Prion protein expression differences in microglia and astroglia influence scrapie-induced neurodegeneration in the retina and brain of transgenic mice

Activated microglia and astroglia are known to be involved in a variety of neurodegenerative diseases, including prion diseases. In the present experiments, we studied activation of astroglia and microglia after intraocular scrapie infection in transgenic mice expressing prion protein (PrP) in multiple cell types (tg7 mice) or in neurons only (tgNSE mice). In this model, scrapie infection and protease-resistant PrP deposition occurs in the retinas of both strains of mice, but retinal degeneration is observed only in tg7 mice. Our results showed that the retinas of tg7 and tgNSE mice both had astroglial activation with increased chemokine expression during the course of infection. However, only tg7 retinas exhibited strong microglial activation compared to tgNSE retinas, which showed little microglial activation by biochemical or morphological criteria. Therefore, microglial PrP expression might be required for scrapie-induced retinal microglial activation and damage. Furthermore, microglial activation preceded retinal neurodegeneration in tg7 mice, suggesting that activated microglia might contribute to the degenerative process, rather than being a response to the damage. Surprisingly, brain differed from retina in that an altered profile of microglial activation markers was upregulated, and the profiles in the two mouse strains were indistinguishable. Microglial activation in the brain was associated with severe brain vacuolation and neurodegeneration, leading to death. Thus, retinal and brain microglia appeared to differ in their requirements for activation, suggesting that different activation pathways occur in the two tissues.     

A primary assembly of a bovine haplotype block map based on a 15,036-single-nucleotide polymorphism panel genotyped in holstein-friesian cattle

Analysis of data on 1000 Holstein-Friesian bulls genotyped for 15,036 single-nucleotide polymorphisms (SNPs) has enabled genomewide identification of haplotype blocks and tag SNPs. A final subset of 9195 SNPs in Hardy-Weinberg equilibrium and mapped on autosomes on the bovine sequence assembly (release Btau 3.1) was used in this study. The average intermarker spacing was 251.8 kb. The average minor allele frequency (MAF) was 0.29 (0.05-0.5). Following recent precedents in human HapMap studies, a haplotype block was defined where 95% of combinations of SNPs within a region are in very high linkage disequilibrium. A total of 727 haplotype blocks consisting of > or =3 SNPs were identified. The average block length was 69.7 +/- 7.7 kb, which is approximately 5-10 times larger than in humans. These blocks comprised a total of 2964 SNPs and covered 50,638 kb of the sequence map, which constitutes 2.18% of the length of all autosomes. A set of tag SNPs, which will be useful for further fine-mapping studies, has been identified. Overall, the results suggest that as many as 75,000-100,000 tag SNPs would be needed to track all important haplotype blocks in the bovine genome. This would require approximately 250,000 SNPs in the discovery phase.     

Insights into molecular interactions between CaM and its inhibitors from molecular dynamics simulations and experimental data

In order to contribute to the structural basis for rational design of calmodulin (CaM) inhibitors, we analyzed the interaction of CaM with 14 classic antagonists and two compounds that do not affect CaM, using docking and molecular dynamics (MD) simulations, and the data were compared to available experimental data. The Ca²⁺-CaM-Ligands complexes were simulated 20 ns, with CaM starting in the “open” and “closed” conformations. The analysis of the MD simulations provided insight into the conformational changes undergone by CaM during its interaction with these ligands. These simulations were used to predict the binding free energies (ΔG) from contributions ΔH and ΔS, giving useful information about CaM ligand binding thermodynamics. The ΔG predicted for the CaM’s inhibitors correlated well with available experimental data as the r² obtained was 0.76 and 0.82 for the group of xanthones. Additionally, valuable information is presented here: I) CaM has two preferred ligand binding sites in the open conformation known as site 1 and 4, II) CaM can bind ligands of diverse structural nature, III) the flexibility of CaM is reduced by the union of its ligands, leading to a reduction in the Ca²⁺-CaM entropy, IV) enthalpy dominates the molecular recognition process in the system Ca²⁺-CaM-Ligand, and V) the ligands making more extensive contact with the protein have higher affinity for Ca²⁺-CaM. Despite their limitations, docking and MD simulations in combination with experimental data continue to be excellent tools for research in pharmacology, toward a rational design of new drugs.     

Exploring trophic niches and parasite communities of sympatric Arctic charr and brown trout populations of southern Norway

Hydrobiologia - Catchment-scale variation between lake habitats has the potential to simultaneously influence the trophic niche and parasite community of fish hosts. In this study, we investigated...     

Comparison of historical bottleneck effects and genetic consequences of re‐introduction in a critically endangered island passerine

Re‐introduction is an important tool for recovering endangered species; however, the magnitude of genetic consequences for re‐introduced populations remains largely unknown, in particular the relative impacts of historical population bottlenecks compared to those induced by conservation management. We characterize 14 microsatellite loci developed for the Seychelles paradise flycatcher and use them to quantify temporal and spatial measures of genetic variation across a 134‐year time frame encompassing a historical bottleneck that reduced the species to ~28 individuals in the 1960s, through the initial stages of recovery and across a second contemporary conservation‐introduction‐induced bottleneck. We then evaluate the relative impacts of the two bottlenecks, and finally apply our findings to inform broader re‐introduction strategy. We find a temporal trend of significant decrease in standard measures of genetic diversity across the historical bottleneck, but only a nonsignificant downward trend in number of alleles across the contemporary bottleneck. However, accounting for the different timescales of the two bottlenecks (~40 historical generations versus <1 contemporary generation), the loss of genetic diversity per generation is greater across the contemporary bottleneck. Historically, the flycatcher population was genetically structured; however, extinction on four of five islands has resulted in a homogeneous contemporary population. We conclude that severe historical bottlenecks can leave a large footprint in terms of sheer quantity of genetic diversity lost. However, severely depleted genetic diversity does not render a species immune to further genetic erosion upon re‐introduction. In some cases, the loss of genetic diversity per generation can, initially at least, be greater across re‐introduction‐induced bottlenecks.     

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

RING‐in‐between‐RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub‐conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT‐type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING‐type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING2 important for its recruitment to RING1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.     

CTCF cis-Regulates Trinucleotide Repeat Instability in an Epigenetic Manner: A Novel Basis for Mutational Hot Spot Determination

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting “instability elements,” and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF—a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.     

Limitations of a morphological criterion of adaptive inference in the fossil record

Experimental analyses directly inform how an anatomical feature or complex functions during an organism's lifetime, which serves to increase the efficacy of comparative studies of living and fossil taxa. In the mammalian skull, food material properties and feeding behaviour have a pronounced influence on the development of the masticatory apparatus. Diet‐related variation in loading magnitude and frequency induce a cascade of changes at the gross, tissue, cellular, protein and genetic levels, with such modelling and remodelling maintaining the integrity of oral structures vis‐à‐vis routine masticatory stresses. Ongoing integrative research using rabbit and rat models of long‐term masticatory plasticity offers unique insight into the limitations of functional interpretations of fossilised remains. Given the general restriction of the palaeontological record to bony elements, we argue that failure to account for the disparity in the hierarchical network of responses of hard versus soft tissues may overestimate the magnitude of the adaptive divergence that is inferred from phenotypic differences. Second, we note that the developmental onset and duration of a loading stimulus associated with a given feeding behaviour can impart large effects on patterns of intraspecific variation that can mirror differences observed among taxa. Indeed, plasticity data are relevant to understanding evolutionary transformations because rabbits raised on different diets exhibit levels of morphological disparity comparable to those found between closely related primate species that vary in diet. Lastly, pronounced variation in joint form, and even joint function, can also characterise adult conspecifics that differ solely in age. In sum, our analyses emphasise the importance of a multi‐site and hierarchical approach to understanding determinants of morphological variation, one which incorporates critical data on performance.     

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2^−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2^−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV_T MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.