Transgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrP^C was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrP^d) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrP^d originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.     

Design, synthesis, and vasorelaxant and platelet antiaggregatory activities of coumarin-resveratrol hybrids

We have synthesized the coumarin-resveratrol hybrid 4 and its dimethoxy derivative 3 by a very direct synthetic route involving a Pechmann procedure. Compound 4 has also been synthesized by an alternative route (Perkin), which also allowed the synthesis of compounds 9-13. In addition, we have evaluated the potential vasorelaxant activity of the new compounds in endothelium-containing rat aorta rings pre-contracted with noradrenaline, as well as the inhibitory effects on platelet aggregation induced by thrombin in washed human platelets. The compounds reported here relaxed vascular smooth muscle and inhibited platelet aggregation with a profile similar to that of trans-resveratrol (t-RESV) and, in some cases, showed activity higher than that of the natural compound. This is the case for compound 13, which has a vasorelaxant activity that is twice as high as that of t-resveratrol and a platelet antiaggregant activity that is six times higher. These results suggest that these novel compounds may have potential as structural templates for the design and subsequent development of new vasodilatory and platelet antiaggregatory drugs.     

Effect of volunteers on maize gene flow

Regulatory approvals for deliberate release of GM maize events into the environment have lead to real situations of coexistence between GM and non-GM, with some fields being cultivated with GM and conventional varieties in successive seasons. Given the common presence of volunteer plants in maize fields in temperate areas, we investigated the real impact of GM volunteers on the yield of 12 non-GM agricultural fields. Volunteer density varied from residual to around 10% of plants in the field and was largely reduced using certain cultural practices. Plant vigour was low, they rarely had cobs and produced pollen that cross-fertilized neighbour plants only at low—but variable—levels. In the worst-case scenario, the estimated content of GMO was 0.16%. The influence of GM volunteers was not enough to reach the 0.9% adventitious GM threshold but it could potentially contribute to adventitious GM levels, especially at high initial densities (i.e. above 1,000 volunteers/ha).     

Seedling recruitment, survival and facilitation in alpine Pinus uncinata tree line ecotones. Implications and potential responses to climate warming

Aims Alpine tree line ecotones are harsh environments where low temperatures constrain tree regeneration and growth. However, the expected upward shift of tree line ecotones in response to climate warming has not been ubiquitous. The lack of coupling between tree line dynamics and climate warming might be explained by factors other than climate variation that determine seedling recruitment in these ecotones. We want to assess how the availability of suitable habitat for establishment and the effects of facilitation on seedling survival and growth affect tree recruitment within tree line ecotones and modulate their responses to climate. Location We evaluate the relevance of these factors for Pinus uncinata tree line ecotones in the Catalan Pyrenees (north‐east Spain) and Andorra. Methods We analysed the microhabitat of naturally established seedlings in rectangular plots at the tree line ecotone, assessing the habitat type and the proximity to potentially protective elements that may improve microsite conditions. We tested whether krummholz individuals influence regeneration at the tree line by performing a transplantation field experiment to evaluate the extent of facilitation on seedling survival and growth in height. A total of 820 seedlings were transplanted at different distances and orientations (resulting in 12 positions) from krummholz mats and monitored over 2 years. Results Safe sites for P. uncinata recruits consisted of sparse vegetation covering bare soil, gravel or litter, and close to protective elements that may ameliorate microsite conditions. The field experiment showed that directional positive interactions enhance seedling survival and growth, altering the spatial patterns of recruit survivorship, especially during harsh winter conditions (shallow and irregular snowpack). Main conclusions Our results suggest that scarce availability of safe sites and uneven facilitation by krummholz control seedling recruitment patterns within alpine tree line ecotones. Such constraints may distort or counter the response of tree line ecotones to climate warming at local and regional scales.     

Systemic inflammation in chronic obstructive pulmonary disease: a population-based study

Background

Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue.

Methods

Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay.

Results

IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.

Conclusion

AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.     

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr⁶⁸⁷ in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr⁶⁸⁷ and association with components of the Tyr¹⁰⁶² signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser⁶⁹⁶, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr⁶⁸⁷ as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.     

Characterisation of the PTEN inhibitor VO-OHpic

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a phosphatidylinositol triphosphate 3-phosphatase that counteracts phosphoinositide 3-kinases and has subsequently been implied as a valuable drug target for diabetes and cancer. Recently, we demonstrated that VO-OHpic is an extremely potent inhibitor of PTEN with nanomolar affinity in vitro and in vivo. Given the importance of this inhibitor for future drug design and development, its mode of action needed to be elucidated. It was discovered that inhibition of recombinant PTEN by VO-OHpic is fully reversible. Both K _m and V _max are affected by VO-OHpic, demonstrating a noncompetitive inhibition of PTEN. The inhibition constants K _ic and K _iu were determined to be 27 ± 6 and 45 ± 11 nM, respectively. Using the artificial phosphatase substrate 3-O-methylfluorescein phosphate (OMFP) or the physiological substrate phosphatidylinositol 3,4,5-triphosphate (PIP₃) comparable parameters were obtained suggesting that OMFP is a suitable substrate for PTEN inhibition studies and PTEN drug screening.