(−)-Reboxetine inhibits muscle nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites

The interaction of (−)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (−)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca²⁺ influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC₅₀ = 3.86 ± 0.49 and 1.92 ± 0.48 μM, respectively, (b) binds to the [³H]TCP site with ∼13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [³H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6′ and 13′ in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9′ is the minimum structural component for (−)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (−)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants.     

Identification of a new membrane-permeable inhibitor against inositol-1,4,5-trisphosphate-3-kinase A

Ectopic expression of the neuron-specific inositol-1,4,5-trisphosphate-3-kinase A (ITPKA) in lung cancer cells increases their metastatic potential because the protein exhibits two actin regulating activities; it bundles actin filaments and regulates inositol-1,4,5-trisphosphate (InsP₃)-mediated calcium signals by phosphorylating InsP₃. Thus, in order to inhibit the metastasis-promoting activity of ITPKA, both its actin bundling and its InsP₃kinase activity has to be blocked. In this study, we performed a high throughput screen in order to identify specific and membrane-permeable substances against the InsP₃kinase activity. Among 341,44 small molecules, 237 compounds (0.7%) were identified as potential InsP₃kinase inhibitors. After determination of IC₅₀-values, the three compounds with highest specificity and highest hydrophobicity (EPPC-3, BAMB-4, MEPTT-3) were further characterized. Only BAMB-4 was nearly completely taken up by H1299 cells and remained stable after cellular uptake, thus exhibiting a robust stability and a high membrane permeability. Determination of the inhibitor type revealed that BAMB-4 belongs to the group of mixed type inhibitors. Taken together, for the first time we identified a highly membrane-permeable inhibitor against the InsP₃kinase activity of ITPKA providing the possibility to partly inhibit the metastasis-promoting effect of ITPKA in lung tumor cells.     

Anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors with sub-micromolar potency in the cell-based replicon assay

Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1 and 2 resulted in the identification of multiple structural elements that contributed to improved cell culture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic (IC₅₀) and cell culture (EC₅₀) potencies of less than 100 nanomolar.     

Structural crown properties of Norway spruce (Picea abies [L.] Karst.) and European beech (Fagus sylvatica [L.]) in mixed versus pure stands revealed by terrestrial laser scanning

How tree morphology develops in mixed-species stands is essential for understanding and modelling mixed-stand dynamics. However, research so far focused on the morphological variation between tree species and neglected the variation within a species depending on intra- and interspecific competition. Our study, in contrast, addresses crown properties of nine mature Norway spruces (Picea abies [L.] Karst.) of a pure stand and compares them with ten spruces growing in mixture with European beech (Fagus sylvatica [L.]). The same was done with 11 pure stand beeches and 12 beeches growing in mixture with spruce. Through application of a terrestrial laser scanner and a new skeletonization approach, we deal with both species’-specific morphological traits such as branch angle, branch length, branch bending, crown volume and space occupation of branches within the crown, some of which were hardly accessible so far. Special attention is paid to distinct differences between trees growing in mixed and pure stands: for spruce, our study reveals significantly longer branches and greater crown volumes in the mixed stand when compared to the pure stand. In case of European beech, individuals growing in mixture show flatter branch angles, more distinct ramification, greater crown volumes and a lower share of a single branch’s space occupation in the total crown volume. The results show that the presented methods yield detailed information on the morphological traits analyzed in this study and that interspecific competition on its own may have a significant impact on crown structures. Implications for production ecology and stand dynamics of mixed-species forests are discussed.     

Coccidioidomycosis and blastomycosis: Advances in molecular diagnosis

Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.     

Loss of TRAIL-Receptors Is a Recurrent Feature in Pancreatic Cancer and Determines the Prognosis of Patients with No Nodal Metastasis after Surgery

Introduction

Agonistic antibodies targeting TRAIL-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are being developed as a novel therapeutic approach in cancer therapy including pancreatic cancer. However, the cellular distribution of these receptors in primary pancreatic cancer samples has not been sufficiently investigated and no study has yet addressed the issue of their prognostic significance in this tumor entity.

Aims and Methods

Applying tissue microarray (TMA) analysis, we performed an immunohistochemical assessment of TRAIL-receptors in surgical samples from 84 consecutive patients affected by pancreatic adenocarcinoma and in 26 additional selected specimens from patients with no lymph nodes metastasis at the time of surgery. The prognostic significance of membrane staining and staining intensity for TRAIL-receptors was evaluated.

Results

The fraction of pancreatic cancer samples with positive membrane staining for TRAIL-R1 and TRAIL-R2 was lower than that of cells from surrounding non-tumor tissues (TRAIL-R1: p<0.001, TRAIL-R2: p = 0.006). In addition, subgroup analyses showed that loss of membrane staining for TRAIL-R2 was associated with poorer prognosis in patients without nodal metastases (multivariate Cox regression analysis, Hazard Ratio: 0.44 [95% confidence interval: 0.22−0.87]; p = 0.019). In contrast, analysis of decoy receptors TRAIL-R3 and -R4 in tumor samples showed an exclusively cytoplasmatic staining pattern and no prognostic relevance.

Conclusion

This is a first report on the prognostic significance of TRAIL-receptors expression in pancreatic cancer showing that TRAIL-R2 might represent a prognostic marker for patients with early stage disease. In addition, our data suggest that loss of membrane-bound TRAIL-receptors could represent a molecular mechanism for therapeutic failure upon administration of TRAIL-receptors-targeting antibodies in pancreatic cancer. This hypothesis should be evaluated in future clinical trials.     

5-Methoxyleoligin,a Lignan from Edelweiss,Stimulates CYP26B1-Dependent Angiogenesis In Vitro and Induces Arteriogenesis in Infarcted Rat Hearts In Vivo

Background

Insufficient angiogenesis and arteriogenesis in cardiac tissue after myocardial infarction (MI) is a significant factor hampering the functional recovery of the heart. To overcome this problem we screened for compounds capable of stimulating angiogenesis, and herein investigate the most active molecule, 5-Methoxyleoligin (5ML), in detail.

Methods and Results

5ML potently stimulated endothelial tube formation, angiogenic sprouting, and angiogenesis in a chicken chorioallantoic membrane assay. Further, microarray- and knock down- based analyses revealed that 5ML induces angiogenesis by upregulation of CYP26B1. In an in vivo rat MI model 5ML potently increased the number of arterioles in the peri-infarction and infarction area, reduced myocardial muscle loss, and led to a significant increase in LV function (plus 21% 28 days after MI).

Conclusion

The present study shows that 5ML induces CYP26B1-dependent angiogenesis in vitro, and arteriogenesis in vivo. Whether or not CYP26B1 is relevant for in vivo arteriogenesis is not clear at the moment. Importantly, 5ML-induced arteriogenesis in vivo makes the compound even more interesting for a post MI therapy. 5ML may constitute the first low molecular weight compound leading to an improvement of myocardial function after MI.     

Blood-Brain Barrier Breakdown after Embolic Stroke in Rats Occurs without Ultrastructural Evidence for Disrupting Tight Junctions

The term blood-brain barrier (BBB) relates to the ability of cerebral vessels to hold back hydrophilic and large molecules from entering the brain, thereby crucially contributing to brain homeostasis. In fact, experimental opening of endothelial tight junctions causes a breakdown of the BBB evidenced as for instance by albumin leakage. This and similar observations led to the conclusion that BBB breakdown is predominantly mediated by damage to tight junction complexes, but evidentiary ultrastructural data are rare. Since functional deficits of the BBB contribute to an increased risk of hemorrhagic transformation and brain edema after stroke, which both critically impact on the clinical outcome, we studied the mechanism of BBB breakdown using an embolic model of focal cerebral ischemia in Wistar rats to closely mimic the essential human pathophysiology. Ischemia-induced BBB breakdown was detected using intravenous injection of FITC-albumin and tight junctions in areas of FITC-albumin extravasation were subsequently studied using fluorescence and electron microscopy. Against our expectation, 25 hours after ischemia induction the morphology of tight junction complexes (identified ultrastructurally and using antibodies against the transcellular proteins occludin and claudin-5) appeared to be regularly maintained in regions where FITC-albumin massively leaked into the neuropil. Furthermore, occludin signals along pan-laminin-labeled vessels in the affected hemisphere equaled the non-affected contralateral side (ratio: 0.966 vs. 0.963; P = 0.500). Additional ultrastructural analyses at 5 and 25 h after ischemia induction clearly indicated FITC-albumin extravasation around vessels with intact tight junctions, while the endothelium exhibited enhanced transendothelial vesicle trafficking and signs of degeneration. Thus, BBB breakdown and leakage of FITC-albumin cannot be correlated with staining patterns for common tight junction proteins alone. Understanding the mechanisms causing functional endothelial alterations and endothelial damage is likely to provide novel protective targets in stroke.