?2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ?2-microglobulin (?2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ?2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ?2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ?2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ?2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ?2m amyloid-associated osteoarticular tissue destruction in DRA

High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein

Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.Publisher PDFPeer reviewe

Gene-Based Analysis of Regionally Enriched Cortical Genes in GWAS Data Sets of Cognitive Traits and Psychiatric Disorders

Background: Despite its estimated high heritability, the genetic architecture leading to differences in cognitive performance remains poorly understood. Different cortical regions play important roles in normal cognitive functioning and impairment. Recently, we reported on sets of regionally enriched genes in three different cortical areas (frontomedial, temporal and occipital cortices) of the adult rat brain. It has been suggested that genes preferentially, or specifically, expressed in one region or organ reflect functional specialisation. Employing a gene-based approach to the analysis, we used the regionally enriched cortical genes to mine a genome-wide association study (GWAS) of the Norwegian Cognitive NeuroGenetics (NCNG) sample of healthy adults for association to nine psychometric tests measures. In addition, we explored GWAS data sets for the serious psychiatric disorders schizophrenia (SCZ) (n = 3 samples) and bipolar affective disorder (BP) (n = 3 samples), to which cognitive impairment is linked. Principal Findings: At the single gene level, the temporal cortex enriched gene RAR-related orphan receptor B (RORB) showed the strongest overall association, namely to a test of verbal intelligence (Vocabulary, P = 7.7E-04). We also applied gene set enrichment analysis (GSEA) to test the candidate genes, as gene sets, for enrichment of association signal in the NCNG GWAS and in GWASs of BP and of SCZ. We found that genes differentially expressed in the temporal cortex showed a significant enrichment of association signal in a test measure of non-verbal intelligence (Reasoning) in the NCNG sample. Conclusion: Our gene-based approach suggests that RORB could be involved in verbal intelligence differences, while the genes enriched in the temporal cortex might be important to intellectual functions as measured by a test of reasoning in the healthy population. These findings warrant further replication in independent samples on cognitive traits

In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors

<p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers.</p> <p>Methods</p> <p>Mice carrying human (FaDu_dd) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared.</p> <p>Results</p> <p>In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDu_dd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker.</p> <p>Conclusions</p> <p>The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.</p

Dietary Vitamin D3 Supplements Reduce Demyelination in the Cuprizone Model

Vitamin D is emerging as a probably important environmental risk factor in multiple sclerosis, affecting both susceptibility and disease progression. It is not known to what extent this effect is due to a modulation of peripheral lymphocyte function, or to intrathecal effects of vitamin D. We investigated the effect of dietary vitamin D3 content on de/remyelination in the cuprizone model, which is a well established toxic model of demyelination, with no associated lymphocyte infiltration. The mice received diets either deficient of (<50 IU/kg), or supplemented with low (500 IU/kg), high (6200 IU/kg) or very high (12500 IU/kg) amounts of vit D3. Cuprizone (0.2%) was added to the diet for six weeks, starting two weeks after onset of the experimental diets. Mouse brain tissue was histopathologically evaluated for myelin and oligodendrocyte loss, microglia/macrophage activation, and lymphocyte infiltration after six weeks of cuprizone exposure, and two weeks after discontinuation of cuprizone exposure. High and very high doses of vitamin D3 significantly reduced the extent of white matter demyelination (p = 0.004) and attenuated microglia activation (p = 0.001). No differences in the density of oligodendrocytes were observed between the diet groups. Two weeks after discontinuation of cuprizone exposure, remyelination was only detectable in the white matter of mice receiving diets deficient of or with low vitamin D3 content. In conclusion, high dietary doses of vitamin D3 reduce the extent of demyelination, and attenuate microglia activation and macrophage infiltration in a toxic model of demyelination, independent of lymphocyte infiltration