Mechanism of thrombin clearance by human astrocytoma cells

Astroglial cells secrete a variety of factors that contribute to the regulation of neurite initiation and continued outgrowth, among them proteases and protease inhibitors. An alteration in the balance between these proteins has been implicated in Alzheimer's disease, resulting in an accumulation of thrombin:protease nexin 1 (PN1) complexes in the brains of these patients. This report aims at providing a biochemical explanation for this phenomenon. We show that human astrocytoma cells bind and internalize thrombin and thrombin:PN1 complexes efficiently by a PN1-dependent mechanism. Binding was potently inhibited by soluble heparin and did not occur with the mutant PN1 (K7E) deficient in heparin binding. Receptor-associated protein, an antagonist of the low-density lipoprotein receptor-related protein (LRP), inhibited internalization of thrombin by the astrocytoma cells, but did not affect cell-surface binding. The results are consistent with a mechanism by which astrocytoma cells clear thrombin in a sequential manner: thrombin is first complexed with PN1, then bound to cell-surface heparins, and finally internalized by LRP. This mechanism provides a link between the neuronal growth regulators thrombin and PN1 and proteins genetically associated with Alzheimer's disease, such as LRP.     

Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism. The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or internalization

We have previously described thrombin (Th)-protease nexin 1 (PN1) inhibitory complex binding to cell surface heparins and subsequent low density lipid receptor-related protein (LRP)-mediated internalization. Our present studies examine the catabolism of urinary plasminogen activator (uPA)-PN1 inhibitory complexes, which, unlike Th.PN1 complexes, bind almost exclusively through the uPA receptor. In addition, the binding site in PN1 required for the LRP-mediated internalization of Th.PN1 complexes is not required for the LRP-mediated internalization of uPA.PN1 complexes. Thus, the protease moiety of the complex partially determines the mechanistic route of entry. Because cell surface heparins are only minimally involved in the binding and internalization of uPA.PN1 complexes, we then predicted that complexes between uPA and the heparin binding-deficient PN1 variant, PN1(K7E), should be catabolized at the same rate as complexes formed with native PN1. Surprisingly, the uPA.PN1(K7E) complexes were degraded at only a fraction of the rate of native complexes. Internalization studies revealed that both uPA. PN1(K7E) and native uPA.PN1 complexes were initially internalized at the same rate, but uPA.PN1(K7E) complexes were rapidly retro-endocytosed in an intact form. By examining the pH dependence of complex binding in the range of 4.0-7.0, it was determined that the uPA.PN1 inhibitory complexes must specifically bind to endosomal heparins at pH 5.5 to be retained and sorted to lysosomes. These studies are the first to document a role for heparins in the catabolism of SERPIN-protease complexes at a point further in the pathway than cell surface binding, and this role may extend to other heparin-binding LRP-internalized ligands.     

Apolipoprotein E levels in cerebrospinal fluid and the effects of ABCA1 polymorphisms

Background

Animal studies suggest that brain apolipoprotein E (apoE) levels influence amyloid-β (Aβ) deposition and thus risk for Alzheimer's disease (AD). We have previously demonstrated that deletion of the ATP-binding cassette A1 transporter (ABCA1) in mice causes dramatic reductions in brain and cerebrospinal fluid (CSF) apoE levels and lipidation. To examine whether polymorphisms in ABCA1 affect CSF apoE levels in humans, we measured apoE in CSF taken from 168 subjects who were 43 to 91 years old and were either cognitively normal or who had mild AD. We then genotyped the subjects for ten previously identified ABCA1 single nucleotide polymorphisms (SNPs).

Results

In all subjects, the mean CSF apoE level was 9.09 μg/ml with a standard deviation of 2.70 μg/ml. Levels of apoE in CSF samples taken from the same individual two weeks apart were strongly correlated (r² = 0.93, p < 0.01). In contrast, CSF apoE levels in different individuals varied widely (coefficient of variation = 46%). CSF apoE levels did not vary according to AD status, APOE genotype, gender or race. Average apoE levels increased with age by ~0.5 μg/ml per 10 years (r² = 0.05, p = 0.003). We found no significant associations between CSF apoE levels and the ten ABCA1 SNPs we genotyped. Moreover, in a separate sample of 1225 AD cases and 1431 controls, we found no association between the ABCA1 SNP rs2230806 and AD as has been previously reported.

Conclusion

We found that CSF apoE levels vary widely between individuals, but are stable within individuals over a two-week interval. AD status, APOE genotype, gender and race do not affect CSF apoE levels, but average CSF apoE levels increase with age. Given the lack of association between CSF apoE levels and genotypes for the ABCA1 SNPs we examined, either these SNPs do not affect ABCA1 function or if they do, they do not have strong effects in the CNS. Finally, we find no evidence for an association between the ABCA1 SNP rs2230806 and AD in a large sample set.     

Rational design of 7-arylquinolines as non-competitive metabotropic glutamate receptor subtype 5 antagonists

Rational replacement of the alkyne linker of mGluR5 antagonist MPEP gave 7-arylquinolines. SAR optimization gave an orally active compound with high affinity for the MPEP binding site.     

Synthesis and SAR of 2-aryl pyrido[2,3-d]pyrimidines as potent mGlu5 receptor antagonists

A novel series of potent 2-aryl pyrido[2,3-d]pyrimidine mGlu5 receptor antagonists are described. The synthesis and pharmacological activities of these analogs are discussed.     

A study to determine the impact of IMPT optimization techniques on prostate synthetic CT image sets dose comparison against CT image sets

BackgroundThe objective of this study is to determine the impact of intensity modulated proton therapty (IMPT) optimization techniques on the proton dose comparison of commercially available magnetic resonance for calculating attenuation (MRCA T) images, a synthetic computed tomography CT (sCT) based on magnetic resonance imaging (MRI) scan against the CT images and find out the optimization technique which creates plans with the least dose differences against the regular CT image sets.Material and methodsRegular CT data sets and sCT image sets were obtained for 10 prostate patients for the study. Six plans were created using six distinct IMPT optimization techniques including multi-field optimization (MFO), single field uniform dose (SFUD) optimization, and robust optimization (RO) in CT image sets. These plans were copied to MRCA T, sCT datasets and doses were computed. Doses from CT and MRCA T data sets were compared for each patient using 2D dose distribution display, dose volume histograms (DVH), homogeneity index (HI), conformation number (CN) and 3D gamma analysis. A two tailed t-test was conducted on HI and CN with 5% significance level with a null hypothesis for CT and sCT image sets.ResultsAnalysis of ten CT and sCT image sets with different IMPT optimization techniques shows that a few of the techniques show significant differences between plans for a few evaluation parameters. Isodose lines, DVH, HI, CN and t-test analysis shows that robust optimizations with 2% range error incorporated results in plans, when re-computed in sCT image sets results in the least dose differences against CT plans compared to other optimization techniques. The second best optimization technique with the least dose differences was robust optimization with 5% range error.ConclusionThis study affirmatively demonstrates the impact of IMPT optimization techniques on synthetic CT image sets dose comparison against CT images and determines the robust optimization with 2% range error as the optimization technique which gives the least dose difference when compared to CT plans.     

Bee Tracker—an open‐source machine learning‐based video analysis software for the assessment of nesting and foraging performance of cavity‐nesting solitary bees

The foraging and nesting performance of bees can provide important information on bee health and is of interest for risk and impact assessment of environmental stressors. While radiofrequency identification (RFID) technology is an efficient tool increasingly used for the collection of behavioral data in social bee species such as honeybees, behavioral studies on solitary bees still largely depend on direct observations, which is very time‐consuming. Here, we present a novel automated methodological approach of individually and simultaneously tracking and analyzing foraging and nesting behavior of numerous cavity‐nesting solitary bees. The approach consists of monitoring nesting units by video recording and automated analysis of videos by machine learning‐based software. This Bee Tracker software consists of four trained deep learning networks to detect bees that enter or leave their nest and to recognize individual IDs on the bees’ thorax and the IDs of their nests according to their positions in the nesting unit. The software is able to identify each nest of each individual nesting bee, which permits to measure individual‐based measures of reproductive success. Moreover, the software quantifies the number of cavities a female enters until it finds its nest as a proxy of nest recognition, and it provides information on the number and duration of foraging trips. By training the software on 8 videos recording 24 nesting females per video, the software achieved a precision of 96% correct measurements of these parameters. The software could be adapted to various experimental setups by training it according to a set of videos. The presented method allows to efficiently collect large amounts of data on cavity‐nesting solitary bee species and represents a promising new tool for the monitoring and assessment of behavior and reproductive success under laboratory, semi‐field, and field conditions.     

Expansion of Brown Bears (Ursus arctos) into the Eastern Alps: A Spatially Explicit Population Model

We present a spatially explicit population model for analysing the expansion of brown bears (Ursus arctos) after the reintroduction program in central Austria. The model is based on field investigations into brown bears in Austria and Slovenia and on current knowledge of brown bears. The landscape of the eastern Alps is represented by a GIS-derived raster map defining local habitat suitability and five major spatial barriers to dispersal. The population model follows the fate of individual bears and simulates reproduction, dispersal, home range establishment, and mortality in annual time steps. We indirectly adjust unknown or uncertain model parameters with 10-year data on the number of females with cubs in central Austria and determine key variables of population dynamics, such as population sizes and growth rates within different population nuclei, dispersal distances, or mortality rates, for model parameterisations that reproduce the data on females with cubs. We estimated a current (1996–2000) growth rate of the population in Austria and adjacent parts of Italy of some 14%; a high proportion of this growth was due toimmigration from Slovenia. Consequently, the growth rate of the subpopulation in central Austria, which probably is isolated functionally (i.e., no exchange of females) from the nuclei along the Austrian–Slovenian border, yielded some 7%. This subpopulation may comprise seven residents, and we estimated for females a 33% risk of extinction during the 1992–2000 period. Validation and confirmation of our model results with data on bear densities that were not used for model construction and parameterisation supported our findings. The high female mortality rates, together with the vulnerability of the small population to chance events (i.e., demographic stochasticity), are the most pressing threat for the population in the eastern Alps. Our approach could be widely applied for analysing dynamics of rare and endangered species in which the paucity of data precludes an appraisal of the state of the population using standard methods.     

An otoprotective role for the apoptosis inhibitor protein survivin

Hearing impairment caused by ototoxic insults, such as noise or gentamicin is a worldwide health problem. As the molecular circuitries involved are not yet resolved, current otoprotective therapies are rather empirical than rational. Here, immunohistochemistry and western blotting showed that the cytoprotective protein survivin is expressed in the human and guinea pig cochlea. In the guinea pig model, moderate noise exposure causing only a temporary hearing impairment transiently evoked survivin expression in the spiral ligament, nerve fibers and the organ of Corti. Mechanistically, survivin upregulation may involve nitric oxide (NO)-induced Akt signaling, as enhanced expression of the endothelial NO synthase and phosphorylated Akt were detectable in some surviving-positive cell types. In contrast, intratympanic gentamicin injection inducing cell damage and permanent hearing loss correlated with attenuated survivin levels in the cochlea. Subsequently, the protective activity of the human and the guinea pig survivin orthologs against the ototoxin gentamicin was demonstrated by ectopic overexpression and RNAi-mediated depletion studies in auditory cells in vitro. These data suggest that survivin represents an innate cytoprotective resistor against stress conditions in the auditory system. The pharmacogenetic modulation of survivin may thus provide the conceptual basis for the rational design of novel therapeutic otoprotective strategies.