Interaction of the TNFR-receptor associated factor TRAF1 with I-kappa B kinase 2 (IKK2, IKK-beta, IKBKB) and TRAF2 indicating a dose dependent regulatory function of TRAF1 for NF-kappa B signaling

IKK2 is one of the most crucial signaling kinases for activation of the transcription factor NF-kappa B. Since many NF-kappa B activating pathways converge at the level of IKK2, we searched for interaction partners of this kinase using the C-terminal part (aa 466-756) as bait in a yeast two-hybrid system. We identified the N-terminal part (aa 1-228) of the TNF-receptor associated factor TRAF1 as putative interaction partner, which was subsequently confirmed in mammalian cells by coimmunoprecipitation experiments. However, this interaction seemed weaker than the interaction between TRAF1 and TRAF2, an important activating adapter molecule of NF-kappa B signaling indicating that relative levels of IKK2, TRAF1 and TRAF2 might be important for the final biological readout. Reporter gene and kinase assays using ectopic expression of TRAF1 indicated that it can have both activating and inhibiting functions for IKK2 and NF-kappa B. Co-expression of fluorescently tagged TRAF1 and TRAF2 at different ratios implied that TRAF1 can affect clustering and presumably the activating function of TRAF2 in a dose dependent manner

The actin-binding protein profilin II in neuronal plasticity

Activity-dependent plasticity in neurons involves changes in synaptic transmission and connectivity. These changes lead to altered neuronal circuit properties and are thought to underlie learning and memory. Transcription and protein synthesis are indispensable in order to maintain changes in neural circuitry over periods of several hours or longer. Therefore signaling from the synapse to the nucleus is required to control activity-dependent expression of RNA and proteins which have to be transported back to the activated synaptic sites. The small actin-binding protein profilin has been shown to accumulate in postsynaptic dendritic spines of pyramidal neurons as a necessary element in activity-dependent stabilization of synaptic morphology, a putative anatomical correlate of changes in transmission strength. In this work I show that profilin also enters the nucleus in an NMDA receptor and Ca2+ dependent manner. However, in contrast to spine targeting, nuclear enrichment is reversible within minutes after removal of the stimulus. Nuclear accumulation of profilin is likely coupled to activity-dependent actin polymerization at the cell cortex which also takes place in response to NMDA receptor stimulation. Nuclear profilin has been implicated in different steps of gene expression including transcription and pre-mRNA splicing. Activity-dependent nuclear and synaptic accumulation suggests profilin to be involved in different aspects of neuronal plasticity. To this end, I introduce approaches to elucidate profilin function in experience-dependent plasticity and gene expression

(A) Vision for 2050 - Context-Based Image Understanding for a Human-Robot Soccer Match

We believe it is possible to create the visual subsystem needed for the RoboCup 2050 challenge - a soccer match between humans and robots - within the next decade.  In this position paper, we argue, that the basic techniques are available, but the main challenge will be to achieve the necessary robustness. We propose to address this challenge through the use of probabilistically modeled context, so for instance a visually indistinct circle is  accepted as the ball, if it fits well with the ball's motion model and vice versa.Our vision is accompanied by a sequence of (partially already conducted) experiments for its verification.  In these experiments, a human soccer player carries a helmet with a camera and an inertial sensor and the vision system has to extract all information from that data, a humanoid robot would need to take the human's place

Steroid-eluting epicardial pacing in children

Background: Permanent cardiac pacing is a known method of the treatment for children with bradycardia. Epicardial pacing is required in small children and in children with some congenital heart defects. Steroid-eluting leads (SEL) have been introduced to reduce implant site fibrosis and to retain permanent low-pacing threshold values. Our aim was to evaluate the pacing characteristic and follow-up of children with epicardial steroid-eluting pacing. Material and methods: We implanted steroid-eluting epicardial pacing systems in 53 children (age at implantation: 2 days - 17.5 years, mean 4.6 years), of whom 37 (70%) had congenital heart disease and 22 of these had already had cardiac surgery. These children formed group I. Group II was the control group and consisted of 29 children (age at implantation: 10 days - 13 years, mean 6.5 years) with non-steroid epicardial pacing systems; 15 patients (51%) had diagnosed congenital heart disease, and 9 of these had had cardiac surgery. The pacing threshold (PT) was obtained during implantation, before discharge, 1-3 and 6 months following implantation and then every 6 months. Results: In group I the mean PT during the implantation procedure was 1.6 V/0.4 ms and decreased significantly before discharge. In group II the PT during the implantation procedure was low and increased before discharge. In group I the PT of the atrial leads was low and stable during the follow-up period, while the PT of the ventricular leads slowly increased and four years following implantation was similar to that of group II. Conclusions: In children with permanent epicardial pacing the ventricular PT was significantly lower when steroid-eluting leads had been used then when these had not been used, but during the follow-up period the PT slowly increased, while the pacing threshold of atrial steroid-eluting leads remained stable

Functional Remodeling of Benign Human Prostatic Tissues In Vivo by Spontaneously Immortalized Progenitor and Intermediate Cells

Tissue remodeling or regeneration is believed to initiate from multipotent stem and progenitor cells. We report here the establishment of two spontaneously immortalized adult non-tumorigenic human prostate epithelial cell lines, NHPrE1 and BHPrE1. NHPrE1 (CD133high/CD44high/OCT4high/PTENhigh) was characterized as a putative progenitor cell, and BHPrE1 (p63high/p53high/p21(WAF1)high/RBhigh) was characterized as a putative epithelial intermediate cell. Genomic analysis demonstrated an abnormal karyotype with genomic rearrangements including PTEN amplification in NHPrE1 and CTNNB1 (β-catenin) amplification in BHPrE1 cells. Embedded three-dimensional culture of NHPrE1 showed greater branching than BHPrE1. A tissue recombination-xenografting model was utilized to compare remodeling of human prostatic tissues in vivo. A series of tissue recombinants, made by mixing different ratios of human prostatic epithelial cells and inductive rat urogenital sinus mesenchyme, were grafted to the renal capsule of severe combined immunodeficient mice. Both cell lines were able to regenerate benign secretory ductal-acinar architecture in vivo, containing intact basal and luminal epithelial layers confirmed by the expression of appropriate CK profiles. Prostate-specific antigen, 15-lipoxygenase-2, androgen receptor, and NKX3.1 proteins were appropriately expressed in the regenerated epithelia. Regeneration of benign prostatic glandular structures could be achieved using as few as 10 NHPrE1 cells, whereas 200,000 BHPrE1 cells were required to achieve prostatic architecture. This suggests a greater proportion of progenitor/stem cells in NHPrE1 than in BHPrE1. These cell lines provide important data on progenitor and intermediate cell phenotypes and represent significant new tools for the elucidation of molecular mechanisms of human prostatic regeneration, pathogenesis, and carcinogenesis

Chronic hypoxemia increases myocardial cytochrome oxidase

ObjectiveCyanotic patients have potentially decreased tissue oxygen tension. Cytochrome oxidase catalyzes the reduction of oxygen and is integral to adenosine triphosphate production. Cytochrome oxidase subunit I, the active site, is encoded by mitochondrial DNA. Using a newborn swine model of chronic hypoxemia, we evaluated ventricular cytochrome oxidase subunit I mRNA and protein expression and assessed cytochrome oxidase activity.MethodsThirty-two newborn piglets underwent thoracotomy and placement of a pulmonary artery–to–left atrium shunt or sham operation. Two weeks later, partial pressure of arterial oxygen, hematocrit, and left ventricular shortening fraction values were compared with baseline values. Northern blot hybridization and protein immunoblotting for ventricular cytochrome oxidase subunit I were performed. Cytochrome oxidase kinetic activity was measured. Heme a,a3 content and turnover number were determined. Significance was assessed with a t test.ResultsBaseline partial pressure of arterial oxygen and hematocrit values were similar. Hypoxemic piglets had a lower partial pressure of arterial oxygen of 38 ± 10 mm Hg (P < .001) and higher hematocrit value of 31.4% ± 2.9% (P < .001) compared with a partial pressure of arterial oxygen of 140 ± 47 mm Hg and hematocrit value of 24.6% ± 3.9% after the sham operation. Baseline and postprocedure left ventricular shortening fraction were similar within and between groups. Chronic hypoxemia increased right ventricular and left ventricular cytochrome oxidase I mRNA and protein by more than 1.4-fold. Cytochrome oxidase activity increased significantly in hypoxemia by 2.5-fold compared with that seen after the sham operation. Heme a,a3 content and turnover number increased by 1.5-fold during hypoxemia.ConclusionsChronic hypoxemia increases cytochrome oxidase I message, protein expression, and activity. The increase in kinetics was due to increased enzyme content and catalytic activity. This is a possible adaptive mechanism that might preserve organ function during chronic hypoxemia

Elektrody nasierdziowe uwalniające sterydy w stałej stymulacji serca u dzieci

Wstęp: Stała stymulacja serca jest uznaną metodą leczenia bradyarytmii u dzieci. U małych dzieci i w niektórych patologiach układu krążenia konieczne jest zastosowanie elektrod nasierdziowych. Przyjmuje się, że sterydy uwalniane na końcu elektrod nasierdziowych, zmniejszając miejscowe włóknienie, przyczyniają się do utrzymania niskiego progu stymulacji serca. Celem pracy była ocena przebiegu stymulacji serca u dzieci z EP z elektrodami uwalniającymi sterydy. Materiał i metody: Pierwszą badaną grupę stanowiło 53 dzieci z EP z elektrodami uwalniającymi sterydy, w wieku od 2 doby życia do 17,5 roku (śr. 4,6 roku) w momencie zabiegu. U 37 (70%) z nich rozpoznano wrodzoną wadę serca, 22 przebyło operację serca. Do grupy II - kontrolnej zakwalifikowano 29 dzieci z EP niesterydowymi wszczepionymi w wieku od 10 doby życia do 13 lat (śr. 6,5 roku). U 15 (51%) rozpoznano wrodzoną wadę serca, 9 leczono kardiochirurgicznie. Przebieg stymulacji analizowano retro- i prospektywnie. Progi stymulacji (PT) mierzono w czasie wszczepienia EP, przy wypisie, po 1&#8211;3 oraz po 6 miesiącach od zabiegu, a następnie co pół roku. Wyniki: U pacjentów z I grupy PT podczas wszczepienia EP wynosił średnio 1,6 V/0,4 ms i istotnie obniżał się już w pierwszych dniach po zabiegu, natomiast u pacjentów z grupy II początkowo niski PT, później narastał. W okresie obserwacji PT elektrod przedsionkowych uwalniających sterydy były stabilne, a komorowych powoli wzrastały i po 4 latach od zabiegu były porównywalne z PT elektrod niesterydowych. Wnioski: U dzieci z EP progi stymulacji komorowej są istotnie niższe po zastosowaniu elektrod uwalniających sterydy w porównaniu z elektrodami niesterydowymi, jednak powoli się podnoszą

Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137

Profilin-1 (PFN1) plays an important role in the control of actin dynamics, and could represent an important therapeutic target in several diseases. We previously identified PFN1 as a huntingtin aggregation inhibitor, and others have implicated it as a tumor-suppressor. Rho-associated kinase (ROCK) directly phosphorylates PFN1 at Ser-137 to prevent its binding to polyproline sequences. This negatively regulates its anti-aggregation activity. However, the phosphatase that dephosphorylates PFN1 at Ser-137, and thus activates it, is unknown. Using a phospho-specific antibody against Ser-137 of PFN1, we characterized PFN1 dephosphorylation in cultured cells based on immunocytochemistry and a quantitative plate reader-based assay. Both okadaic acid and endothall increased pS137-PFN1 levels at concentrations more consistent with their known IC50s for protein phosphatase 1 (PP1) than protein phosphatase 2A (PP2A). Knockdown of the catalytic subunit of PP1 (PP1Cα), but not PP2A (PP2ACα), increased pS137-PFN1 levels. PP1Cα binds PFN1 in cultured cells, and this interaction was increased by a phosphomimetic mutation of PFN1 at Ser-137 (S137D). Together, these data define PP1 as the principal phosphatase for Ser-137 of PFN1, and provide mechanistic insights into PFN1 regulation by phosphorylation

Preserved Morphology and Physiology of Excitatory Synapses in Profilin1-Deficient Mice

Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity