Identification of proteins involved in intracellular ubiquinone trafficking in Saccharomyces cerevisiae using artificial ubiquinone probe.

Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ6, exogenously supplied UQs can be taken up and delivered to mitochondria through an unknown mechanism, restoring the growth of UQ6-deficient yeast in non-fermentable medium. Since elucidating the mechanism responsible may markedly contribute to therapeutic strategies for patients with UQ deficiency, many attempts have been made to identify the machinery involved in UQ trafficking in the yeast model. However, definite experimental evidence of the direct interaction of UQ with a specific protein(s) has not yet been demonstrated. To gain insight into intracellular UQ trafficking via a chemistry-based strategy, we synthesized a hydrophobic UQ probe (pUQ5), which has a photoreactive diazirine group attached to a five-unit isoprenyl chain and a terminal alkyne to visualize and/or capture the labeled proteins via click chemistry. pUQ5 successfully restored the growth of UQ6-deficient S. cerevisiae (Δcoq2) on a non-fermentable carbon source, indicating that this UQ was taken up and delivered to mitochondria, and served as a UQ substrate of respiratory enzymes. Through photoaffinity labeling of the mitochondria isolated from Δcoq2 yeast cells cultured in the presence of pUQ5, we identified many labeled proteins, including voltage-dependent anion channel 1 (VDAC1) and cytochrome c oxidase subunit 3 (Cox3). The physiological relevance of UQ binding to these proteins is discussed.

Intermittent face cooling reduces perceived exertion during exercise in a hot environment.

BACKGROUND: Facial cooling (FC) is effective in improving endurance exercise performance in hot environments. In this study, we evaluated the impact of intermittent short-lasting FC on the ratings of perceived exertion (RPE) during exercise. METHODS: Ten healthy men performed 40 continuous minutes of ergometric cycle exercise at 65% of the peak heart rate in a climatic chamber controlled at an ambient temperature of 35 °C and a relative humidity of 50%. In the control (CONT) trial, the participants performed the exercise without FC. In two cooling trials, each participant underwent 10 s of FC at 2- (FC2) and 4-min (FC4) intervals while continuing to exercise. FC was achieved by applying two soft-gel packs (cooled to 0 °C) directly and bilaterally on the forehead, eyes, and cheeks. In another cooling trial, 10 s of FC was performed at 2-min intervals using two soft-gel packs cooled to 20 °C (FC2-20). RESULTS: The RPE values in the FC4 trial were significantly lower than those in the CONT trial at 20 min (FC4, 11.6 ± 2.2 points; CONT, 14.2 ± 1.3 points; P < 0.01). Further, significant differences in the RPE values were observed between the FC4 and CONT trials at 5-15 min and 25-40 min (P < 0.05). RPE values were also significantly lower in the FC2 trial than in the CONT trial (5-40 min). Although the RPE values in the FC2-20 trial were significantly lower (5-10 min; 15-20 min) than those in the CONT trial, there were no significant differences in the RPE between the FC2-20 and CONT trials at 25-40 min. At 35 min, the RPE values were significantly higher in the FC2-20 trial than in the FC2 trial (P < 0.05). CONCLUSION: Intermittent short-lasting FC was associated with a decrease in RPE, with shorter intervals and lower temperatures eliciting greater attenuation of increase in the RPE.

External mechanical work done during the acceleration stage of maximal sprint running and its association with running performance.

This study aimed to elucidate how external mechanical work done during maximal acceleration sprint running changes with increasing running velocity and is associated with running performance. In twelve young males, work done at each step over 50 m from the start was calculated from mechanical energy changes in horizontal anterior-posterior and vertical directions and was divided into braking (-Wkap and -Wv, respectively) and propulsive (+Wkap and +Wv, respectively) phases. The maximal running velocity (Vmax) appeared at 35.87±7.76 m and the time required to run 50 m (T50 m) was 7.11±0.54 s. At 80% Vmax or higher, +Wkap largely decreased and -Wkap abruptly increased. The change in the difference between +Wkap and |-Wkap| (ΔWkap) at every step was relatively small at 70% Vmax or lower. Total work done over 50 m was 82.4±7.5 J kg-1 for +Wkap, 36.2±4.4 J kg-1 for |-Wkap|, 14.3±1.9 J kg-1 for +Wv, and 10.4±1.2 J kg-1 for |-Wv|. The total ΔWkap over 50 m was more strongly correlated with T50 m (r=-0.946, P<0.0001) than the corresponding associations for the other work variables. These results indicate that in maximal sprint running over 50 m, work done during the propulsive phase in the horizontal anterior-posterior direction accounts for the majority of the total external work done during the acceleration stage, and maximizing it while suppressing work done during the braking phase is essential to achieve a high running performance.

Age-Related Differences in Spatiotemporal Variables and Ground Reaction Forces During Sprinting in Boys.

PURPOSE: We aimed to elucidate age-related differences in spatiotemporal and ground reaction force variables during sprinting in boys over a broad range of chronological ages. METHODS: Ground reaction force signals during 50-m sprinting were recorded in 99 boys aged 6.5-15.4 years. Step-to-step spatiotemporal variables and mean forces were then calculated. RESULTS: There was a slower rate of development in sprinting performance in the age span from 8.8 to 12.1 years compared with younger and older boys. During that age span, mean propulsive force was almost constant, and step frequency for older boys was lower regardless of sprinting phase. During the ages younger than 8.8 years and older than 12.1 years, sprint performance rapidly increased with increasing mean propulsive forces during the middle acceleration and maximal speed phases and during the initial acceleration phase. CONCLUSION: There was a stage of temporal slower development of sprinting ability from age 8.8 to 12.1 years, being characterized by unchanged propulsive force and decreased step frequency. Moreover, increasing propulsive forces during the middle acceleration and maximal speed phases and during the initial acceleration phase are probably responsible for the rapid development of sprinting ability before and after the period of temporal slower development of sprinting ability.

Association of Sprint Performance With Ground Reaction Forces During Acceleration and Maximal Speed Phases in a Single Sprint.

We aimed to clarify the mechanical determinants of sprinting performance during acceleration and maximal speed phases of a single sprint, using ground reaction forces (GRFs). While 18 male athletes performed a 60-m sprint, GRF was measured at every step over a 50-m distance from the start. Variables during the entire acceleration phase were approximated with a fourth-order polynomial. Subsequently, accelerations at 55%, 65%, 75%, 85%, and 95% of maximal speed, and running speed during the maximal speed phase were determined as sprinting performance variables. Ground reaction impulses and mean GRFs during the acceleration and maximal speed phases were selected as independent variables. Stepwise multiple regression analysis selected propulsive and braking impulses as contributors to acceleration at 55%-95% (ß > 0.72) and 75%-95% (ß > 0.18), respectively, of maximal speed. Moreover, mean vertical force was a contributor to maximal running speed (ß = 0.48). The current results demonstrate that exerting a large propulsive force during the entire acceleration phase, suppressing braking force when approaching maximal speed, and producing a large vertical force during the maximal speed phase are essential for achieving greater acceleration and maintaining higher maximal speed, respectively.

Step-to-step spatiotemporal variables and ground reaction forces of intra-individual fastest sprinting in a single session.

We aimed to investigate the step-to-step spatiotemporal variables and ground reaction forces during the acceleration phase for characterising intra-individual fastest sprinting within a single session. Step-to-step spatiotemporal variables and ground reaction forces produced by 15 male athletes were measured over a 50-m distance during repeated (three to five) 60-m sprints using a long force platform system. Differences in measured variables between the fastest and slowest trials were examined at each step until the 22nd step using a magnitude-based inferences approach. There were possibly-most likely higher running speed and step frequency (2nd to 22nd steps) and shorter support time (all steps) in the fastest trial than in the slowest trial. Moreover, for the fastest trial there were likely-very likely greater mean propulsive force during the initial four steps and possibly-very likely larger mean net anterior-posterior force until the 17th step. The current results demonstrate that better sprinting performance within a single session is probably achieved by 1) a high step frequency (except the initial step) with short support time at all steps, 2) exerting a greater mean propulsive force during initial acceleration, and 3) producing a greater mean net anterior-posterior force during initial and middle acceleration.

Corticosteroid-resistant inflammatory signalling in Pseudomonas-infected bronchial cells.

Decreasing the inflammatory response that leads to tissue damage during cystic fibrosis (CF) lung disease has been a long-standing goal of CF therapy. While corticosteroids are widely used anti-inflammatory drugs, their efficacy in CF lung disease remains debated. The complex interaction between the colonising bacteria and the host environment may impact corticosteroid responsiveness. In this study, sputum samples from adult CF patients were collected at baseline and during pulmonary exacerbation episodes. Lung function measurements and sputum microbiological analyses were performed. In parallel, the inflammatory response and corticosteroid sensitivity of airway epithelial cells to Pseudomonas-derived exoproducts was investigated. We report that adult CF patients colonised with mucoid Pseudomonas aeruginosa have higher levels of baseline inflammation, more frequent exacerbations and worse lung function compared with patients colonised with nonmucoid P. aeruginosa. Moreover, mucoid P. aeruginosa activates NF-κB via Toll-like receptor (TLR) 2, which acts in an additive manner to TLR5 to drive inflammation in airway epithelial cells. Furthermore, TLR2-mediated intracellular signalling is more resistant to the anti-inflammatory effects of corticosteroid when compared with other TLR signalling pathways. Overall, these results suggest that airway inflammation triggered by mucoid P. aeruginosa is less responsive to the anti-inflammatory action of corticosteroids. Whether this translates into a diminished response of CF patients to corticosteroid therapy should be examined in future clinical studies.

Association of Step Width with Accelerated Sprinting Performance and Ground Reaction Force.

This study aimed to describe changes in step width (SW) during accelerated sprinting, and to clarify the relationship of SW with sprinting performance and ground reaction forces. 17 male athletes performed maximal-effort 60 m sprints. The SW and other spatiotemporal variables, as well as ground reaction impulses, over a 52 m distance were calculated. Average values for each 4 steps during acceleration were calculated to examine relationships among variables in different sections. The SW rapidly decreased up to the 13th step and slightly afterward during accelerated sprinting, showing a bilinear phase profile. The ratio of SW to the stature was significantly correlated with running speed based on average values over the 52 m distance and in the 9th-12th step section during accelerated sprinting. The SW ratio positively correlated with medial, lateral and mediolateral impulses in all step sections, except for medial impulse in the 17th-20th step section. These results indicate the importance of wider SW for better sprinting performance, especially in the 9th-12th step section. Moreover, the wider SW was associated with larger medial impulse and smaller lateral impulse, suggesting that a wide SW contributes to the production of greater mediolateral body velocity during accelerated sprinting.

Reliability and Validity of Kinetic and Kinematic Parameters Determined With Force Plates Embedded Under a Soil-Filled Baseball Mound.

We developed a force measurement system in a soil-filled mound for measuring ground reaction forces (GRFs) acting on baseball pitchers and examined the reliability and validity of kinetic and kinematic parameters determined from the GRFs. Three soil-filled trays of dimensions that satisfied the official baseball rules were fixed onto 3 force platforms. Eight collegiate pitchers wearing baseball shoes with metal cleats were asked to throw 5 fastballs with maximum effort from the mound toward a catcher. The reliability of each parameter was determined for each subject as the coefficient of variation across the 5 pitches. The validity of the measurements was tested by comparing the outcomes either with the true values or the corresponding values computed from a motion capture system. The coefficients of variation in the repeated measurements of the peak forces ranged from 0.00 to 0.17, and were smaller for the pivot foot than the stride foot. The mean absolute errors in the impulses determined over the entire duration of pitching motion were 5.3 N˙s, 1.9 N˙s, and 8.2 N˙s for the X-, Y-, and Z-directions, respectively. These results suggest that the present method is reliable and valid for determining selected kinetic and kinematic parameters for analyzing pitching performance.

KF24345, an adenosine uptake inhibitor, ameliorates the severity and mortality of lethal acute pancreatitis via endogenous adenosine in mice.

Adenosine protects against cellular damage and dysfunction under several adverse conditions including inflammation and ischemia. In this study, we examined the effects of 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), an adenosine uptake inhibitor, on experimental acute pancreatitis induced by choline-deficient and ethionine-supplemented diet in mice. KF24345, administered with the diet onset and every 24 h thereafter, prevented hyperamylasemia, acinar cell injury and serum tumor necrosis factor-alpha elevation and ultimately decreased mortality. Therapeutic treatment with KF24345, which started 32 h after the diet onset, also decreased mortality. The beneficial effect of KF24345 on mortality was abolished by the pretreatment with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), a selective adenosine A(2A) receptor antagonist. An intravenous injection of KF24345 at 48 h after the diet onset increased plasma adenosine concentrations in mice with acute pancreatitis. These results suggest that KF24345 shows anti-pancreatitis effects via endogenous adenosine and adenosine A(2A) receptors. The adenosine uptake inhibition could be a new therapeutic approach for acute pancreatitis.

Adenosine uptake inhibition ameliorates cerulein-induced acute pancreatitis in mice.

INTRODUCTION AND AIMS: Adenosine shows protective effects against cellular damage and dysfunction under several adverse conditions such as inflammation and ischemia. In the current study, we examined the effects of 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1,3 )-quinazolinedione hydrochloride (KF24345), an adenosine uptake inhibitor, on cerulein-induced acute pancreatitis in mice to investigate whether inhibition of adenosine uptake could ameliorate the severity of acute pancreatitis. METHODOLOGY: Acute pancreatitis was induced in mice with six intraperitoneal injections of cerulein (50 microg/kg each) at hourly intervals. RESULTS: The cerulein injection increased activities of serum amylase and lipase and caused pathologic changes such as interstitial edema, polymorphonuclear cell infiltration, and acinar cell necrosis in the pancreas. KF24345 (10 mg/kg p.o.) ameliorated all these changes observed in mice with acute pancreatitis, and the suppressing effect of KF24345 on the elevation in serum amylase activity was abolished by the treatment with 8-(p-sulfophenyl)theophylline, an adenosine receptor antagonist. In addition, 2-(aminocarbonyl)- -(4-amino-2,6-dichlorophenyl)-4-[5,5-bis-(4-fluorophenyl)pentyl]-1-piperazineacetamide (R75231) and dipyridamole, other adenosine uptake inhibitors, also decreased the elevated serum amylase activity. CONCLUSIONS: These are the first demonstrations that the adenosine uptake inhibitors ameliorate cerulein-induced acute pancreatitis in mice, and these data suggest that adenosine uptake inhibition could ameliorate the severity of acute pancreatitis in vivo.

KF24345, an adenosine uptake inhibitor, suppresses lipopolysaccharide-induced tumor necrosis factor-alpha production and leukopenia via endogenous adenosine in mice.

3-[1-(6,7-Diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345) is a novel potent adenosine uptake inhibitor. KF24345 inhibited [(3)H]adenosine uptake into erythrocytes from human, mouse, rabbit, and hamster with IC(50) values of 59.5, 130.1, 104.2, and 30.9 nM, respectively. In mice, oral administration of KF24345 at 10 mg/kg almost completely inhibited the [(3)H]adenosine uptake into sampled blood cells at least up to 10 h of the administration. In this study, to examine whether the adenosine uptake inhibition exhibits anti-inflammatory effects, we determined the effects of KF24345 on lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) production and leukopenia in mice. KF24345 (10 mg/kg p.o.) significantly suppressed the elevation of serum TNF-alpha concentration after the LPS injection, and the suppressing effect of KF24345 was abolished by the treatment with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, a selective adenosine A(2) receptor antagonist, but not with 8-(noradamantan-3-yl)-1,3-dipropylxanthine, a selective adenosine A(1) receptor antagonist. KF24345 (10 mg/kg p.o.) also inhibited the decrease of leukocytes after the LPS injection, and 8-(p-sulfophenyl)theophylline, a nonselective adenosine receptor antagonist, completely reversed the inhibitory effect of KF24345. These results demonstrate that KF24345 inhibits LPS-induced TNF-alpha production and leukopenia via enhancing the effect of endogenous adenosine. It is thus suggested that the adenosine uptake inhibitor has anti-inflammatory effects in vivo and represents a novel therapeutic approach to the treatment of various inflammatory diseases.