Effects of medially posted insoles on foot and lower limb mechanics across walking and running in overpronating men

Anti-pronation orthoses, like medially posted insoles (MPI), have traditionally been used to treat various of lower limb problems. Yet, we know surprisingly little about their effects on overall foot motion and lower limb mechanics across walking and running, which represent highly different loading conditions. To address this issue, multi-segment foot and lower limb mechanics was examined among 11 overpronating men with normal (NORM) and MPI insoles during walking (self-selected speed 1.70 ± 0.19 m/s vs 1.72 ± 0.20 m/s, respectively) and running (4.04 ± 0.17 m/s vs 4.10 ± 0.13 m/s, respectively). The kinematic results showed that MPI reduced the peak forefoot eversion movement in respect to both hindfoot and tibia across walking and running when compared to NORM (p < 0.05–0.01). No differences were found in hindfoot eversion between conditions. The kinetic results showed no insole effects in walking, but during running MPI shifted center of pressure medially under the foot (p < 0.01) leading to an increase in frontal plane moments at the hip (p < 0.05) and knee (p < 0.05) joints and a reduction at the ankle joint (p < 0.05). These findings indicate that MPI primarily controlled the forefoot motion across walking and running. While kinetic response to MPI was more pronounced in running than walking, kinematic effects were essentially similar across both modes. This suggests that despite higher loads placed upon lower limb during running, there is no need to have a stiffer insoles to achieve similar reduction in the forefoot motion than in walking.     

Evaluation of plantar flexion contracture contribution during the gait of children with Duchenne muscular dystrophy

Because of extensor weakness, children with Duchenne muscular dystrophy (DMD) maintain internal flexion moments at the joints of the lower extremities when they walk. We believe that at the ankle, the plantar flexion moments caused by contractures may contribute significantly to the production of the net ankle flexion moment during the gait in these children. The goal of the present study is to quantify ankle plantar flexion passive moments that may be associated with the presence of flexion contractures and to estimate their contribution to the net moment during the gait of children with DMD. Kinematic and kinetic parameters were collected during gait of eleven subjects with DMD. Ankle plantar flexion passive moments were also measured experimentally during the same session. Fourteen control children participated in the study in order to have normal reference values. The presence of ankle plantar flexion contractures in children with DMD was reflected by a rigidity coefficient obtained at a common moment of ?7 Nm that was higher for these children (0.75 Nm/° vs. 0.48 Nm/°; p < 0.05). The relative passive moment contribution to the net plantar flexion moments was higher for the children with DMD at the end of the lengthening phase of the plantar flexors (25% vs. 18%; p < 0.05). We believe that the passive moments can compensate for the presence of progressive muscle weakness in the children with DMD and help these children with gait.     

Serum progesterone concentrations,follicular development and time of ovulation using a new progesterone releasing device (DICO®) in sheep

Four experiments were conducted to evaluate the effectiveness of a new controlled drug releasing device containing 0.3 g progesterone (DICO^®) on ovarian control in sheep. In experiment 1, serum progesterone concentrations induced by a 14 days treatment of DICO^® (n = 9) and CIDR-G^® (n = 9) were compared in ovariectomized ewes. Both devices induced similar responses and no differences were recorded. In experiment 2, the onset of oestrus and the time of ovulation obtained after 14 days treatment with DICO^® (n = 8) and CIDR-G^® (n = 7) were compared in cyclic ewes. Both devices induced oestrus and ovulation in all of the ewes. The onset of oestrus (34.5 ± 2.8 and 30.0 ± 7.7 h), the time of ovulation (60.0 ± 9.1 and 54.9 ± 6.4 h), the ovulation rate (1.3 ± 0.5 and 1.4 ± 0.5), the follicular diameter at ovulation (7.0 ± 0.8 and 7.3 ± 1.1 mm), and the lifespan of the ovulatory follicles (8.6 ± 2.2 and 10.0 ± 2.9 days) were similar for the DICO^® and CIDR-G^® devices, respectively. In Experiment 3, the re-utilization of DICO^® devices inserted for 6 days (i.e. short-term protocol) was evaluated in ovariectomized ewes. The females received a re-used (previously used for 6 days; n = 11) or a new DICO^® (n = 11) for a period of 6 days. The re-used DICO^® devices induced a lower serum progesterone concentration than the new devices (P < 0.05). However, the re-used DICO^® device maintained serum progesterone concentrations above 7.1 nmol/L (i.e. >2 ng/ml) throughout treatment. In Experiment 4, the administration of eCG treatment at DICO^® withdrawal was evaluated in cyclic ewes. The short-term protocol using DICO^® devices for 6 days was applied with (n = 8) or without (n = 7) 300 IU eCG at the time of device withdrawal. The administration of eCG advanced ovarian follicular development, synchronizing the onset of oestrus at 36 h and the time of ovulation at 60 h from device withdrawal. In conclusion, data from these experiments show the use of DICO^® or CIDR-G^® devices containing 0.3 g of progesterone to have a similar efficiency in controlling serum progesterone concentrations, follicular development and the time of ovulation in sheep. The re-use of the devices, associated with the short-term protocol for 6 days is possible, although further studies on induced fertility rates are warranted.     

Effects of plyometric and pneumatic explosive strength training on neuromuscular function and dynamic balance control in 60–70 year old males

The present study compared neuromuscular adaptations to 12 weeks of plyometric (PLY) or pneumatic (PNE) power training and their effects on dynamic balance control. Twenty-two older adults aged 60–70 (PLY n = 9, PNE n = 11) participated in the study. Measurements were conducted at Pre, 4, 8 and 12 weeks. Dynamic balance was assessed as anterior–posterior center of pressure (COP) displacement in response to sudden perturbations. Explosive isometric knee extension and plantar flexion maximal voluntary contractions (MVCs) were performed. Maximal drop jump performance from optimal dropping height was measured in a sledge ergometer. Increases in knee extensor and ankle plantar flexor torque and muscle activity were higher and occurred sooner in PNE, whereas in drop jumping, PLY showed a clearer increase in optimal drop height (24%, p < 0.01) after 8 weeks of training and soleus muscle activity after 12 weeks of training. In spite of these training mode specific adaptations, both groups showed similar improvements in dynamic balance control after 4 weeks of training (PLY 38%, p < 0.001; PNE 31%, p < 0.001) and no change thereafter. These results show that although power and plyometric training may involve different neural adaptation mechanisms, both training modes can produce similar improvements in dynamic balance control in older individuals. As COP displacement was negatively correlated with rapid knee extension torque in both groups (PLY r = −0.775, p < 0.05; PNE r = −0.734, p < 0.05) after training, the results also highlight the importance of targeting rapid force production when training older adults to improve dynamic balance.     

Preference and motivation for different diet forms and their effect on motivation for a foraging enrichment in captive Orange-winged Amazon parrots (Amazona amazonica)

Motivation tests were conducted to assess preference strength for diet form and a cage enrichment device by Orange-winged Amazon parrots (Amazona amazonica) held in individual cages, N = 10. Each cage was equipped with two trough-type feeders, one of which had a hinged lid that required lifting to access feeder content; cost of lifting the lid could be increased by the addition of up to 480 g upon it, ～1.5 times the mass of an Amazon parrot. Motivation tests were conducted using three different diet forms of pellets (regular-sized, 0.16 g/pellet (cylindrical-shaped); large-sized, 3.4 g/pellet (cylindrical); and over-sized, 3–5 g/pellet (cuboid-to-spheroid)) manufactured from the same diet formulation. When regular pellets were concomitantly freely available, birds were still highly motivated to gain access to either large-sized or over-sized pellets, with 7 of 10 birds lifting the maximum weight of 480 g. In motivation tests comparing over-sized vs. large-sized pellets, birds worked more for over-sized pellets (when large-sized pellets were freely available) than vice versa: motivation for over-sized pellets exceeded that for large-sized pellets by approximately 242.67 ± 64.4 g (F = 14.2, P = 0.0055; Sign Test, P = 0.0078). Additional tests assessed motivation to access 2.5 cm/side wooden cube enrichment devices when regular or over-sized pellets were freely available. Birds removed more cubes when fed regular pellets (Sign Test, P = 0.0078) and lifted an average of 221.33 ± 64.62 g more to access them (F = 11.73, P = 0.009; Sign Test, P = 0.0063), than when over-sized pellets were freely available, suggesting that enrichment devices may act as foraging substitutes. Likewise, both wooden cubes and over-sized pellets elicited comparable podomandibulation (handling with beak and foot) behavior, and podomandibulation was reduced when parrots were fed regular as opposed to over-sized pellets (58 ± 10 s [mean ± SE] vs. 4.27 min ± 31 s; P < 0.0001). Finally, there was no evidence of contrafreeloading: in control experiments, birds only removed pellets which were freely available, when the same pellets were concomitantly available in weighted-lid feeders (F = 120.20, P < 0.0001). In summary, these results show that captive Orange-winged Amazon parrots strongly prefer pellet forms that are substantially larger than what is commercially recommended for them; preferred forms more closely resemble that of native wild foods, such as palm fruits.     

Unchanged H-reflex during a sustained isometric submaximal plantar flexion performed with an EMG biofeedback

The aim of this study was to assess H-reflex plasticity and activation pattern of the plantar flexors during a sustained contraction where voluntary EMG activity was controlled via an EMG biofeedback. Twelve healthy males (28.0 ± 4.8 yr) performed a sustained isometric plantar flexion while instructed to maintain summed EMG root mean square (RMS) of gastrocnemius lateralis (GL) and gastrocnemius medialis (GM) muscles fixed at a target corresponding to 80% maximal voluntary contraction torque via an EMG biofeedback. Transcutaneous electrical stimulation of the posterior tibial nerve was evoked during the contraction to obtain the maximal H-reflex amplitude to maximal M-wave amplitude ratio (H_sup/M_sup ratio) from GL, GM and soleus (SOL) muscles. Neuromuscular function was also assessed before and immediately after exercise. Results showed a decrease in SOL activation during sustained flexion (from 65.5 ± 6.4% to 42.3 ± 3.8% maximal EMG, p < 0.001), whereas summed EMG RMS of GL and GM remained constant (59.7 ± 4.8% of maximal EMG on average). No significant change in the H_sup/M_sup ratio was found for SOL, GL and GM muscles. Furthermore, it appears that the decrease in maximal voluntary contraction torque (?20.4 ± 2.9%, p < 0.001) was related to both neural and contractile impairment. Overall, these findings indicate that the balance between excitation and inhibition affecting the motoneuron pool remains constant during a sustained contraction where myoelectrical activity is controlled via an EMG biofeedback or let free to vary.     

How symmetric are metal-on-metal hip resurfacing patients during gait? Insights for the rehabilitation

Metal-on-metal hip resurfacing patients demonstrate hip biomechanics closer to normal in comparison to total hip arthroplasty during gait. However, it is not clear how symmetric is the gait of hip resurfacing patients. Biomechanical data of 12 unilateral metal-on-metal hip resurfacing participants were collected during gait at a mean time of 45 months (SD 24) after surgery. Ankle, knee, hip, pelvis and trunk kinematics and kinetics of both sides were measured with a motion and force-capture system. Principal component analysis and mean hypothesis’ tests were used to compare the operated and healthy sides. The operated side had prolonged ankle eversion angle during late stance and delayed increased ankle inversion angle during early swing (p = 0.008; effect size = 0.70), increased ankle inversion moment during late stance (p = 0.001; effect size = 0.78), increased knee adduction angle during swing (p = 0.044; effect size = 0.57), decreased knee abduction moment during stance (p = 0.05; effect size = 0.40), decreased hip range of motion in the sagittal plane (p = 0.046; effect size = 0.56), decreased range of hip abduction moment during stance (p = 0.02; effect size = 0.63), increased hip range of motion in the transverse plane (p = 0.02; effect size = 0.62), decreased hip internal rotation moment during the transition from loading response to midstance (p = 0.001; effect size = 0.81) and increased trunk ipsilateral lean (p = 0.03; effect size = 0.60). Therefore, hip resurfacing patients have some degree of asymmetry in long term, which may be related to hip weakness and decreased range of motion, to foot misalignments and to strategies implemented to reduce loading on the operated hip. Interventions such as muscle strengthening and stretching, insoles and gait feedback training may help improving symmetry following hip resurfacing.     

The effect of deep hypothermia on the human pulmonary circulation

ObjectiveAcute rises in pulmonary artery pressures following complex cardiac surgery are associated with high morbidity and mortality. We hypothesised that periods of deep hypothermia predispose to elevated pulmonary pressures upon rewarming. We investigated the effect of this hypothermic preconditioning on isolated human pulmonary arteries and isolated perfused lungs.MethodsIsometric tension was measured in human pulmonary artery rings (n=24). We assessed the constriction and dilation of these arteries at 37 °C and 17 °C. Isolated perfused human lung models consisted of lobes ventilated via a bronchial cannula and perfused with Krebs via a pulmonary artery cannula. Bronchial and pulmonary artery pressures were recorded. We investigated the effect of temperature using a heat exchanger.ResultsRewarming from 17 °C to 37 °C caused a 1.3 fold increase in resting tension (p<0.05). Arteries constricted 8.6 times greater to 30 nM KCl, constricted 17 times greater to 1 nM Endothelin-1 and dilated 30.3 times greater to 100 μM SNP at 37 °C than at 17 °C (p<0.005). No difference was observed in the responses of arteries originally maintained at 37 °C compared to those arteries maintained at 17 °C and rewarmed to 37 °C. Hypothermia blunted the increase in pulmonary artery pressures to stimulants such as potassium chloride as well as to H-R but did not precondition arteries to higher pulmonary artery pressures upon re-warming.ConclusionsDeep hypothermia reduces the responsiveness of human pulmonary arteries but does not, however, precondition an exaggerated response to vasoactive agents upon re-warming.     

Abdominal muscle activity during breathing with and without inspiratory and expiratory loads in healthy subjects

Central Nervous System modulates the motor activities of all trunk muscles to concurrently regulate the intra-abdominal and intra-thoracic pressures. The study aims to evaluate the effect of inspiratory and expiratory loads on abdominal muscle activity during breathing in healthy subjects. Twenty-three higher education students (21.09 ± 1.56 years; 8 males) breathed at a same rhythm (inspiration: two seconds; expiration: four seconds) without load and with 10% of the maximal inspiratory or expiratory pressures, in standing. Surface electromyography was performed to assess the activation intensity of rectus abdominis, external oblique and transversus abdominis/internal oblique muscles, during inspiration and expiration. During inspiration, transversus abdominis/internal oblique activation intensity was significantly lower with inspiratory load when compared to without load (p = 0.009) and expiratory load (p = 0.002). During expiration, the activation intensity of all abdominal muscles was significantly higher with expiratory load when compared to without load (p < 0.05). The activation intensity of external oblique (p = 0.036) and transversus abdominis/internal oblique (p = 0.022) was significantly higher with inspiratory load when compared to without load. Transversus abdominis/internal oblique activation intensity was significantly higher with expiratory load when compared to inspiratory load (p < 0.001).Transversus abdominis/internal oblique seems to be the most relevant muscle to modulate the intra-abdominal pressure for the breathing mechanics.     

Identification of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC₅₀ = 11 nM; PC-3 antiproliferative IC₅₀ = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC₅₀ = 10 nM; A2780 antiproliferative IC₅₀ = 7 nM) in complex with the NAMPT protein was also determined.     

Ligand-based CoMFA and CoMSIA studies on chromone derivatives as radical scavengers

The antioxidant activity for a series of chromone compounds, evaluated by DPPH free radical scavenging assay, were subjected to 3D-QSAR studies using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). All 48 chromone derivatives were geometry optimized by AM1 and HF/6-31G^* calculations. The CoMFA and CoMSIA results were compared between different alignment strategies. The best CoMFA model obtained from HF/6-31G^* optimization with field fit alignment gave cross-validated r² (q²) = 0.821, noncross-validated r² = 0.987, S = 0.095, and F = 388.255. The best CoMSIA model derived from AM1 optimized structures and superimposition alignment gave q² = 0.876, noncross-validated r² = 0.976, S = 0.129, and F = 208.073, including electrostatic, hydrophobic, hydrogen bond donor and acceptor fields. The contour maps provide the fruitful structure–radical scavenging activity relationships which are useful for designing new compounds with higher activity.     

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70 l bioreactor level

A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l⁻¹ h⁻¹. Scale-up factors were measured as volumetric oxygen transfer coefficient (k_La) i.e., 56 h⁻¹ and impeller tip velocity (V_tip) i.e., 7.065 m s⁻¹, respectively. The kinetic parameters K_m, k_cat, and k_cat/K_m of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min⁻¹, and 31.49 μM min⁻¹, respectively, when IPTG induced recombinant E. coli culture was used.     

In vivo moment generation and architecture of the human plantar flexors after different shortening–stretch cycles velocities

The purpose of this study was to examine the moment generation of the human plantar flexors and the architecture of the gastrocnemius medialis muscle during and after shortening–stretch cycles in vivo. Fourteen male subjects (30 ± 7 years, 177 ± 7 cm, 80 ± 9 kg) performed a series of electro-stimulated shortening–stretch plantar flexion contractions. The shortening–stretch cycles were performed at three constant angular velocities (25°/s, 50°/s, 100°/s), two amplitudes (15° and 25° ankle angle changes) and at two different stimulation frequencies (30 Hz and 85 Hz). The resultant ankle joint moments were calculated through inverse dynamics. Pennation angle and fascicle length of the m. gastrocnemius medialis at rest and during contractions were measured using ultrasonography. The corresponding ankle moments, kinematics and changes in muscle architecture were analysed at seven time intervals. A three-way analysis of variance (amplitude × velocity × stimulation frequency) and post-hoc test with Bonferroni correction were used to check the amplitude, velocity and stimulation level related effects on moment enhancement (α = 0.05). The results show an ankle joint moment enhancement after shortening–stretch cycles influenced by muscle architectural changes. We found 2–3% isometric ankle joint moment enhancement at steady state, 1.5–2.0 s after the shortening–stretch cycle. However, the observed alteration in muscle architecture after the imposed perturbation, could lead to an underestimation (1–3%) of joint moment enhancement due to the force–length relationship of the triceps surae. Furthermore, the enhancement observed was independent of the shortening–stretch amplitude, velocity and stimulation frequency.     

Differences in proprioception,muscle force control and comfort between conventional and new-generation knee and ankle orthoses

The aim of this study was to compare muscle force control and proprioception between conventional and new-generation experimental orthoses. Sixteen healthy subjects participated in a single-blind controlled trial in which two different types of orthosis were applied to the dominant knee or ankle, while the following variables were evaluated: muscle force control (accuracy), joint position sense, kinesthesia, static balance as well as subjective outcomes. The use of experimental orthoses resulted in better force accuracy during isometric knee extensions compared to conventional orthoses (P = 0.005). Moreover, the use of experimental orthoses resulted in better force accuracy during concentric (P = 0.010) and eccentric (P = 0.014) ankle plantar flexions and better knee joint kinesthesia in the flexed position (P = 0.004) compared to conventional orthoses. Subjective comfort (P < 0.001) and preference scores were higher with experimental orthoses compared to conventional ones. In conclusion, orthosis type affected static and dynamic muscle force control, kinesthesia, and perceived comfort in healthy subjects. New-generation experimental knee and ankle orthoses may thus be recommended for prophylactic joint bracing during physical activity and to improve the compliance for orthosis use, particularly in patients who require long-term bracing.     

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add l-Ala, d-Glu, meso-A₂pm or l-Lys, and d-Ala-d-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute ‘Diversity Set’ on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC₅₀ = 10 μM) and one novel MurF inhibitor (IC₅₀ = 63 μM).     

Acute effect of heel-drop exercise with varying ranges of motion on the gastrocnemius aponeurosis-tendon’s mechanical properties

The objectives of this study was to investigate the acute effects of various magnitudes of tendon strain on the mechanical properties of the human medial gastrocnemius (MG) in vivo during controlled heel-drop exercises. Seven male and seven female volunteers performed two different exercises executed one month apart: one was a heel-drop exercise on a block (HDB), and the other was a heel-drop exercise on level floor (HDL). In each regimen, the subjects completed a session of 150 heel-drop exercises (15 repetitions × 10 sets; with a 30 s rest following each set). Before and immediately after the heel-drop exercise, the ankle plantar flexor torque and elongation of the MG were measured using a combined measurement system of dynamometry and ultrasonography and then the MG tendon strain and stiffness were evaluated in each subject. The tendon stiffness measured prior to the exercises was not significantly different between the two groups 23.7 ± 10.6 N/mm and 24.1 ± 10.0 N/mm for the HDB and HDL, respectively (p > .05). During the heel-drop exercise, it was found that the tendon strain during the heel-drop exercise on a block (8.4 ± 3.7%) was significantly higher than the strain measured on the level floor (5.4 ± 3.8%) (p < .05). In addition, the tendon stiffness following the heel-drop exercise on a block (32.3 ± 12.2 N/mm) was significantly greater than the tendon stiffness measured following the heel-drop exercise on the level floor (25.4 ± 11.4 N/mm) (p < .05). The results of this study suggest that tendon stiffness immediately following a heel-drop exercise depends on the magnitude of tendon strain.     

Effect of 14 days of bed rest in older adults on parameters of the body sway and on the local ankle function

This study explored the effects of a 14-day horizontal bed rest (BR) without countermeasures on postural sway, maximal voluntary torque and precision of voluntary torque matching. Sixteen subjects were tested before, immediately after and two weeks after BR. The increase in frequency and amplitude after BR was comparable for both sway subcomponents (rambling and trembling) in medial-lateral direction. But in anterior–posterior direction, rambling increased more in frequency (?7% vs. +31%, p < 0.05) while trembling increased more in amplitude (+35% vs. +84%, p < 0.05). The drop in maximal voluntary torque after BR was present for plantar flexion (p < 0.05) but not for dorsal flexion. After the BR, the subjects were less precise in the dorsal flexion torque matching task (p < 0.05). All the observed parameters, except the dorsal flexion torque matching error, returned back to the pre-BR values after the two weeks of re-conditioning. Results of this study indicate that body sway subcomponents responded differently to BR. Based on these findings, it was not possible to draw clear assumptions on the effects of neural and structural changes on body sway.     

High hydrostatic pressure increased stability and activity of immobilized lipase in hexane

Lipases are important to high value product synthesis, modification, and enhancement. However, they are often unstable above 40 °C. While most current applications of high hydrostatic pressure (HHP) are for inactivating deleterious enzymes, there is evidence that HHP can stabilize and increase activity of some enzymes. This study examines the apparent kinetics of immobilized lipase-catalyzed synthesis of isoamyl acetate at HHP in hexane. HHP reduced thermal inactivation of lipase by up to 152% after 4 h at 80 °C and 400 MPa when compared to incubations at low pressure. No significant differences were found in activation energy (E_a) at different pressures, irrespectively of the pressurization and heating sequence, and were between 35.7 ± 3.5 and 47.8 ± 8.2 kJ mol^?1, depending on the method. In all methods utilized, activity at 63.5 and 80 °C at 400 MPa was greater (from about 20 to 96% increase) than at low pressure. Activity increased by 110% at low pressure versus a 239% increase at 350 MPa when the temperature was increased from 40 to 80 °C. Increasing pressure up to 350 MPa increased lipase activity while pressures greater than 350 MPa maintained or decreased lipase activity. Activation volume (ΔV^≠) appeared negative between ambient pressure and 200 MPa in contrast to a positive ΔV^≠ between 300 and 600 MPa. Apparent ΔV^≠ was 14.3 ± 1.7 or 15.2 ± 2.2 cm³ mol^?1 at 40 or 80 °C, respectively, between 300 and 500 MPa.     

Characterization of the N-oxygenase AurF from Streptomyces thioletus

AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing ∼10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a K_m of 14.7 ± 1.1 μM, a k_cat of 47.5 ± 5.4 min⁻¹ and a k_cat/K_m of 3.2 ± 0.4 μM⁻¹ min⁻¹. PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H₂O₂ produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the K_m is the main determinant of AurF activity toward alternative substrates.