Reliability, familiarization effect, and comparisons between a predetermined and a self-determined isometric-squat testing protocol

Purpose: This study examined the interday reliability of a predetermined and a self-determined isometric-squat test among youth soccer players. Familiarization effects were evaluated to determine the minimum number of trials necessary to obtain consistent outputs. Finally, differences between protocols were evaluated.Methods: Thirty-one youth soccer players (mean [SD] age: 13.2 [1.0] y; body mass: 54.1 [3.4] kg; stature: 166.3 [11.2] cm; percentage of estimated adult height: 92.6% [3.6%]) from a top-tier professional academy completed 4 experimental sessions for each protocol: familiarization 1, familiarization 2, test, and retest sessions. Peak force; relative peak force; impulse from 0 to 50 milliseconds, 0 to 100 milliseconds, 0 to 150 milliseconds, and 0 to 200 milliseconds; and rate of force development from 0 to 50 milliseconds, 0 to 100 milliseconds, 0 to 150 milliseconds, and 0 to 200 milliseconds were measured. Results: Both protocols displayed acceptable (intraclass correlation coefficient >=.75 and coefficient of variation ≤10%) reliability statistics for all metrics apart from rate of force development of any time epoch. Differences were found between familiarization 2 and both test and retest sessions for peak force (P = .034 and .021, respectively) and relative peak force (P = .035 and .005, respectively) across both protocols. Conclusions: The isometric-squat test is a reliable test among youth soccer players. Two familiarization sessions seem to be sufficient to ensure data stabilization. Outputs between the self-determined and predetermined are comparable; however, the latter seems preferable due to improved testing time efficiency

Differentiation-inducing-factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum

Differentiation-inducing-factor 1 (DIF-1) is a dichlorinated alkyl phenone {1-[(3,5-dichloro-2,6-dihydroxy-4-methoxy)phenyl]hexan-1-one} from Dictyostelium discoideum, that induces amoebae to differentiate into stalk cells. It was shown previously that DIF-1 is rapidly metabolized into a series of more polar compounds by living cells [Traynor, D. & Kay, R. R. (1991) J. Biol. Chem. 266, 5291–5297]. The first step in DIF-1 metabolism is the formation of DIF metabolite 1 (now known to be DIF-3) by a monodechlorination. We report here the discovery of the enzyme activity catalyzing this dechlorination. A very sensitive enzyme assay was developed, using [3H]DIF-1 and a TLC system to separate DIF-1 from the product, DIF-3. DIF-1 3(5)-dechlorinase is present in the high-speed supernatant of cell lysates, and uses glutathione, at physiological concentrations, as cofactor. Kinetic measurements indicate a Km for DIF-1 of about 70 nM. The enzyme activity is inhibited by DIF-2 (the pentan-1-one analogue of DIF-1), with a median inhibitor concentration (IC50) of 1 μM, and DIF-3 (IC50= 5 μM), which presumably act as substrates, but other compounds structurally related to DIF-1 were much less effective. Aurothioglucose, an inhibitor of selenocysteine enzymes, inhibited DIF-1 3(5)-dechlorinase with IC50= 100 nM. DIF-1 3(5)-dechlorinase activity is developmentally regulated. It is essentially absent from growing cells and increases at the end of aggregation to reach a first peak of activity at the first finger stage, with a further rise at culmination

Slow receptor dissociation kinetics differentiate macitentan from other endothelin receptor antagonists in pulmonary arterial smooth muscle cells.

Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are currently approved for the treatment of pulmonary arterial hypertension (PAH), a devastating disease involving an activated endothelin system and aberrant contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). The novel ERA macitentan has recently concluded testing in a Phase III morbidity/mortality clinical trial in PAH patients. Since the association and dissociation rates of G protein-coupled receptor antagonists can influence their pharmacological activity in vivo, we used human PASMC to characterize inhibitory potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan using calcium release and inositol-1-phosphate (IP(1)) assays. In calcium release assays macitentan, ambrisentan and bosentan were highly potent ERAs with K(b) values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not ambrisentan and bosentan, displayed slow apparent receptor association kinetics as evidenced by increased antagonistic potency upon prolongation of antagonist pre-incubation times. In compound washout experiments, macitentan displayed a significantly lower receptor dissociation rate and longer receptor occupancy half-life (ROt(1/2)) compared to bosentan and ambrisentan (ROt(1/2):17 minutes versus 70 seconds and 40 seconds, respectively). Because of its lower dissociation rate macitentan behaved as an insurmountable antagonist in calcium release and IP(1) assays, and unlike bosentan and ambrisentan it blocked endothelin receptor activation across a wide range of endothelin-1 (ET-1) concentrations. However, prolongation of the ET-1 stimulation time beyond ROt(1/2) rendered macitentan a surmountable antagonist, revealing its competitive binding mode. Bosentan and ambrisentan behaved as surmountable antagonists irrespective of the assay duration and they lacked inhibitory activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with significantly slower receptor dissociation kinetics than the currently approved ERAs. Slow dissociation caused insurmountable antagonism in functional PASMC-based assays and this could contribute to an enhanced pharmacological activity of macitentan in ET-1-dependent pathologies