Surface exposed epitopes and structural heterogeneity of in vivo formed transthyretin amyloid fibrils

We have investigated the structure of in vivo formed transthyretin (TTR) amyloid deposits by using antisera raised against short linear sequences of the TTR molecule. In immunohistochemistry, antisera anti-TTR41-50 and anti-TTR115-124-a reacted specifically with both wildtype ATTR and ATTR V30M material, whereas only anti-TTR41-50 recognized ATTR Y114C material. Similar results were obtained by ELISA analysis of ATTR V30M and ATTR Y114C vitreous amyloid, where the anti-TTR115-124-a antiserum failed to react with ATTR Y114C material. Moreover, neither of the antisera recognized natively structured TTR present in pancreatic alpha cells. Our results strongly indicate that the TTR molecule undergoes structural changes during fibrillogenesis in vivo. The finding of a structural difference between wildtype ATTR and ATTR V30M material on one hand and ATTR Y114C material on the other suggests that the fibril formation pathway of these ATTR variants may differ in vivo.     

Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine

Alpha-1 acid glycoprotein (AGP, orosomucoid), a major acute phase protein in plasma, displays potent cytoprotective and anti-inflammatory activities whose molecular mechanisms are largely unknown. Because AGP binds various exogenous drugs, we have searched for endogenous ligands for AGP. We found that AGP binds lysophospholipids in a manner discernible from albumin in several ways. First, mass spectrometric analyses showed that AGP isolated from plasma and serum contained lysophosphatidylcholine (LPC) enriched in mono and polysaturated acyl chains, whereas albumin contained mostly saturated LPC. Second, AGP bound LPC in a 1:1 molar ratio and with a higher affinity than free fatty acids, whereas albumin bound LPC in a 3:1 ratio but with a lower affinity than that of free fatty acids. Consequently, free fatty acids displaced LPC more avidly from albumin than from AGP. Competitive ligand displacement indicated the highest affinity for AGP to LPC20:4, 18:3, 18:1, and 16:0 (150-180 nM), lysophosphatidylserine (Kd 190 nM), and platelet activating factor (PAF) (Kd 235 nM). The high affinity of AGP to LPC in equilibrium was verified by stopped-flow kinetics, which implicated slow dissociation after fast initial binding, being consistent with an induced-fit mechanism. AGP also bound pyrene-labeled phospholipids directly from vesicles and more efficiently than albumin. AGP prevented LPC-induced priming and PAF-induced activation of human granulocytes, thus indicating scavenging of the cellular effects of the lipid ligands. The results suggest that AGP complements albumin as a lysophospholipid scavenging protein, particularly in inflammatory conditions when the capacity of albumin to sequester LPC becomes impaired.     

Valine Pyrrolidide Preserves Intact Glucose-Dependent Insulinotropic Peptide and Improves Abnormal Glucose Tolerance in Minipigs With Reduced β-Cell Mass

The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important in blood glucose regulation.However, both incretin hormones are rapidly degraded by the enzyme dipeptidyl peptidase IV (DPPIV). The concept of DPPIV inhibition as a treatment for type 2 diabetes was evaluated in a new large animal model of insulin-deficient diabetes and reduced β-cell mass, the nicotinamide (NIA) (67 mg/kg) and streptozotocin (STZ) (125 mg/kg)–treated minipig, using the DPPIV inhibitor, valine pyrrolidide (VP) (50 mg/kg).VP did not significantly affect levels of intact GLP-1 but increased levels of intact GIP (from 4543 ± 1880 to 9208 ± 3267 pM × min; P<.01), thus improving glucose tolerance (area under the curve [AUC] for glucose reduced from 1904 ± 480 to 1582 ± 353 mM × min;P = .05).VP did not increase insulin levels during the oral glucose tolerance test (OGTT) but increased the insulinogenic index in normal animals (from 83 ± 42 to 192 ± 108; P < .05), but not after NIA + STZ, possibly because of less residual insulin secretory capacity in these animals. GIP seems to contribute to the antihyperglycemic effect of VP in this model; however, additional mechanisms for the effect of DPPIV inhibition cannot be excluded. The authors conclude that DPPIV inhibitors may be useful to treat type 2 diabetes, even when this is due to reduced β-cell mass.     

How do immobilised cell-adhesive Arg–Gly–Asp-containing peptides behave at the PAA brush surface?

Bio-engineered surfaces that aim to induce normal cell behaviour in vitro need to ‘mimic’ the extracellular matrix in a way that allows cell adhesion. In this computational work, several model cell-binding peptides with a minimal cell-adhesive Arg–Gly–Asp sequence are investigated in the bulk as well as immobilised on a soft surface. For this reason, a combination of density functional theory and all-atom MD simulations is applied. The major goal of the modelling is to characterise the accessibility of the cell-recognition motif on the functionalised soft polymer surface. As a reference system, the behaviour of three peptide sequences is preliminarily studied in explicit water simulations. From the analysis of the MD trajectories, the solvent accessible surface area, the distribution of water molecules around peptide groups, the secondary structure and the thermodynamics of hydration are evaluated. Furthermore, each peptide is immobilised on the surface of a homopolymer poly(acrylic acid) brush. During MD simulations, all three peptides approach closely toward PAA brush, and their surface accessibility is characterised. Although the peptides are adsorbed onto the brush, they are not hidden by the polymer strands, with RGD unit accessible on the surface and available for guided cell adhesion.     

Environmental Change and Extended Phenotypes: Does Eutrophication Influence Nest Building in Sticklebacks?

Many species use extended phenotypes, such as purpose‐built nests, to increase their reproductive success. These traits have to be adjusted to local environmental conditions to maximize fitness. An important question is whether species are able to adjust their extended phenotypes to human‐induced rapid environmental changes. We investigated whether populations of threespine stickleback, Gasterosteus aculeatus, exposed to different degrees of human‐induced eutrophication during the last decades, have differentiated phenotypically in their nest‐building behaviour. Stickleback males build nests that they use both in mate attraction and for offspring protection and whose characteristics vary with environmental conditions. We allowed males from parallel pairs of mildly and severely eutrophied habitats to build nests under standardized conditions in the laboratory. We recorded the time it took the males to build a nest, the size and neatness of the completed nest and the use of nest ornaments. We found eutrophication at the site of capture to influence nest‐building time – males from eutrophied habitats built faster. However, eutrophication did not alter nest structure or the use of nest ornaments. This is probably because the nests are concealed in vegetation or under stones and females cannot evaluate them before they have followed the male to the nest, and predators cannot detect them before close to the nest. Thus, reduced long‐range visibility does not influence the use of nests as mate choice cues or for offspring protection. This contrasts with a recorded effect of eutrophication on courtship behaviour, whose efficiency depends on long‐range visibility. This suggests that traits are adjusted to eutrophication depending on the influence of eutrophication on the function of the traits.     

Insulin analog with additional disulfide bond has increased stability and preserved activity

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.     

Overweight in Childhood,Adolescence and Adulthood and Cardiovascular Risk in Later Life: Pooled Analysis of Three British Birth Cohorts

Background

Overweight and obesity in adulthood are established risk factors for adverse cardiovascular outcomes, but the contribution of overweight in childhood to later cardiovascular risk is less clear. Evidence for a direct effect of childhood overweight would highlight early life as an important target for cardiovascular disease prevention. The aim of this study was to assess whether overweight and obesity in childhood and adolescence contribute to excess cardiovascular risk in adults.

Methods and findings

Data from three British birth cohorts, born in 1946, 1958 and 1970, were pooled for analysis (n = 11,447). Individuals were categorised, based on body mass index (BMI), as being of normal weight or overweight/obese in childhood, adolescence and adulthood. Eight patterns of overweight were defined according to weight status at these three stages. Logistic regression models were fitted to assess the associations of patterns of overweight with self-reported type 2 diabetes, hypertension, and coronary heart disease (CHD) in adulthood (34–53 years). Compared to cohort members who were never overweight, those who were obese in adulthood had increased risk of all outcomes. For type 2 diabetes, the odds ratio was higher for obese adults who were also overweight or obese in childhood and adolescence (OR 12.6; 95% CI 6.6 to 24.0) than for those who were obese in adulthood only (OR 5.5; 95% CI 3.4 to 8.8). There was no such effect of child or adolescent overweight on hypertension. For CHD, there was weak evidence of increased risk among those with overweight in childhood. The main limitations of this study concern the use of self-reported outcomes and the generalisability of findings to contemporary child populations.

Conclusions

Type 2 diabetes and to a lesser extent CHD risk may be affected by overweight at all stages of life, while hypertension risk is associated more strongly with weight status in adulthood.     

Is R2* a New MRI Biomarker for the Progression of Parkinson’s Disease? A Longitudinal Follow-Up

Purpose

To study changes of iron content in basal ganglia in Parkinson’s disease (PD) through a three-year longitudinal follow-up of the effective transverse relaxation rate R₂*, a validated MRI marker of brain iron content which can be rapidly measured under clinical conditions.

Methods

Twenty-seven PD patients and 26 controls were investigated by a first MRI (t₀). Longitudinal analysis was conducted among the 18 controls and 14 PD patients who underwent a second MRI (t₁) 3 years after. The imaging protocol consisted in 6 gradient echo images obtained at different echo-times for mapping R₂*. Quantitative exploration of basal ganglia was performed by measuring the variation of R₂* [R₂*(t₁) – R₂*(t₀)] in several regions of interest.

Results

During the three-year evolution of PD, R₂* increased in Substantia nigra (SN) (by 10.2% in pars compacta, p = 0.001, and 8.1% in pars reticulata, p = 0.013) and in the caudal putamen (11.4%, p = 0.011), without significant change in controls. Furthermore, we showed a positive correlation between the variation of R₂* and the worsening of motor symptoms of PD (p = 0.028).

Conclusion

Significant variation of R₂* was longitudinally observed in the SN and caudal putamen of patients with PD evolving over a three-year period, emphasizing its interest as a biomarker of disease progression. Our results suggest that R₂* MRI follow-up could be an interesting tool for individual assessment of neurodegeneration due to PD, and also be useful for testing the efficiency of disease-modifying treatments.     

Novel route for elimination of brain oxysterols across the blood-brain barrier: conversion into 7alpha-hydroxy-3-oxo-4-cholestenoic acid

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4-5 mg/day. As the steady-state levels of this sterol are only 1-2 mug/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C(27) steroidal acid 7alpha-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7alpha-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7alpha-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.