C. elegans expressing D76N ß2-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis.

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of ß2-microglobulin (D76N ß2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N ß2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N ß2-m expressing worms. We also demonstrated the specificity of the ß2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when ß2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates.

Radiographic findings of femoroacetabular impingement in capoeira players.

PURPOSE: Capoeira is a Brazilian martial art that requires extreme movements of the hip to perform jumps and kicks. This study evaluated a group of capoeira players to assess the prevalence of femoroacetabular impingement (FAI) in athletes practicing this martial art. METHODS: Twenty-four experienced capoeira players (14 men, 10 women) underwent a diagnostic assessment, including clinical examination and standard radiographs of the pelvis and hips. The α-angle, head-neck offset, crossover sign, acetabular index, lateral centre-edge angle, and the Tönnis grade were assessed using the radiographs. Clinical relationships for any radiographic abnormalities indicating FAI were also evaluated. RESULTS: Four subjects (17 %) reported pain in their hips. Forty-four hips (91.7 %) had at least one radiographic sign of CAM impingement, and 22 (45.8 %) had an α-angle of more than 60°. Eighteen hips (37.5 %) had at least one sign of pincer impingement and 16 (33.3 %) a positive crossover sign. Sixteen hips (33.3 %) had mixed impingement. There was a significant positive association between having an α-angle of more than 60° and the presence of groin pain (P = 0.002). A reduced femoral head-neck offset (P < 0.001) and an increased α-angle on the anteroposterior radiograph (P = 0.008) were independently associated with a higher Tönnis grade. CONCLUSION: High prevalence of radiographic CAM-type FAI among these skilled capoeira players was found. In these subjects, a negative clinical correlation for an increased α-angle was also detected. Additional caution should be exercised whenever subjects with past or present hip pain engage in capoeira.

Effect of tetracyclines on the dynamics of formation and destructuration of beta2-microglobulin amyloid fibrils.

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of ß2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits ß2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble ß2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.

Search of ligands for the amyloidogenic protein beta2-microglobulin by capillary electrophoresis and other techniques.

Beta2-microglobulin (beta2-m) is a small amyloidogenic protein normally present on the surface of most nucleated cells and responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, and consequent inhibition of protein misfolding and amyloid fibril formation. A few compounds have been described to weakly bind beta2-m, including the drug suramin. The lack of a binding site for nonpolypeptidic ligands on the beta2-m structure makes it difficult for both the identification of functional groups responsible for the binding and the search of hits to be optimized. The characterization of the binding properties of suramin for beta2-m by using three different techniques (surface plasmon resonance, affinity CE (ACE), ultrafiltration) is here described and the results obtained are compared. The common features of the chemical structures of the compounds known to bind the protein led us to select 200 sulfonated/suramin-like molecules from a wider chemical library on the basis of similarity rules, so as to possibly single out some interesting hits and to gain more information on the functional groups involved in the binding. The development of screening methods to test the compounds by using ultrafiltration and ACE is described.