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Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel alpha-conotoxin (alpha-TxIA) in the venom of Conus textile. alpha-TxIA bound with high affinity to AChBPs from different species and selectively targeted the alpha3beta2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.
Background The purpose of the trial was to evaluate the safety and performance of the new Protégé stent in the treatment of common and/or internal carotid artery stenoses. Methods The Protégé® GPS stent is a self-expanding Nitinol stent system. It is mounted on a 6 Fr 0.018” (6-9mm stent) or 7 Fr, 0.035” (10mm stent) over-the-wire-delivery system and includes a new stent release system which allows exact placement of the stent. Seventyseven patients were enrolled in the trial. Study patient assessments were conducted clinically and by duplex scan at baseline, peri-procedure, discharge, one and six months post procedure. Results Seventyseven lesions were treated. Thirtyone lesions were symptomatic, 46 lesions were asymptomatic. The procedure was technically successful in 76 (99%) lesions. The percentage of stenosis was reduced from 86 ± 7 % to 16 ± 8 %. One procedure failed because the embolic protection device could not be retrieved and the patient was sent to surgery. Within 30 days there were 4 (5.2%) Major Adverse Neurological Events (MANEs). Three of the MANEs were major strokes (3.9%), one a minor stroke. The fifth MANE occurred prior to the six month follow-up visit; this patient had a major stroke 75 days after the procedure and died 36 days later. One additional death occurred due to urosepsis. Conclusions The trial shows that the Protégé stent satisfies safety and performance criteria for the treatment of carotid artery stenosis. The complication rate was comparable to the incidence of these events in other recent carotid stent and endarterectomy studies.
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
In the European Union (EU), health policy and the institutional reform of health systems have been treated primarily as national affairs, and health care systems within the EU thus differ considerably. However, the health policy field is undergoing a dynamic process of Europeanization. This process is stimulated by the orientation towards a more competitive economy, recently inaugurated and known as the Lisbon Strategy, while the regulatory requirements of the European Economic and Monetary Union are stimulating the Europeanization of health policy. In addition, the so-called open method of coordination, representing a new mode of regulation within the European multi-level system, is applied increasingly to the health policy area. Diverse trends are thus emerging. While the Lisbon Strategy goes along with a strategic upgrading of health policy more generally, health policy is increasingly used to strengthen economic competitiveness. Pressure on Member States is expected to increase to contain costs and promote market-based health care provision.
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: The evaluation of local mental health care remains difficult. For this reason systematic development of appropriate services is barely possible.
Methods: We examined involuntary hospitalization in the city of Frankfurt/Main with regard to diagnoses, socio-demographic data, complementary psychosocial outpatient care, and circumstances of hospitalization. There are four psychiatric clinics, each serving a catchment area of more than 165.000 inhabitants. These clinics are responsible for all psychiatric in-patient treatments regardless of the admission modus. During a one year period, 677 patients were involuntarily hospitalized. Statistical analyses were performed subsequent to pooling the data.
Results: During a period of one year, 103 out of 100.000 inhabitants of Frankfurt/Main were admitted involuntarily. The rate of involuntary admissions related to all admissions was 10.98 percent. Any complementary psychosocial care was missing in more than 70 percent of patients admitted involuntarily. Only about 10 percent of patients were examined by a physician before reaching the hospital and in disappointing 1.3 percent the municipal mental health service had been consulted prior to involuntarily admission.
Conclusion: Our results show that a systematic improvement of precautionary complementary psychosocial care for risk patients is needed as well as the obligation of psychiatric emergency consultation before involuntary hospitalization.
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: The purpose of this study was to evaluate the feasibility and short-term efficacy of transcatheter paravalvular leak closure using different occlusion devices. Methods and Results: Twenty one patients underwent transcatheter closure of either aortic or mitral paravalvular leak from June 2002 to February 2006 using the Amplatzer PDA, ASD or VSD occluder. All patients had symptoms and signs of haemolysis and/or cardiac decompensation with dyspnoea. Implantation of a device was technically successful in twenty patients (95 %). Immediate residual leak was found in seventeen patients (85 %). Significant shunting persisted in nine cases during follow up (45 %). Permanent leaflet obstruction was observed in one patient. Severe complications during follow up led to early death in one patient and surgical intervention in three. A successful second catheter treatment was performed in another three patients. The event-free survival from reoperation, death and stroke at the end of the observation period was 80 %. Conclusion: Transcatheter closure of paravalvular leaks is a technically feasible, but demanding procedure. Residual leaks are common and may worsen pre-existing haemolysis. Due to the significant ongoing morbidity in this group of patients and the complexity of follow up individual patient results differ considerably. Nevertheless, it is possible to achieve some symptomatic relief, thus an interventional approach should be discussed as a potential treatment option for those patients with a limited defect and who are not deemed suitable for another operation.
Background: False aneurysms at the puncture site develop in up to 8 % after catheter procedures. They can be treated surgically or by ultrasound guided manual compression. A new method is to inject thrombin into the aneurysm under ultrasound guidance. We evaluated safety and efficacy of this approach in a multicenter registry. Methods: In 595 consecutive patients (age: 31-94 years, median 70) a pseudoaneurysm (593 femoral arteries, 2 brachial arteries) was diagnosed 0 to 250 days (median 3 days) after a catheter procedure. The diameter of the aneurysm ranged from 0.5 x 0.5 x 0.5 (L x W x D) to 8x11x16 cm (median 2 x 2 x1.6 cm). 20 U to 4000 U of thrombin solution (median 400 U) were injected percutaneously into the aneurysm under ultrasound guidance. Results: The procedure was technically successful in 587/595 (99%) patients. The aneurysms were thrombosed after the first injection in 531 patients (89 %). Thirty-eight (6%) patients needed a second injection and 8 (1%) patients a third injection because residual flow in the aneurysm was visible at follow-up. In 4 additional patients (0.7%) the thrombosis of the aneurysms was delayed and occurred only after 24 hours to 7 days. 6 (1%) patients surgery was performed after successful closure of the aneurysm to remove the resulting haematoma. The overall technical success rate was 99% (587/595) and clinical success was achieved in 572/595 (96%) patients. Eight (1%) other patients underwent surgery due to thrombin injection failure. Complications occurred in 9 patients (1.5%): Intravascular thrombus formation (n=3), deep venous thrombosis (n=3), pulmonary embolism due to deep venous thrombosis (n=1), transient paresthesia in the leg during injection (n=3). Conclusion: Ultrasound guided thrombin injection is a safe, painless, effective and rapid alternative to treat false aneurysms. Complications and recurrent pseudoaneurysms are very rare. It has become the treatment of choice in our institution.
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.
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.
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.
Background Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-alpha) and interleukin-1beta (IL-1beta). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo. Methods Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured. Results In WT mice, CpG-ODN induced a strong activation of pulmonary NFKB as well as a significant increase in pulmonary TNF-alpha and IL-1beta mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice. Conclusion This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.
Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP, thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an amino-terminal region, two regulatory GAF domains, and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B; however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze whether PDE2A could affect the function of XAP2, we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. Furthermore, PDE2A attenuated TCDD-induced transcription in reporter gene assays. We conclude that XAP2 targets PDE2A to the AhR complex, thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.
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.
The majority of cells are equipped to detect and decipher physical stimuli, and then react to these stimuli in a cell type-specific manner. Ultimately, these cellular behaviors are synchronized to produce a tissue response, but how this is achieved remains enigmatic. Here, we investigated the genetic basis for mechanotransduction using the bone marrow as a model system. We found that physical stimuli produced a pattern of principal strain that precisely corresponded to the site-specific expression of sox9 and runx2, two transcription factors required for the commitment of stem cells to a skeletogenic lineage, and the arrangement and orientation of newly deposited type I collagen fibrils. To gain insights into the genetic basis for skeletal mechanotransduction we conditionally inactivated focal adhesion kinase (FAK), an intracellular component of the integrin signaling pathway. By doing so we abolished the mechanically induced osteogenic response and thus identified a critical genetic component of the molecular machinery required for mechanotransduction. Our data provide a new framework in which to consider how physical forces and molecular signals are synchronized during the program of skeletal regeneration.
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.
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.
Visual selective attention and visual working memory (WM) share the same capacity-limited resources. We investigated whether and how participants can cope with a task in which these 2 mechanisms interfere. The task required participants to scan an array of 9 objects in order to select the target locations and to encode the items presented at these locations into WM (1 to 5 shapes). Determination of the target locations required either few attentional resources (“popout condition”) or an attention-demanding serial search (“non pop-out condition”). Participants were able to achieve high memory performance in all stimulation conditions but, in the non popout conditions, this came at the cost of additional processing time. Both empirical evidence and subjective reports suggest that participants invested the additional time in memorizing the locations of all target objects prior to the encoding of their shapes into WM. Thus, they seemed to be unable to interleave the steps of search with those of encoding. We propose that the memory for target locations substitutes for perceptual pop-out and thus may be the key component that allows for flexible coping with the common processing limitations of visual WM and attention. The findings have implications for understanding how we cope with real-life situations in which the demands on visual attention and WM occur simultaneously. Keywords: attention, working memory, interference, encoding strategies