Attenuated Abeta42 responses to low potency gamma-secretase modulators can be overcome for many pathogenic presenilin mutants by second-generation compounds

Sequential processing of the β-amyloid precursor protein by β- and γ-secretase generates the amyloid β-peptide (Aβ), which is widely believed to play a causative role in Alzheimer disease. Selective lowering of the pathogenic 42-amino acid variant of Aβ by γ-secretase modulators (GSMs) is a promising therapeutic strategy. Here we report that mutations in presenilin (PS), the catalytic subunit of γ-secretase, display differential responses to non-steroidal anti-inflammatory drug (NSAID)-type GSMs and more potent second-generation compounds. Although many pathogenic PS mutations resisted lowering of Aβ(42) generation by the NSAID sulindac sulfide, the potent NSAID-like second-generation compound GSM-1 was capable of lowering Aβ(42) for many but not all mutants. We further found that mutations at homologous positions in PS1 and PS2 can elicit differential Aβ(42) responses to GSM-1, suggesting that a positive GSM-1 response depends on the spatial environment in γ-secretase. The aggressive pathogenic PS1 L166P mutation was one of the few pathogenic mutations that resisted GSM-1, and Leu-166 was identified as a critical residue with respect to the Aβ(42)-lowering response of GSM-1. Finally, we found that GSM-1-responsive and -resistant PS mutants behave very similarly toward other potent second-generation compounds of different structural classes than GSM-1. Taken together, our data show that a positive Aβ(42) response for PS mutants depends both on the particular mutation and the GSM used and that attenuated Aβ(42) responses to low potency GSMs can be overcome for many PS mutants by second generation GSMs.     

Proteomic profile of culture filtrate from the Brazilian vaccine strain <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG Moreau compared to <Emphasis Type="Italic">M. bovis</Emphasis> BCG Pasteur

Background  

Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.     

T cells potentiate PTH-induced cortical bone loss through CD40L signaling

Parathyroid hormone (PTH) promotes bone catabolism by targeting bone marrow (BM) stromal cells (SCs) and their osteoblastic progeny. Here we show that a continuous infusion of PTH that mimics hyperparathyroidism fails to induce osteoclast formation, bone resorption, and cortical bone loss in mice lacking T cells. T cells provide proliferative and survival cues to SCs and sensitize SCs to PTH through CD40 ligand (CD40L), a surface molecule of activated T cells that induces CD40 signaling in SCs. As a result, deletion of T cells or T cell-expressed CD40L blunts the bone catabolic activity of PTH by decreasing bone marrow SC number, the receptor activator of nuclear factor-κB ligand (RANKL)/OSTEOPROTEGERN (OPG) ratio, and osteoclastogenic activity. Therefore, T cells play an essential permissive role in hyperparathyroidism as they influence SC proliferation, life span, and function through CD40L. T cell-SC crosstalk pathways may thus provide pharmacological targets for PTH-induced bone disease.     

Effects of Hardwood Reduction Techniques on Longleaf Pine Sandhill Vegetation in Northwest Florida

We tested whether the intensity of hardwood midstory reduction causes commensurate improvements of herbaceous groundcover in fire‐suppressed Pinus palustris (longleaf pine) sandhills. Using a complete randomized block design, we compared the effects of three hardwood reduction techniques (spring burning, application of the ULW^® form of the herbicide hexazinone, chainsaw felling/girdling) and a no‐treatment control on plant species richness, and on life form and common species densities at Eglin Air Force Base, Florida, U.S.A., from 1995 to 1998. ULW^® and felling/girdling plots were burned for fuel reduction two years after initial treatment application. We also sampled the same variables in frequently‐burned reference sandhills to establish targets for restoration. Spring burns achieved partial topkill of oaks (17.6–41.1% from 1995 to 1998) compared to reductions of 69.1–94% accomplished by ULW^® and of 93.2–67.8% by felling/girdling treatments. We predicted that plant species richness and densities of herbaceous groundcover life forms would increase according to the percent hardwood reductions. Predictions were not supported by treatment effects for species richness because positive responses to fire best explained increases in plant richness, whereas ULW^® effects accounted for the largest initial decreases. Legumes, non‐legume forbs, and graminoids did not respond to treatments as predicted by the hypothesis. Again, positive responses to fire dominated the results, which was supported by greater herbaceous densities observed in reference plots. Overall, we found that the least effective and least expensive hardwood midstory reduction method, fire, resulted in the greatest groundcover improvements as measured by species richness and herbaceous groundcover plant densities.     

Randomised controlled trial of gabapentin in Complex Regional Pain Syndrome type 1 [ISRCTN84121379]

Background  

Complex Regional Pain Syndrome type one (CRPS I) or formerly Reflex Sympathetic Dystrophy (RSD) is a disabling syndrome, in which a painful limb is accompanied by varying symptoms. Neuropathic pain is a prominent feature of CRPS I, and is often refractory to treatment. Since gabapentin is an anticonvulsant with a proven analgesic effect in various neuropathic pain syndromes, we sought to study the efficacy of the anticonvulsant gabapentin as treatment for pain in patients with CRPS I.     

Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wld s ) gene

Background

The strain of MeCP2^tm1.1Bird mice is a broadly used model for Rett syndrome. Because males carrying the invalidated MeCP2 locus are sterile, this strain has to be maintained in a heterozygous state. All animals therefore have to be genotyped at every generation to discriminate those carrying the invalidated allele (+/- females and y/- males) from those that do not. This is conveniently carried out by PCR on tail genomic DNA but because the primer pairs described initially for this purpose yield very similar size DNA bands on the WT and the KO alleles, this requires to carry out two independent PCR reactions on tail DNA preparations from all animals.

Results

After cloning and sequencing the PCR fragment amplified on the KO allele, we tested several sets of primers that were designed to yield PCR fragments of different sizes on the KO and WT alleles.

Conclusion

We have thus identified a set of three primers that allows for efficient genotyping of the animals by a single PCR reaction. Furthermore, using of this set of primers also resolves a recurrent problem related to the tendency of one of the initial primers to give rise to a non specific band because of its capacity to anneal at both ends of a repeated genomic element which we have identified as MurvyLTR.