High-Frequency Stimulation of the Subthalamic Nucleus Counteracts Cortical Expression of Major Histocompatibility Complex Genes in a Rat Model of Parkinson’s Disease

High-frequency stimulation of the subthalamic nucleus (STN-HFS) is widely used as therapeutic intervention in patients suffering from advanced Parkinson’s disease. STN-HFS exerts a powerful modulatory effect on cortical motor control by orthodromic modulation of basal ganglia outflow and via antidromic activation of corticofugal fibers. However, STN-HFS-induced changes of the sensorimotor cortex are hitherto unexplored. To address this question at a genomic level, we performed mRNA expression analyses using Affymetrix microarray gene chips and real-time RT-PCR in sensorimotor cortex of parkinsonian and control rats following STN-HFS. Experimental parkinsonism was induced in Brown Norway rats by bilateral nigral injections of 6-hydroxydopamine and was assessed histologically, behaviorally, and electrophysiologically. We applied prolonged (23h) unilateral STN-HFS in awake and freely moving animals, with the non-stimulated hemisphere serving as an internal control for gene expression analyses. Gene enrichment analysis revealed strongest regulation in major histocompatibility complex (MHC) related genes. STN-HFS led to a cortical downregulation of several MHC class II (RT1-Da, Db1, Ba, and Cd74) and MHC class I (RT1CE) encoding genes. The same set of genes showed increased expression levels in a comparison addressing the effect of 6-hydroxydopamine lesioning. Hence, our data suggest the possible association of altered microglial activity and synaptic transmission by STN-HFS within the sensorimotor cortex of 6-hydroxydopamine treated rats.     

Crucial Role for Neuronal Nitric Oxide Synthase in Early Microcirculatory Derangement and Recipient Survival following Murine Pancreas Transplantation

Objective

Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation.

Background

Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined.

Methods

Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days.

Results

Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin.

Conclusion

We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.     

Expression of Eukaryotic Initiation Factor 5A and Hypusine Forming Enzymes in Glioblastoma Patient Samples: Implications for New Targeted Therapies

Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA) could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity.     

Predictive Mapping of Plant Species and Communities Using GIS and Landsat Data in a Southern Mongolian Mountain Range

We assessed presence/absence prediction of plant species and communities in a southern Mongolian mountain range from geospatial data using a randomized sampling approach. One hundred randomized vegetation samples (3?×?3 m) were collected within the 2?×?2 km summit region of the Dund Saykhan range, which forms part of the core zone of the Gobi Gurvan Saykhan National Park in arid southern Mongolia. Using logistic regression, habitat preference models for all abundant species (n?=?52) and communities (n?=?5) were constructed; predictors were derived from Landsat 5 imagery and a digital elevation model. Nagelkerkes r ² was used for an initial data mining, and all significant models were validated by splitting the data and using one half for accuracy assessment based on the AUC (Area Under the receiver operating characteristic Curve)-values. Significant models could be built for half of the species. Altitude proved to be the most important predictor followed by variables derived from Landsat data. The clear altitudinal distribution patterns most definitely reflect precipitation; overall biodiversity in this arid environment is widely controlled by moisture availability. The chosen approach may prove valuable for applied studies wherever spatial data on species distributions are required for conservation efforts.     

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse,a model of Huntington’s disease

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca²⁺ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca²⁺ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca²⁺ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca²⁺ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca²⁺ removal from the myoplasm and Ca²⁺ release flux from the sarcoplasmic reticulum were characterized using a Ca²⁺ binding and transport model, which indicated a significant reduction in slow Ca²⁺ removal activity and Ca²⁺ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca²⁺ channel conductance. These results indicate profound changes of Ca²⁺ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.     

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

Phytochromes are photoreceptors using a bilin tetrapyrrole as chromophore, which switch in canonical phytochromes between red (P_r) and far red (P_fr) light-absorbing states. Cph2 from Synechocystis sp., a noncanonical phytochrome, harbors besides a cyanobacteriochrome domain a second photosensory module, a P_r/P_fr-interconverting GAF-GAF bidomain (SynCph2(1-2)). As in the canonical phytochromes, a unique motif of the second GAF domain, the tongue region, seals the bilin-binding site in the GAF1 domain from solvent access. Time-resolved spectroscopy of the SynCph2(1-2) module shows four intermediates during P_r → P_fr phototransformation and three intermediates during P_fr → P_r back-conversion. A mutation in the tongue''s conserved PRXSF motif, S385A, affects the formation of late intermediate R3 and of a P_fr-like state but not the back-conversion to P_r via a lumi-F-like state. In contrast, a mutation in the likewise conserved WXE motif, W389A, changes the photocycle at intermediate R2 and causes an alternative red light-adapted state. Here, back-conversion to P_r proceeds via intermediates differing from SynCph2(1-2). Replacement of this tryptophan that is ∼15 Å distant from the chromophore by another aromatic amino acid, W389F, restores native P_r → P_fr phototransformation. These results indicate large scale conformational changes within the tongue region of GAF2 during the final processes of phototransformation. We propose that in early intermediates only the chromophore and its nearest surroundings are altered, whereas late changes during R2 formation depend on the distant WXE motifs of the tongue region. Ser-385 within the PRXSF motif affects only late intermediate R3, when refolding of the tongue and docking to the GAF1 domain are almost completed.     

2,5-Diketopiperazines as potent and selective oxytocin antagonists 1: Identification, stereochemistry and initial SAR

This paper covers efforts to discover orally active potent and selective oxytocin antagonists. Screening pooled libraries identified a novel series of 2,5-diketopiperazine derivatives with antagonist activity at the human oxytocin receptor. We report the initial structure-activity relationship investigations and the determination of the stereochemistry of the most potent compounds.