Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

gamma-Secretase-dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase-3beta, Neprilysin and APP, we found no consistent effects of gamma-secretase inhibitors or of genetic deficiencies in the gamma-secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD-binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Abeta generation in Alzheimer's disease will not interfere significantly with the function of these genes.     

Identification of a small molecule that modulates platelet glycoprotein Ib-von Willebrand factor interaction

The von Willebrand factor (VWF) A1-glycoprotein (GP) Ibα interaction is of major importance during thrombosis mainly at sites of high shear stress. Inhibitors of this interaction prevent platelet-dependent thrombus formation in vivo, without major bleeding complications. However, the size and/or protein nature of the inhibitors currently in development limit oral bioavailability and clinical development. We therefore aimed to search for a small molecule protein-protein interaction inhibitor interfering with the VWF-GPIbα binding. After determination of putative small molecule binding pockets on the surface of VWF-A1 and GPIbα using site-finding algorithms and molecular dynamics, high throughput molecular docking was performed on both binding partners. A selection of compounds showing good in silico docking scores into the predicted pockets was retained for testing their in vitro effect on VWF-GPIbα complex formation, by which we identified a compound that surprisingly stimulated the VWF-GPIbα binding in a ristocetin cofactor ELISA and increased platelet adhesion in whole blood to collagen under arterial shear rate but in contrast inhibited ristocetin-induced platelet aggregation. The selected compound adhering to the predicted binding partner GPIbα could be confirmed by saturation transfer difference NMR spectroscopy. We thus clearly identified a small molecule that modulates VWF-GPIbα binding and that will now serve as a starting point for further studies and chemical modifications to fully characterize the interaction and to manipulate specific activity of the compound.     

Romulus and Remus,Two Phage Isolates Representing a Distinct Clade within the Twortlikevirus Genus,Display Suitable Properties for Phage Therapy Applications

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.     

Crystal structure of the outer membrane protein RcsF, a new substrate for the periplasmic protein-disulfide isomerase DsbC

The bacterial Rcs phosphorelay is a stress-induced defense mechanism that controls the expression of numerous genes, including those for capsular polysaccharides, motility, and virulence factors. It is a complex multicomponent system that includes the histidine kinase (RcsC) and the response regulator (RcsB) and also auxiliary proteins such as RcsF. RcsF is an outer membrane lipoprotein that transmits signals from the cell surface to RcsC. The physiological signals that activate RcsF and how RcsF interacts with RcsC remain unknown. Here, we report the three-dimensional structure of RcsF. The fold of the protein is characterized by the presence of a central 4-stranded β sheet, which is conserved in several other proteins, including the copper-binding domain of the amyloid precursor protein. RcsF, which contains four conserved cysteine residues, presents two nonconsecutive disulfides between Cys(74) and Cys(118) and between Cys(109) and Cys(124), respectively. These two disulfides are not functionally equivalent; the Cys(109)-Cys(124) disulfide is particularly important for the assembly of an active RcsF. Moreover, we show that formation of the nonconsecutive disulfides of RcsF depends on the periplasmic disulfide isomerase DsbC. We trapped RcsF in a mixed disulfide complex with DsbC, and we show that deletion of dsbC prevents the activation of the Rcs phosphorelay by signals that function through RcsF. The three-dimensional structure of RcsF provides the structural basis to understand how this protein triggers the Rcs signaling cascade.     

Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.     

Panton-Valentine leukocidin does play a role in the early stage of Staphylococcus aureus skin infections: a rabbit model

Despite epidemiological data linking necrotizing skin infections with the production of Panton-Valentine leukocidin (PVL), the contribution of this toxin to the virulence of S. aureus has been highly discussed as a result of inconclusive results of in vivo studies. However, the majority of these results originate from experiments using mice, an animal species which neutrophils--the major target cells for PVL--are highly insensitive to the action of this leukocidin. In contrast, the rabbit neutrophils have been shown to be as sensitive to PVL action as human cells, making the rabbit a better experimental animal to explore the PVL role. In this study we examined whether PVL contributes to S. aureus pathogenicity by means of a rabbit skin infection model. The rabbits were injected intradermally with 10(8) cfu of either a PVL positive community-associated methicillin-resistant S. aureus isolate, its isogenic PVL knockout or a PVL complemented knockout strain, and the development of skin lesions was observed. While all strains induced skin infection, the wild type strain produced larger lesions and a higher degree of skin necrosis compared to the PVL knockout strain in the first week after the infection. The PVL expression in the rabbits was indirectly confirmed by a raise in the serum titer of anti-LukS-PV antibodies observed only in the rabbits infected with PVL positive strains. These results indicate that the rabbit model is more suitable for studying the role of PVL in staphylococcal diseases than other animal models. Further, they support the epidemiological link between PVL producing S. aureus strains and necrotizing skin infections.     

Female lizards ignore the sweet scent of success: Male characteristics implicated in female mate preference

Sexual selection molds the morphology, physiology and behavior of males in many animals. At first glance, it seems reasonable to assume that females would use the same male traits and signals in mate choice as males do during male-male competition. However, intra- and intersexual competition may affect traits in the same or the opposite direction, with differing strength. We investigated which color, morphometric and performance traits are selected for through male-male competition and whether female mate preference is based on these same traits and/or dominance status in the three male color morphs of the lizard Podarcis melisellensis. Males with relatively bigger heads and relatively higher bite forces were more likely to win fights and orange males were always dominant over the other morphs. Females, however, preferred scents of bigger males that were in better body condition, and surprisingly had lower bite force capacities. They did not show a preference for scents of any particular color morph or for scents of the more dominant males. These results indicate that intra- and intersexual competition may result in selection for different secondary sexual traits in P. melisellensis.     

Cancer gene prioritization for targeted resequencing using FitSNP scores

Background

Although the throughput of next generation sequencing is increasing and at the same time the cost is substantially reduced, for the majority of laboratories whole genome sequencing of large cohorts of cancer samples is still not feasible. In addition, the low number of genomes that are being sequenced is often problematic for the downstream interpretation of the significance of the variants. Targeted resequencing can partially circumvent this problem; by focusing on a limited number of candidate cancer genes to sequence, more samples can be included in the screening, hence resulting in substantial improvement of the statistical power. In this study, a successful strategy for prioritizing candidate genes for targeted resequencing of cancer genomes is presented.

Results

Four prioritization strategies were evaluated on six different cancer types: genes were ranked using these strategies, and the positive predictive value (PPV) or mutation rate within the top-ranked genes was compared to the baseline mutation rate in each tumor type. Successful strategies generate gene lists in which the top is enriched for known mutated genes, as evidenced by an increase in PPV. A clear example of such an improvement is seen in colon cancer, where the PPV is increased by 2.3 fold compared to the baseline level when 100 top fitSNP genes are sequenced.

Conclusions

A gene prioritization strategy based on the fitSNP scores appears to be most successful in identifying mutated cancer genes across different tumor entities, with variance of gene expression levels as a good second best.     

Analgesia induced by combined action of Tramadol and microwave irradiation in somatic pain in mice

In experiments on albino male mice weighing 24 to 32 g, we studied the suppression of the pain reaction induced by injection of formalin into the dorsal surface of the foot, using a combination of injection of tramadol hydrochloride (Tramal) in a dose of 0.42 mg/kg and irradiation of the acupuncture point (AP) E-36 with low-intensity microwaves (frequency 30 to 300 GHz and power rate density 3·10⁻⁹ W/cm²). The combined use of tramadol injection and the action of microwaves on the AP led to a decrease in the duration of the pain reaction (licking of the pain nidus), on average, by 58.2%, as compared with that in the control animal group. The analgesic effect was more strong in the case of such a combination than after isolated injection of tramadol in doses of 0.83 and 0.42 mg/kg (by 14.4 and by 10.0%, respectively) and isolated microwave irradiation of the AP TA E-36 (by 48.7%). The data obtained demonstrate the possibility of effective analgesia using a combination of microwave irradiation of the AP and the pharmacological influence with a significant decrease in the dose of the analgesic. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 314–319, July–August, 2006.