Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates

Abstract Phospholipid flippases (P4-ATPases) translocate specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. While there is good evidence that the overall molecular structure of flippases is similar to that of P-type ATPase ion-pumps, the transport pathway for the “giant” lipid substrate has not been determined. ATP8A2 is a flippase with selectivity toward phosphatidylserine (PS), possessing a net negatively charged head group, whereas ATP8B1 exhibits selectivity toward the electrically neutral phosphatidylcholine (PC). Setting out to elucidate the functional consequences of flippase disease mutations, we have identified residues of ATP8A2 that are critical to the interaction with the lipid substrate during the translocation process. Among the residues pinpointed are I91 and L308, which are positioned near proposed translocation routes through the protein. In addition we pinpoint two juxtaposed oppositely charged residues, E897 and R898, in the exoplasmic loop between transmembrane helices 5 and 6. The glutamate is conserved between PS and PC flippases, whereas the arginine is replaced by a negatively charged aspartate in ATP8B1. Our mutational analysis suggests that the glutamate repels the PS head group, whereas the arginine minimizes this repulsion in ATP8A2, thereby contributing to control the entry of the phospholipid substrate into the translocation pathway

State-building, war and violence : evidence from Latin America

In European history, war has played a major role in state‐building and the state monopoly on violence. But war is a very specific form of organized political violence, and it is decreasing on a global scale. Other patterns of armed violence now dominate, ones that seem to undermine state‐building, thus preventing the replication of European experiences. As a consequence, the main focus of the current state‐building debate is on fragility and a lack of violence control inside these states. Evidence from Latin American history shows that the specific patterns of the termination of both war and violence are more important than the specific patterns of their organization. Hence these patterns can be conceptualized as a critical juncture for state‐building. While military victories in war, the subordination of competing armed actors and the prosecution of perpetrators are conducive for state‐building, negotiated settlements, coexistence, and impunity produce instability due to competing patterns of authority, legitimacy, and social cohesion

Accounting for International War: The State of the Discipline

In studies of war it is important to observe that the processes leading to so frequent an event as conflict are not necessarily those that lead to so infrequent an event as war. Also, many models fail to recognize that a phenomenon irregularly distributed in time and space, such as war, cannot be explained on the basis of relatively invariant phenomena. Much research on periodicity in the occurrence of war has yielded little result, suggesting that the direction should now be to focus on such variables as diffusion and contagion. Structural variables, such as bipolarity, show contradictory results with some clear inter-century differences. Bipolarity, some results suggest, might have different effects on different social entities. A considerable number of studies analysing dyadic variables show a clear connection between equal capabilities among contending nations and escalation of conflict into war. Finally, research into national attributes often points to strength and geographical location as important variables. In general, the article concludes, there is room for modest optimism, as research into the question of war is no longer moving in non-cumulative circles. Systematic research is producing results and there is even a discernible tendency of convergence, in spite of a great diversity in theoretical orientations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69148/2/10.1177_002234338101800101.pd

A Highly Sensitive Biomarker of Type II Collagen C-Terminal Pro-Peptide Associated with Cartilage Formation

The type II collagen C-terminal pro-peptide is one of the most abundant polypeptides in cartilage. The purpose of this study was to develop a competitive chemiluminescence enzyme-linked immunosorbent assay, CALC2, targeting this pro-peptide as a marker of cartilage formation. Technical assay parameters were evaluated. CALC2 level was measured after in vitro cleavage of recombinant type II collagen with bone morphogenetic protein-1 (BMP-1) and treatment of ex vivo human osteoarthritis (OA) cartilage explant model (HEX) with insulin-like growth factor-1 (IGF-1). Serum CALC2 levels were assessed in 18 patients with rheumatoid arthritis (RA), 19 patients with ankylosing spondylitis (AS), and 18 age- and sex-matched controls in cohort 1 and 8 patients with OA and 14 age- and sex-matched controls in cohort 2. Type II collagen cleavage with BMP-1 increased the CALC2 level. IGF-1 treatment increased the CALC2 levels in HEX compared with the untreated explants (p p = 0.01 and p = 0.02, respectively). These findings indicate that CALC2 may be a novel biomarker of type II collagen formation. However, further preclinical and clinical studies are required to validate these findings

The activation fragment of PAR2 is elevated in serum from patients with rheumatoid arthritis and reduced in response to anti-IL6R treatment

Osteoarthritis (OA) and rheumatoid arthritis (RA) are serious and painful diseases. Protease-activated receptor 2 (PAR2) is involved in the pathology of both OA and RA including roles in synovial hyperplasia, cartilage destruction, osteophyogenesis and pain. PAR2 is activated via cleavage of its N-terminus by serine proteases. In this study a competitive ELISA assay was developed targeting the 36-amino acid peptide that is cleaved and released after PAR2 activation (PRO-PAR2). Technical assay parameters including antibody specificity, intra- and inter-assay variation (CV%), linearity, accuracy, analyte stability and interference were evaluated. PRO-PAR2 release was confirmed after in vitro cleavage of PAR2 recombinant protein and treatment of human synovial explants with matriptase. Serum levels of 22 healthy individuals, 23 OA patients and 15 RA patients as well as a subset of RA patients treated with tocilizumab were evaluated. The PRO-PAR2 antibody was specific for the neo-epitope and intra-inter assay CV% were 6.4% and 5.8% respectively. In vitro cleavage and matriptase treated explants showed increased PRO-PAR2 levels compared to controls. In serum, PRO-PAR2 levels were increased in RA patients and decreased in RA patients treated with tocilizumab. In conclusion, PRO-PAR2 may be a potential biomarker for monitoring RA disease and pharmacodynamics of treatment