A Src homology 3-binding sequence on the C terminus of Sprouty2 is necessary for inhibition of the Ras/ERK pathway downstream of fibroblast growth factor receptor stimulation

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.     

Familial, structural, and environmental correlates of MRI-defined bone marrow lesions: a sibpair study

The aim of this study was to estimate the heritability and describe the correlates of bone marrow lesions in knee subchondral bone. A sibpair design was used. T2- and T1-weighted MRI scans were performed on the right knee to assess bone marrow lesions at lateral tibia and femora and medial tibia and femora, as well as chondral defects. A radiograph was taken on the same knee and scored for individual features of osteoarthritis (radiographic osteoarthritis; ROA) and alignment. Other variables measured included height, weight, knee pain, and lower-limb muscle strength. Heritability was estimated with the program SOLAR (Sequential Oligogenetic Linkage Analysis Routines). A total of 115 siblings (60 females and 55 males) from 48 families, representing 95 sib pairs, took part. The adjusted heritability estimates were 53 ± 28% (mean ± SEM; p = 0.03) and 65 ± 32% (p = 0.03) for severity of bone marrow lesions at lateral and medial compartments, respectively. The estimates were reduced by 8 to 9% after adjustment for chondral defects and ROA (but not alignment). The adjusted heritability estimate was 99% for prevalent bone marrow lesions at both lateral and medial compartments. Both lateral and medial bone marrow lesions were significantly correlated with age, chondral defects, and ROA of the knee (all p < 0.05). Medial bone marrow lesions were also more common in males and were correlated with body mass index (BMI). Thus, bone marrow lesions have a significant genetic component. They commonly coexist with chondral defects and ROA but only share common genetic mechanisms to a limited degree. They are also more common with increasing age, male sex, and increasing BMI.     

Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications

Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.     

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.     

Is the involvement of opinion leaders in the implementation of research findings a feasible strategy?

Background

Although numerous therapies have been shown to be beneficial in the prevention of myocardial infarction and/or death in patients with coronary disease, these therapies are under-used and this gap contributes to sub-optimal patient outcomes. To increase the uptake of proven efficacious therapies in patients with coronary disease, we designed a multifaceted quality improvement intervention employing patient-specific reminders delivered at the point-of-care, with one-page treatment guidelines endorsed by local opinion leaders ("Local Opinion Leader Statement"). This trial is designed to evaluate the impact of these Local Opinion Leader Statements on the practices of primary care physicians caring for patients with coronary disease. In order to isolate the effects of the messenger (the local opinion leader) from the message, we will also test an identical quality improvement intervention that is not signed by a local opinion leader ("Unsigned Evidence Statement") in this trial.

Methods

Randomized trial testing three different interventions in patients with coronary disease: (1) usual care versus (2) Local Opinion Leader Statement versus (3) Unsigned Evidence Statement. Patients diagnosed with coronary artery disease after cardiac catheterization (but without acute coronary syndromes) will be randomly allocated to one of the three interventions by cluster randomization (at the level of their primary care physician), if they are not on optimal statin therapy at baseline. The primary outcome is the proportion of patients demonstrating improvement in their statin management in the first six months post-catheterization. Secondary outcomes include examinations of the use of ACE inhibitors, anti-platelet agents, beta-blockers, non-statin lipid lowering drugs, and provision of smoking cessation advice in the first six months post-catheterization in the three treatment arms. Although randomization will be clustered at the level of the primary care physician, the design effect is anticipated to be negligible and the unit of analysis will be the patient.

Discussion

If either the Local Opinion Leader Statement or the Unsigned Evidence Statement improves secondary prevention in patients with coronary disease, they can be easily modified and applied in other communities and for other target conditions.     

How can automimicry persist when predators can preferentially consume undefended mimics?

It is common for species that possess toxins or other defences to advertise these defences to potential predators using aposematic ("warning") signals. There is increasing evidence that within such species, there are individuals that have reduced or non-existent levels of defence but still signal. This phenomenon (generally called automimicry) has been a challenge to evolutionary biologists because of the need to explain why undefended automimics do not gain such as a fitness advantage by saving the physiological costs of defence that they increase in prevalence within the population, hence making the aposematic signal unreliable. The leading theory is that aposematic signals do not stop all predatory attacks but rather encourage predators to attack cautiously until they have identified the defence level of a specific individual. They can then reject defended individuals and consume the undefended. This theory has recently received strong empirical support, demonstrating that high-accuracy discrimination appears possible. However, this raises a new evolutionary problem: if predators can perfectly discriminate the defended from the undefended and preferentially consume the latter, then how can automimicry persist? Here, we present four different mechanisms that can allow non-trivial levels of automimics to be retained within a population, even in the extreme case where predators can differentiate defended from undefended individuals with 100% accuracy. These involve opportunity costs to the predator of sampling carefully, temporal fluctuation in predation pressure, predation pressure being correlated with the prevalence of automimicry, or developmental or evolutionary constraints on the availability of defence. These mechanisms generate predictions as to the conditions where we would expect aposematically signalling populations to feature automimicry and those where we would not.     

Evolution of anti-predator traits in response to a flexible targeting strategy by predators

Anti-predator benefits increase with vigilance rate and group size in many species of animal, while simultaneously resource intake rates usually decrease. This implies that there is an optimal group size and vigilance rate that will maximize individual fitness. While this basic theory of vigilance has been modelled and tested extensively, it has often been assumed that the predator represents a 'fixed-risk' such that groups of prey are essentially independent entities that exert little or no effect on one another either directly or indirectly. We argue that this is an over-simplification, and propose that the behaviour of one group of prey will likely affect the fitness of another local group of prey if the predator preferentially attacks the most vulnerable group-rather than attack both with constant rates. Using a numerical simulation model, we make the first examination of this game and allow the prey to dynamically evolve both optimal group size distributions between two habitats and vigilance rates in response to a predator with a preference for whichever group is the more vulnerable. We show that the density of prey in the population and the sensitivity of a predator to differences in prey vulnerability are likely to drive the dynamics of such a game. This novel approach to vigilance theory opens the door to several challenging lines of future research, both experimental and theoretical.     

Discovery of 3-arylpropionic acids as potent agonists of sphingosine-1-phosphate receptor-1 (S1P1) with high selectivity against all other known S1P receptor subtypes

A series of 3-arylpropionic acids were synthesized as S1P1 receptor agonists. Structure-activity relationship studies on the pendant phenyl ring revealed several structural features offering selectivity of S1P1 binding against S1P2-5. These highly selective S1P1 agonists induced peripheral blood lymphocyte lowering in mice and one of them was found to be efficacious in a rat skin transplantation model, supporting that S1P1 agonism is primarily responsible for the immunosuppressive efficacy observed in preclinical animal models.