Nitrated plasma albumin as a marker of nitrative stress and neonatal encephalopathy in perinatal asphyxia

Reactive nitrogen species (RNS) have been shown to play a major role in the pathophysiology of hypoxic–ischemic cerebral injury. Using a novel sensitive ELISA allowing the quantification of nitrated albumin (nitroalbumin) in plasma, we tested the hypothesis that perinatal asphyxia increases nitrating RNS generation by verifying whether the concentration of one of its target proteins is correlated with the clinical outcome. We assayed nitroalbumin in 114 plasma samples collected during the first hour, at day 1, and at day 4 of life from 48 term newborns suffering from perinatal asphyxia and correlated this marker with neurological and systemic neonatal outcomes. Nitroalbumin levels at day 1, but not at days 0 and 4, were significantly increased in patients who developed moderate or severe encephalopathy compared to those who had a normal neurological evolution or developed mild encephalopathy (median: 14.4 ng/ml versus 7.3 ng/ml, respectively). In contrast, nitroalbumin concentration at day 1 was not associated with systemic complications. First-hour and fourth-day nitroalbumin concentrations did not differ with respect to the neonatal neurological course. At day 0, nitroalbumin levels also correlated with circulating leukocytes. We conclude that plasma nitroalbumin seems to be a specific marker of neurological injury after perinatal asphyxia and may serve as a secondary end-point in neuroprotective clinical trials.     

Insight into early events in the aggregation of the prion protein on lipid membranes

The key molecular event underlying prion diseases is the conversion of the monomeric and α-helical cellular form of the prion protein (PrP^C) to the disease-associated state, which is aggregated and rich in β-sheet (PrP^Sc). The molecular details associated with the conversion of PrP^C into PrP^Sc are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces β-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the α-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.     

Enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source

Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrP^C) is required for prion replication, the influence of PrP^C abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrP^C was overexpressed. Tg(MoPrP)4112 mice overexpressing PrP^C supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrP^C-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrP^C is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrP^Sc amplification by using concentrated PrP^C source and expands the use of this methodology.     

Human Neuroserpin: Structure and Time-Dependent Inhibition

Human neuroserpin (hNS) is a protein serine protease inhibitor expressed mainly in the nervous system, where it plays key roles in neural development and plasticity by primarily targeting tissue plasminogen activator (tPA). Four hNS mutations are associated to a form of autosomal dominant dementia, known as familial encephalopathy with neuroserpin inclusion bodies. The medical interest in and the lack of structural information on hNS prompted us to study the crystal structure of native and cleaved hNS, reported here at 3.15 and 1.85 Å resolution, respectively. In the light of the three-dimensional structures, we focus on the hNS reactive centre loop in its intact and cleaved conformations relative to the current serpin polymerization models and discuss the protein sites hosting neurodegenerative mutations. On the basis of homologous serpin structures, we suggest the location of a protein surface site that may stabilize the hNS native (metastable) form. In parallel, we present the results of kinetic studies on hNS inhibition of tPA. Our data analysis stresses the instability of the hNS-tPA complex with a dissociation half-life of minutes compared to a half-life of weeks observed for other serpin-cognate protease complexes.     

Scrapie-Infected Transgenic Mice Expressing a Laminin Receptor Decoy Mutant Reveal a Prolonged Incubation Time Associated with Low Levels of PrPres

The 37-kDa/67-kDa laminin receptor (LRP/LR) was identified as a cell surface receptor for prion proteins. The laminin receptor mutant LRP102-295∷FLAG interfered with PrP^Sc propagation in murine neuronal cells presumably acting as a decoy in a transdominant negative fashion by trapping PrP molecules in the extracellular matrix. Here, we generated hemizygous transgenic mice expressing LRP102-295∷FLAG in the brain. Scrapie-infected transgenic mice exhibit a significantly prolonged incubation time in comparison to scrapie-infected wild-type (FVB) mice. At the terminal stage, transgenic mice revealed significantly reduced proteinase-K-resistant PrP levels by 71% compared to wild-type mice. Our results recommend the laminin receptor decoy mutant as an alternative therapeutic tool for treatment of transmissible spongiform encephalopathies.     

纳米锁阳对肝性脑病肠道菌群及免疫功能的调整

目的研究纳米中药锁阳对内毒素诱发肝硬化大鼠发生肝性脑病的肠道菌群与免疫功能的影响。方法肝硬化大鼠行小剂量内毒素腹腔注射造成肝性脑病模型。观察常态、纳米中药锁阳对肠道菌群,血清IL-2水平及血浆中血氨含量影响。结果肝性脑病大鼠血浆内毒素及血氨明显升高,正常对照组与模型组比较差异有显著性（P〈0．05）。常态锁阳治疗组与纳米锁阳治疗组血浆中血氨、内毒素均明显降低,肠道菌群失调症有明显改变。常态锁阳治疗组与自然恢复组比较差异有显著性（P〈0．05）,纳米中药治疗组与常态中药治疗组比较差异有显著性（P〈0．05）。结论高血氨是内毒素诱发肝性脑病发生的主要诱因。应用中药锁阳从调整微生态失调角度来治疗肝性脑病,取得较好疗效,纳米中药锁阳的效果更佳。     

Nanopore detection of antibody prion interactions

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. A prion peptide, PrP(143-169), can readily translocate through the pore, but on the addition of monoclonal antibody M2188, which binds the peptide, the number of translocations is reduced because the complex is too large to translocate. At a peptide-to-immunoglobulin G (IgG) ratio of 2:1, only bumping events were observed. The event profile of a control peptide that does not bind the antibody was unchanged. Similarly, the presence of the antibody prevents translocation of the full-length prion protein. Because a nanopore can detect a single molecule, these experiments represent an important first step towards the development of a sensitive prion detector.     

Refolding and Polymerization Pathways of Neuroserpin

Neuroserpin is a member of the serpin superfamily, and its mutants are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. It has been proposed that neuroserpin polymers are formed by a conformational change in the folded protein. However, an alternative model whereby polymers are formed during protein folding rather than from the folded protein has recently been proposed. We investigated the refolding and polymerization pathways of wild-type neuroserpin (WT) and of the pathogenic mutants S49P and H338R. Upon refolding, denatured WT immediately formed an initial refolding intermediate I_IN and then underwent further refolding to the native form through a late refolding intermediate, I_R. The late-onset mutant S49P was also able to refold to the native form through I_IN and I_R, but the final refolding step proceeded at a slower rate and with a lower refolding yield as compared with WT. The early-onset mutant H338R formed I_R through the same pathway as S49P, but the protein could not attain the native state and remained as I_R. The I_Rs of the mutants had a long lifespan at 4 °C and thus were purified and characterized. Strikingly, when incubated under physiological conditions, I_R formed ordered polymers with essentially the same properties as the polymers formed from the native protein. The results show that the mutants have a greater tendency to form polymers during protein folding than to form polymers from the folded protein. Our finding provides insights into biochemical approaches to treating serpinopathies by targeting a polymerogenic folding intermediate.     

Molecular analysis of carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) as a candidate gene for bovine spongiform encephalopathy susceptibility

Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N -acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP^C into the pathogenic PrP^d. Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.