The Effects of an Acute Dose of New Zealand Blackcurrant Extract on 5-km Running Performance.

This study investigated the effects of an acute dose (900 mg) of New Zealand Blackcurrant (NZBC) extract on 5-km running performance, alongside associated physiological and metabolic responses. Sixteen trained male runners (age 26 ± 5 years, stature 173.4 ± 7.3 cm, body mass 73.7 ± 6.9 kg, maximal oxygen consumption [V˙O2max] 55.4 ± 6.1 ml·kg-1·min-1) ingested either capsules containing NZBC extract (3 × 300 mg CurraNZ, 315 mg anthocyanins) or a matched placebo (3 × 300 mg gluten-free flour) 2 hr before exercise in a double-blind, randomized, crossover design. Performance time, physiological, and metabolic responses were assessed in a 5-km time trial, preceded by 10-min exercise at the lactate threshold on a treadmill. NZBC extract did not alter the physiological or metabolic responses to exercise at the lactate threshold (oxygen uptake, respiratory exchange ratio, minute ventilation, carbohydrate oxidation, fat oxidation, heart rate, blood lactate, or rating of perceived exertion, p > .05). The 5-km time trial was completed in a faster time in the NZBC extract condition compared with placebo (NZBC: 1,308.96 ± 122.36 s, placebo: 1,346.33 ± 124.44, p = .001, d = -0.23, confidence interval range = [-0.46, 0.00 s]). No differences in physiological or metabolic responses were apparent between conditions for the 5-km time trial (p > .05). Ingesting 900 mg of NZBC extract as an acute dose improves performance in trained male runners without altering physiological or metabolic responses to exercise. Further research is needed to assess a wider range of possible mechanisms (e.g., cardiovascular function, metabolite profiles) to advance insight into improved performance following supplementation.

The physiological, perceptual and neuromuscular responses of team sport athletes to a running and cycling high intensity interval training session.

PURPOSE: The acute physiological, perceptual and neuromuscular responses to volume-matched running and cycling high intensity interval training (HIIT) were studied in team sport athletes. METHODS: In a randomized cross-over design, 11 male team sport players completed 3 × 6 min (with 5 min between sets) repeated efforts of 15 s exercising at 120% speed (s[Formula: see text]O2max) or power (p[Formula: see text]O2max) at [Formula: see text]O2max followed by 15 s passive recovery on a treadmill or cycle ergometer, respectively. RESULTS: Absolute mean [Formula: see text]O2 (ES [95% CI] = 1.46 [0.47-2.34], p < 0.001) and heart rate (ES [95% CI] = 1.53 [0.53-2.41], p = 0.001) were higher in running than cycling HIIT. Total time at > 90% [Formula: see text]O2max during the HIIT was higher for running compared to cycling (ES [95% CI] = 1.21 [0.26-2.07], p = 0.015). Overall differential RPE (dRPE) (ES [95% CI] = 0.55 [- 0.32-1.38], p = 0.094) and legs dRPE (ES [95% CI] = - 0.65 [- 1.48-0.23], p = 0.111) were similar, whereas breathing dRPE (ES [95% CI] = 1.01 [0.08-1.85], p = 0.012) was higher for running. Maximal isometric knee extension force was unchanged after running (ES [95% CI] = - 0.04 [- 0.80-0.8], p = 0.726) compared to a moderate reduction after cycling (ES [95% CI] = - 1.17 [- 2.02-0.22], p = 0.001). CONCLUSION: Cycling HIIT in team sport athletes is unlikely to meet the requirements for improving run-specific metabolic adaptation but might offer a greater lower limb neuromuscular load.

Bi-exponential modelling of [Formula: see text] reconstitution kinetics in trained cyclists.

PURPOSE: The aim of this study was to investigate the individual [Formula: see text] reconstitution kinetics of trained cyclists following repeated bouts of incremental ramp exercise, and to determine an optimal mathematical model to describe [Formula: see text] reconstitution. METHODS: Ten trained cyclists (age 41 ± 10 years; mass 73.4 ± 9.9 kg; [Formula: see text] 58.6 ± 7.1 mL kg min-1) completed three incremental ramps (20 W min-1) to the limit of tolerance with varying recovery durations (15-360 s) on 5-9 occasions. [Formula: see text] reconstitution was measured following the first and second recovery periods against which mono-exponential and bi-exponential models were compared with adjusted R2 and bias-corrected Akaike information criterion (AICc). RESULTS: A bi-exponential model outperformed the mono-exponential model of [Formula: see text] reconstitution (AICc 30.2 versus 72.2), fitting group mean data well (adjR2 = 0.999) for the first recovery when optimised with parameters of fast component (FC) amplitude = 50.67%; slow component (SC) amplitude = 49.33%; time constant (τ)FC = 21.5 s; τSC = 388 s. Following the second recovery, W' reconstitution reduced by 9.1 ± 7.3%, at 180 s and 8.2 ± 9.8% at 240 s resulting in an increase in the modelled τSC to 716 s with τFC unchanged. Individual bi-exponential models also fit well (adjR2 = 0.978 ± 0.017) with large individual parameter variations (FC amplitude 47.7 ± 17.8%; first recovery: (τ)FC = 22.0 ± 11.8 s; (τ)SC = 377 ± 100 s; second recovery: (τ)FC = 16.3.0 ± 6.6 s; (τ)SC = 549 ± 226 s). CONCLUSIONS: W' reconstitution kinetics were best described by a bi-exponential model consisting of distinct fast and slow phases. The amplitudes of the FC and SC remained unchanged with repeated bouts, with a slowing of W' reconstitution confined to an increase in the time constant of the slow component.

Physiological and anthropometric determinants of critical power, W' and the reconstitution of W' in trained and untrained male cyclists.

PURPOSE: This study examined the relationship of physiological and anthropometric characteristics with parameters of the critical power (CP) model, and in particular the reconstitution of W' following successive bouts of maximal exercise, amongst trained and untrained cyclists. METHODS: Twenty male adults (trained nine; untrained 11; age 39 ± 15 year; mass 74.7 ± 8.7 kg; V̇O2max 58.0 ± 8.7 mL kg-1 min-1) completed three incremental ramps (20 W min-1) to exhaustion interspersed with 2-min recoveries. Pearson's correlation coefficients were used to assess relationships for W' reconstitution after the first recovery (W'rec1), the delta in W' reconstituted between recoveries (∆W'rec), CP and W'. RESULTS: CP was strongly related to V̇O2max for both trained (r = 0.82) and untrained participants (r = 0.71), whereas W' was related to V̇O2max when both groups were considered together (r = 0.54). W'rec1 was strongly related to V̇O2max for the trained (r = 0.81) but not untrained (r = 0.18); similarly, ∆W'rec was strongly related to V̇O2max (r = - 0.85) and CP (r = - 0.71) in the trained group only. CONCLUSIONS: Notable physiological relationships between parameters of aerobic fitness and the measurements of W' reconstitution were observed, which differed among groups. The amount of W' reconstitution and the maintenance of W' reconstitution that occurred with repeated bouts of maximal exercise were found to be related to key measures of aerobic fitness such as CP and V̇O2max. This data demonstrates that trained cyclists wishing to improve their rate of W' reconstitution following repeated efforts should focus training on improving key aspects of aerobic fitness such as V̇O2max and CP.

Slowing the Reconstitution of W' in Recovery With Repeated Bouts of Maximal Exercise.

PURPOSE: This study examined the partial reconstitution of the work capacity above critical power (W') following successive bouts of maximal exercise using a new repeated ramp test, against which the fit of an existing W' balance ( Wbal' ) prediction model was tested. METHODS: Twenty active adults, consisting of trained cyclists (n = 9; age 43 [15] y, V˙O2max 61.9 [8.5] mL·kg-1·min-1) and untrained cyclists (n = 11; age 36 [15] y, V˙O2max 52.4 [5.8] mL·kg-1·min-1) performed 2 tests 2 to 4 d apart, consisting of 3 incremental ramps (20 W·min-1) to exhaustion interspersed with 2-min recoveries. RESULTS: Intratrial differences between recoveries demonstrated significant reductions in the amount of W' reconstituted for the group and both subsets (P < .05). The observed minimal detectable changes of 475 J (first recovery) and 368 J (second recovery) can be used to monitor changes in the rate of W' reconstitution in individual trained cyclists. Intertrial relative reliability of W' reconstitution was evaluated by intraclass correlation coefficients for the group (≥.859) and the trained (≥.940) and untrained (≥.768) subsets. Absolute reliability was evaluated with typical error (TE) and coefficient of variation (CV) for the group (TE ≤ 559 J, CV ≤ 9.2%), trained (TE ≤ 301 J, CV ≤ 4.7%), and untrained (TE ≤ 720 J, CV ≤ 12.4%). CONCLUSIONS: The reconstitution of W' is subject to a fatiguing effect hitherto unaccounted for in Wbal' prediction models. Furthermore, the Wbal' model did not provide a good fit for the repeated ramp test, which itself proved to be a reliable test protocol.

Three-dimensional structure of an intact glycoside hydrolase family 15 glucoamylase from Hypocrea jecorina.

The three-dimensional structure of a complete Hypocrea jecorina glucoamylase has been determined at 1.8 A resolution. The presented structure model includes the catalytic and starch binding domains and traces the course of the 37-residue linker segment. While the structures of other fungal and yeast glucoamylase catalytic and starch binding domains have been determined separately, this is the first intact structure that allows visualization of the juxtaposition of the starch binding domain relative to the catalytic domain. The detailed interactions we see between the catalytic and starch binding domains are confirmed in a second independent structure determination of the enzyme in a second crystal form. This second structure model exhibits an identical conformation compared to the first structure model, which suggests that the H. jecorina glucoamylase structure we report is independent of crystal lattice contact restraints and represents the three-dimensional structure found in solution. The proposed starch binding regions for the starch binding domain are aligned with the catalytic domain in the three-dimensional structure in a manner that supports the hypothesis that the starch binding domain serves to target the glucoamylase at sites where the starch granular matrix is disrupted and where the enzyme might most effectively function.

Structure of a novel enzyme that catalyzes acyl transfer to alcohols in aqueous conditions.

The unusual architecture of the enzyme (MsAcT) isolated from Mycobacterium smegmatis forms the mechanistic basis for favoring alcoholysis over hydrolysis in water. Unlike hydrolases that perform alcoholysis only under anhydrous conditions, MsAcT demonstrates alcoholysis in substantially aqueous media and, in the presence of hydrogen peroxide, has a perhydrolysis:hydrolysis ratio 50-fold greater than that of the best lipase tested. The crystal structures of the apoenzyme and an inhibitor-bound form have been determined to 1.5 A resolution. MsAcT is an octamer in the asymmetric unit and forms a tightly associated aggregate in solution. Relative to other structurally similar monomers, MsAcT contains several insertions that contribute to the oligomerization and greatly restrict the shape of the active site, thereby limiting its accessibility. These properties create an environment by which MsAcT can catalyze transesterification reactions in an aqueous medium and suggests how a serine hydrolase can be engineered to be an efficient acyltransferase.

A silicone-based controlled-release device for accelerated proteolytic debridement of wounds.

A new device for rapid enzymatic debridement of cutaneous wounds has been developed using a controlled-release, silicone-based, dried emulsion. A dehydrated serine protease of the subtilisin family, previously untested for wound debridement, was incorporated into the emulsion. This device exhibited excellent storage stability. Moisture from the wound triggered an even, reproducible, and complete release of the enzyme within the first 8 hours. The device maintains a moist wound environment that allows the enzyme to achieve nearly complete digestion of the hardened eschar of full-thickness burns in a porcine model after an exposure period of 24 hours. Debridement was faster than in untreated wounds or wounds treated with a currently available enzyme ointment. Following rapid enzymatic debridement, healing appeared to progress normally, with no histological evidence of damage to adjacent healthy tissue.

Comparative NMR study on the impact of point mutations on protein stability of Pseudomonas mendocina lipase.

In this work we compare the dynamics and conformational stability of Pseudomonas mendocina lipase enzyme and its F180P/S205G mutant that shows higher activity and stability for use in washing powders. Our NMR analyses indicate virtually identical structures but reveal remarkable differences in local dynamics, with striking correspondence between experimental data (i.e., (15)N relaxation and H/D exchange rates) and data from Molecular Dynamics simulations. While overall the cores of both proteins are very rigid on the pico- to nanosecond timescale and are largely protected from H/D exchange, the two point mutations stabilize helices alpha1, alpha4, and alpha5 and locally destabilize the H-bond network of the beta-sheet (beta7-beta9). In particular, it emerges that helix alpha5, undergoing some fast destabilizing motions (on the pico- to nanosecond timescale) in wild-type lipase, is substantially rigidified by the mutation of Phe180 for a proline at its N terminus. This observation could be explained by the release of some penalizing strain, as proline does not require any "N-capping" hydrogen bond acceptor in the i+3 position. The combined experimental and simulated data thus indicate that reduced molecular flexibility of the F180P/S205G mutant lipase underlies its increased stability, and thus reveals a correlation between microscopic dynamics and macroscopic thermodynamic properties. This could contribute to the observed altered enzyme activity, as may be inferred from recent studies linking enzyme kinetics to their local molecular dynamics.

Glycodendriproteins: a synthetic glycoprotein mimic enzyme with branched sugar-display potently inhibits bacterial aggregation.

The continuing ability of bacteria to resist current antibiotic treatments highlights the need for alternative strategies for inhibiting their pathogenicity. Bacterial attachment is a major factor in infectivity and virulence. This key binding phase of bacteria to any potential host is mediated by adhesin proteins and so these present an attractive therapeutic target for antiinfective blocking strategies. However, the natural ligands to adhesins are large, typically complex molecules that are difficult to mimic with small molecules. We describe here a method that creates precise synthetic mimics of glycoproteins that are designed to bind adhesins. By using protein-degrading enzymes as the basis for these mimics we have created large-molecule protein ligands that inhibit aggregation of pathogenic bacteria at levels greater than a million-fold higher than small-molecule inhibitors of adhesins.

Do enzymes change the nature of transition states? Mapping the transition state for general acid-base catalysis of a serine protease.

The properties of the transition state for serine protease-catalyzed hydrolysis of an amide bond were determined for a series of subtilisin variants from Bacillus lentus. There is no significant change in the structure of the enzyme upon introduction of charged mutations S156E/S166D, suggesting that changes in catalytic activity reflect global properties of the enzyme. The effect of charged mutations on the pK(a) of the active site histidine-64 N(epsilon)(2)-H was correlated with changes in the second-order rate constant k(cat)/K(m) for hydrolysis of tetrapeptide anilides at low ionic strength with a Brønsted slope alpha = 1.1. The solvent isotope effect (D)2(O)(k(cat)/K(m))(1) = 1.4 +/- 0.2. These results are consistent with a rate-limiting breakdown of the tetrahedral intermediate in the acylation step with hydrogen bond stabilization of the departing amine leaving group. There is an increase in the ratio of hydrolysis of succinyl-Ala-Ala-Pro-Phe-anilides for p-nitroaniline versus aniline leaving groups with variants with more basic active site histidines that can be described by the interaction coefficient p(xy) = delta beta(lg)/delta pK(a) (H64) = 0.15. This is attributed to increased hydrogen bonding of the active site imidazolium N-H to the more basic amine leaving group as well as electrostatic destabilization of the transition state. A qualitative characterization of the transition state is presented in terms of a reaction coordinate diagram that is defined by the structure-reactivity parameters.