circuits interact negatively, possible due to presynaptic GABAB receptor mediated inhibition (Sanger et al., 2001; Daskalakis et al., 2002). Which neuronal populations exactly underlie SICI, LICI and IHI, is not completely clear and by which mechanism these inhibitory circuits interact has never been tested pharmacologically so far. Thus, the effects of a single oral dose of Diazepam (DZP), a specific positive allosteric modulator at the GABAA receptor, and of Baclofen (BAC), a specific GABAB receptor agonist, on SICI, LICI and IHI as well as their interactions were tested here in a randomized, placebo controlled, double-blinded crossover study. SICI significantly increased after intake of DZP whereas BAC did not change SICI. Conversely, LICI significantly increased after intake of BAC but did not change after intake of DZP. IHI showed only a trend towards a decrease after intake of DZP but no change after intake of BAC. The interactions IHI-SICI, LICI-IHI and LICI-SICI were all negative at baseline. SICI and IHI were partially suppressed in the presence of IHI and LICI, respectively, and SICI in the presence of LICI was almost completely blocked. BAC did not change any of these interactions, whereas DZP significantly increased SICI in the presence of LICI. This study is the first to examine by means of pharmacological testing the complex interactions between different inhibitory circuits in the human motor cortex. The effects of DZP and BAC on SICI and LICI confirmed the notion that SICI is a GABAA receptor mediated intracortical inhibition whereas LICI depends on GABAB receptor mediated neurotransmission. The pharmacology of IHI at short interstimulus intervals of < 20 ms (12 ms in this study) remains still inconclusive and warrants further investigation. Findings further suggest that SICI, LICI and IHI represent three different inhibitory neuronal circuits which can be tested non-invasively by means of paired-pulse TMS. Furthermore, the data support the idea that the negative interactions IHI-SICI, LICI-IHI and LICI-SICI are most likely due to presynaptic GABAB receptor mediated autoinhibition.
Poster presentation: Hyperphosphorylation of tau is a characteristic of Alzheimer's disease (AD). Our group has established a model for tau hyperphosphorylation by mutating 10 residues from Ser/Thr to Glu to simulate the negative charge of phosphorylated residues ("pseudohyperphosphorylated (PHP)-tau"). In order to analyze temporal and spatial effects of hyperphosphorylation of tau in a systemic context, we have established transgenic mouse lines that express human wild-type (wt)- or PHP-tau under the control of the CamKIIalpha-promoter that leads to a forebrain specific moderate expression in neurons, i.e. the region where also tau-pathology in AD is abundant. For the evaluation of tau-induced changes in the transgenic mice, we quantified spine densities in the neocortex and hippocampus of transgenic mice. The spine densitiy was significantly increased in PHP-tau compared to wt-tau expressing mice. It is known that AD is associated with aberrant pre- and postsynaptic sprouting. Axonal sprouting is also observed in transgenic mice expressing mutated amyloid precursor protein (APP), which suggests that Abeta plays a significant role in this process. We deduce from our results, that (pseudo)-hyperphosphorylation of tau is sufficient to induce aberrant sprouting in the absence of Abeta. Analyses whether this sprouting is induced by pre- or postsynaptic changes and if functionally active synapses are formed are in progress. It will be interesting to determine if stabilization of these newly formed synapses slows or – in contrary – accelerates the progression of the disease. Sprouting as observed in our PHP-tau expressing mice is part of neuronal differentiation. One family of enzymes that is involved in cell differentiation are mitogen-acitvated protein kinases (MAPK). Western blot analysis was performed with brain lysates from transgenic mice to check whether PHP-tau induced sprouting is associated with MAPK activation. In fact, we also observed an increased activation of the MAPK ERK1/2 evident by phosphorylation of the residues Thr202 and Tyr204. ERK1/2 is also known to phosphorylate tau at sites characteristic for AD. Our results suggest the presence of a vicious circle by which (pseudo)-hyperphosphorylated tau activates ERK1/2 which in turn phosphorylates tau.
Poster presentation: The transcription factor NF-kappaB plays a central role in the development and maintenance of the central nervous system and its constitutive activation in neurons has been repeatedly reported. Previous work from our laboratories (poster presentation: Compartimentalized NF-kappaB activity in the axon initial segment) had revealed an intriguing clustering of activated IKKalpha/beta and other downstream elements of an activated NF-kappaB cascade (phospho-IkappaBalpha, phospho-p65(Ser536)) in the axon initial segment (AIS). Accumulation of certain voltage-gated sodium channels (Na(v)1.2), M-type potassium channels (KCNQ2) as well as cytoskeletal anchoring proteins (AnkyrinG) characterise the AIS. However, it is not yet clear how AIS-localized IKK gets activated and whether this can be connected to the constitutive activation of NF-kappaB. Long-term blockade of sodium channels with tetrodotoxin, potassium-channels with linopirdine or NMDA-receptors with MK-801 did not elicit any change upon the constitutive activation of the pathway. Strikingly, the occurrence of phosphorylated IkappaBalpha was even unaltered by 24 h of incubation with protein synthesis inhibitors. Others have reported that impairment of NF-kappaB inhibits neuritogenesis. In this line we observed that the early initiation of IkappaBalpha phosphorylation was susceptible to inhibition of IKK in DIV1–2 neurons. We therefore aim to identify the interaction partners of the activated IKK complex in the AIS. Proteomic methods such as co-immunoprecipitation analyses and mass-spectrometry will help us to identify the key players in the initiation of constitutive IKK phosphorylation and activation in neurons.
Poster presentation: The mammalian pineal organ is a peripheral oscillator, depending on afferent information from the so-called master clock in the suprachiasmatic nuclei of the hypothalamus. One of the best studied outputs of the pineal gland is the small and hydrophobic molecule melatonin. In all vertebrates, melatonin is synthesized rhythmically with high levels at night, signalling the body the duration of the dark period. Changes or disruptions of melatonin rhythms in humans are related to a number of pathophysiological disorders, like Alzheimer's disease, seasonal affective disorder or the Smith-Magenis-Syndrome. To use melatonin in preventive or curative interferences with the human circadian system, a complete understanding of the generation of the rhythmic melatonin signal in the human pineal gland is essential. Melatonin biosynthesis is best studied in the rodent pineal gland, where the activity of the penultimate and rate-limiting enzyme, the arylalkylamine N-acetyltransferase (AA-NAT), is regulated on the transcriptional level, whereas the regulatory role of the ultimate enzymatic step, achieved by the hydroxyindole O-methyltransferase (HIOMT), is still under debate. In rodents, Aa-nat mRNA is about 100-fold elevated during the night in response to adrenergic stimulation of the cAMP-signalling pathway, with AA-NAT protein levels closely following this dynamics. In contrast, in all ungulates studied so far (cow, sheep), a post-transcriptional regulation of the AA-NAT is central to determine rhythmic melatonin synthesis. AA-NAT mRNA levels are constantly elevated, and lead to a constitutive up-regulation of AA-NAT protein, which is, however, rapidly degraded via proteasomal proteolysis during the day. AA-NAT proteolysis is only terminated upon the nocturnal increase in cAMP levels. Similar to ungulates, a post-transcriptional control of this enzyme seems evident in the pineal gland of the primate Macaca mulatta. Studies on the molecular basis of melatonin synthesis in the human being are sparse and almost exclusively based on phenomenological data, derived from non-invasive investigations. Yet the molecular mechanisms underlying the generation of the hormonal message of darkness can currently only be deciphered using autoptic material. We therefore analyzed in human post-mortem pineal tissue Aa-nat and Hiomt mRNA levels, AA-NAT and HIOMT enzyme activity, and melatonin levels for the first time simultaneously within tissue samples of the same specimen. Here presented data show the feasibility of this approach. Our results depict a clear diurnal rhythm in AA-NAT activity and melatonin content, despite constant values for Aa-nat and Hiomt mRNA, and for HIOMT activity. Notably, the here elevated AA-NAT activity during the dusk period does not correspond to a simultaneous elevation in melatonin content. It is currently unclear whether this finding may suggest a more important role of the ultimate enzyme in melatonin synthesis, the HIOMT, for rate-limiting the melatonin rhythm, as reported recently for the rodent pineal gland. Thus, our data support for the first time experimentally that post-transcriptional mechanisms are responsible for the generation of rhythmic melatonin synthesis in the human pineal gland.
Poster presentation: Light is the main phase-adjusting stimulus of the circadian clock located in the suprachiasmatic nucleus (SCN). A candidate pathway transmitting photic information at the postsynaptic site in the SCN is the extracellular signal-regulated kinase (ERK 1/2) which has been previously shown to be an essential element in the photoentrainment of the circadian rhythm. An upstream activator of the ERK signalling route is the small intracellular GTPase Ras. Here we observed that endogenous Ras activity in the SCN was subjected to rhythmic changes, reaching maximum levels at the late subjective day and minimum levels at the late subjective night (CT22). In order to investigate if Ras would modulate the circadian cycle, we used transgenic mice expressing constitutively activated Val-12 Ha-Ras selectively in neurons (synRas mice). In these mice Ras activity was also cycling during the circadian rhythm yet, Ras activities were up-regulated at each time point measured. We investigated if this change in Ras activity translates into a behavioral phenotype by monitoring free-running activity rhythms under conditions of constant darkness. SynRas mice exhibited circadian rhythms in locomotor activities similar to WT mice. However, when challenged by applying a 15 minutes light pulse at CT22 to promote phase advance shifts, synRas mice were completely non-responsive. As a first step towards the possible intracellular mechanism of this behavioral change we analyzed ERK1/2 activities in more details: We found a 1,7-fold increase of circadian peak levels of ERK 1/2 activities at CT10 and CT14 in synRas mice, while at minimum levels (CT18, CT22) no differences were found between ERK1/2 activities of WT and synRas mice. In WT animals the 15 minutes light pulse at CT22 resulted in rapid up regulations of Ras, ERK1/2 and CREB activities as described previously by others. However, in correlation with the lack of a behavioral response, ERK1/2 but not Ras and CREB activities remained unchanged in synRas mice, suggesting that Ras-dependent and Ras-independent pathways may co-exist to regulate ERK1/2 and behavioral phase shifts in response to the acute light treatment. Next we investigated the length "tau" of the locomotor activity rhythm during constant darkness and found a slight shortening by about 10 minutes in synRas mice as compared to the WT littermates. Recently, "tau" has been discussed to be modulated by the interaction between glycogen synthase 3beta (GSK3beta) and a clock gene product (Per 2) that is involved in the determination of circadian phase durations. We describe here a down-regulation of GSK3beta phosphorylation in synRas mice as a possible mechanism of "tau" shortening. Taken together, cycling of Ras activity at elevated levels in the SCN during the circadian rhythm results in a distinct pattern of behavioral phenotype changes correlating with de-regulated ERK1/2 or GSK3beta activities.
Poster presentation: The transcription factor NF-kappaB plays a pivotal role in the development and maintenance of the central nervous system and its constitutive activation in neurons has been previously reported. NF-kappaB is post-translationally activated upon phosphorylation of the IkappaBalpha inhibitory protein by the activated IkappaB kinase (IKKalpha/beta) and the subsequent degradation of IkappaBalpha by the proteasome. Recently, we had demonstrated an unexpected accumulation of three components of the NF-kappaB cascade in the axon initial segment (AIS): Activated IKK, phosphorylated IkappaBalpha and phosphorylated-p65(Ser536). These are all associated with detergent-insoluble cytoskeletal components of the AIS. We observed further compartimentalization as pIKKalpha/beta primarily associated with the membrane cytoskeleton, whereas pIkappaBalpha was sequestered to fasciculated microtubules. Colchicine-induced depolymerization of microtubules was associated with reduced sequestration of pIkappaBalpha in the AIS, which could be blocked by use of proteasome inhibitors like Mg-132 or Lactacystin. Concurrently, enhanced nuclear immunoreactivity for the NF-kappaB subunit p65 was noted. Using NF-kappaB-dependent reporter gene assays, a significant increase in NF-kappaB activity was observed after depolymerization of microtubules and this was inhibited by the microtubule-stabilizing drug paclitaxel. The use of transiently transfected, photoactivatable-GFP p65 fusion proteins will allow us to specifically analyse the compartimentalized signal transduction pathways in unique spatial and temporal resolution. Taken together, these observations provide strong evidence for compartmentalized activation of NF-kappaB in the AIS and modulation of neuronal NF-kappaB activity by microtubule dynamics.
Poster presentation: Here we investigated the role of the amyloid precursor protein (APP) in regulation of Ca2+ store depletion-induced neural cell death. Ca2+ store depletion from the endoplasmic reticulum (ER) was induced by the SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) inhibitor thapsigargin which led to a rapid induction of the unfolded protein response (UPR) and a delayed activation of executioner caspases in the cultures. Overexpression of APP potently enhanced cytosolic Ca2+ levels and cell death after ER Ca2+ store depletion in comparison to vector-transfected controls. GeneChipR and RT-PCR analysis revealed that the expression of classical UPR chaperone genes was not altered by overexpression of APP.Interestingly, the induction of the ER stress-responsive pro-apoptotic transcription factor CHOP was significantly upregulated in APP-overexpressing cells in comparison to vectortransfected controls. Chelation of intracellular Ca2+ with BAPTA-AM revealed that enhanced CHOP expression after store depletion occured in a Ca2+-dependent manner in APPoverexpressing cells. Prevention of CHOP induction by BAPTA-AM and by RNA interference was also able to abrogate the potentiating effect of APP on thapsigargin-induced apoptosis. Application of the store-operated channel (SOC)-inhibitors SK F96365 and 2-APB downmodulated APP-triggered potentiation of cytosolic Ca2+ levels and apoptosis after treatment with thapsigargin. Our data demonstrate that APP-mediated regulation of ER Ca2+ homeostasis significantly modulates Ca2+ store depletion-induced cell death in a SOC- and CHOP-dependent manner, but independent of the UPR.
Within the large variety of subtypes of chronic cough, either defined by their clinical or pathogenetic causes, occupational chronic cough may be regarded as one of the most preventable forms of the disease. Next to obstructive airway diseases such as asthma or chronic obstructive pulmonary disease, which are sometimes concomitant with chronic cough, this chronic airway disease gains importance in the field of occupational medicine since classic fiber-related occupational airway diseases will decrease in the future.
Apart from acute accidents and incidental exposures which may lead to an acute form of cough, there are numerous sources for the development of chronic cough within the workplace. Over the last years, a large number of studies has focused on occupational causes of respiratory diseases and it has emerged that chronic cough is one of the most prevalent work-related airway diseases. Best-known examples of occupations related to the development of cough are coal miners, hard-rock miners, tunnel workers, or concrete manufacturing workers.
As chronic cough is often based on a variety of non-occupational factors such as tobacco smoke, a distinct separation into either occupational or personally -evoked can be difficult. However, revealing the occupational contribution to chronic cough and to the symptom cough in general, which is the commonest cause for the consultation of a physician, can significantly lead to a reduction of the socioeconomic burden of the disease.
Hypersecretion and chronic phlegm are major symptoms of chronic obstructive pulmonary disease (COPD) but animal models of COPD with a defined functional hypersecretion have not been established so far. To identify an animal model of combined morphological signs of airway inflammation and functional hypersecretion, rats were continuously exposed to different levels of sulfur dioxide (SO2, 5 ppm, 10 ppm, 20 ppm, 40 ppm, 80 ppm) for 3 (short-term) or 20–25 (long-term) days. Histology revealed a dose-dependent increase in edema formation and inflammatory cell infiltration in short-term-exposed animals. The submucosal edema was replaced by fibrosis after long-term-exposure. The basal secretory activity was only significantly increased in the 20 ppm group. Also, stimulated secretion was significantly increased only after exposure to 20 ppm. BrdU-assays and AgNOR-analysis demonstrated cellular metaplasia and glandular hypertrophy rather than hyperplasia as the underlying morphological correlate of the hypersecretion.
In summary, SO2-exposure can lead to characteristic airway remodeling and changes in mucus secretion in rats. As only long-term exposure to 20 ppm leads to a combination of hypersecretion and airway inflammation, only this mode of exposure should be used to mimic human COPD. Concentrations less or higher than 20 ppm or short term exposure do not induce the respiratory symptom of hypersecretion. The present model may be used to characterize the effects of new compounds on mucus secretion in the background of experimental COPD.
Cadmium (Cd) has been in industrial use for a long period of time. Its serious toxicity moved into scientific focus during the middle of the last century. In this review, we discuss historic and recent developments of toxicological and epidemiological questions, including exposition sources, resorption pathways and organ damage processes.