Effect of Calcium Pre-rinse and Fluoride Dentifrice on Enamel and on Dental Plaque Formed In Situ

Purpose: The aim of this in situ double-blind randomised crossover study was to investigate the effect of calcium (Ca) pre-rinse on the composition of plaque and on enamel prior to the use of fluoride (F) dentifrice. Materials and Methods: During four phases (14 days each) of this study, 10 volunteers had agreed to wear dental appliances containing two healthy bovine enamel blocks. A fresh solution containing 20% weight/volume (w/v) sucrose was dripped on the enamel blocks ex vivo for 5 min three times a day. Subsequently, the appliances were replaced in the mouth, and the volunteers rinsed their mouth with 10 mL of a Ca (150 mmol/L) or a placebo rinse (1 min). In sequence, a slurry (1:3 w/v) of F (1030 ppm) or placebo dentifrice was dripped onto the blocks ex vivo for 1 min. During this time, the volunteers brushed their teeth with the respective dentifrice. The appliances were replaced in the mouth, and the volunteers rinsed their mouth with water. The plaque formed on the blocks was analysed for F and Ca. The enamel demineralisation as well as the incorporation of F on enamel was evaluated by cross-sectional microhardness and alkali-soluble F analysis, respectively. Data were tested using analysis of variance (P < 0.05). Results: The Ca pre-rinse prior to the use of the F dentifrice led to a three- and sixfold increase in the plaque F and Ca concentrations, respectively. It also did not have any additive effect on the F content on the enamel and the demineralisation of the enamel, in comparison with the use of F dentifrice alone. Conclusions: A Ca lactate rinse used prior to the F dentifrice was able to change the mineral content in the plaque, but it was unable to prevent enamel demineralisation.

Fabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner electrophoresis microchips

In this report, we describe the microfabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner (PT) electrophoresis microchips using toner masks. Planar electrodes were fabricated by three simple steps: (i) drawing and laser-printing the electrode geometry on polyester films, (ii) sputtering deposition onto substrates, and (iii) removal of toner layer by a lift-off process. The polyester film with anchored electrodes was integrated to PT electrophoresis microchannels by lamination at 120 degrees C in less than 1 min. The electrodes were designed in an antiparallel configuration with 750 mu m width and 750 gm gap between them. The best results were recorded with a frequency of 400 kHz and 10 V-PP using a sinusoidal wave. The analytical performance of the proposed microchip was evaluated by electrophoretic separation of potassium, sodium and lithium in 150 mu m wide x 6 mu m deep microchannels. Under an electric field of 250 V/cm the analytes were successfully separated in less than 90 s with efficiencies ranging from 7000 to 13 000 plates. The detection limits (S/N = 3) found for K+, Na+, and Li+ were 3.1, 4.3, and 7.2 mu mol/L, respectively. Besides the low-cost and instrumental simplicity, the integrated PT chip eliminates the problem of manual alignment and gluing of the electrodes, permitting more robustness and better reproducibility, therefore, more suitable for mass production of electrophoresis microchips.

Role of P-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of Glycyrrhiza glabra.

Purpose. Glabridin is a major active constituent of Glycyrrhiza glabra which is commonly used in the treatment of cardiovascular and central nervous system (CNS) diseases. Recently, we have found that glabridin is a substrate of P-glycoprotein (PgP/MDR1). This study aimed to investigate the role of PgP in glabridin penetration across the blood–brain barrier (BBB) using several in vitro and in vivo models.
Materials and Methods. Cultured primary rat brain microvascular endothelial cells (RBMVECs) were used in the uptake, efflux and transcellular transport studies. A rat bilateral in situ brain perfusion model was used to investigate the brain distribution of glabridin. The brain and tissue distribution of glabridin in rats with or without coadministered verapamil or quinidine were examined with correction for the tissue residual blood. In addition, the brain distribution of glabridin in mdr1a(-/-) mice was compared with the wild-type mice. Glabridin in various biological matrices was determined by a validated liquid chromatography mass spectrometric method.
Results. The uptake and efflux of glabridin in cultured RBMVECs were ATP-dependent and significantly altered in the presence of a PgP or multi-drug resistance protein (Mrp1/2) inhibitor (e.g. verapamil or MK-571). A polarized transport of glabridin was found in RBMVEC monolayers with
facilitated efflux from the abluminal (BL) to luminal (AP) side. Addition of a PgP or Mrp1/2 inhibitor in both luminal and abluminal sides attenuated the polarized transport across RBMVECs. In a bilateral in situ brain perfusion model, the uptake of glabridin into the cerebrum increased from 0.42 T 0.09% at 1 min to 9.27 T 1.69% (ml/100 g tissue) at 30 min and was significantly greater than that for sucrose. Coperfusion of a PgP or Mrp1/2 inhibitor significantly increased the brain distribution of glabridin by 33.6j142.9%. The rat brain levels of glabridin were only about 27% of plasma levels when corrected by tissue residual blood and it was increased to up to 44% when verapamil or quinidine was coadministered. The area under the brain concentration-time curve (AUC) of glabridin in mdr1a(-/-) mice was 6.0-fold higher than the wild-type mice.
Conclusions. These findings indicate that PgP limits the brain penetration of glabridin through the BBB and PgP may cause drug resistance to glabridin (licorice) therapy for CNS diseases and potential drugglabridin interactions. However, further studies are needed to explore the role of other drug transporters (e.g. Mrp1-4) in restricting the brain penetration of glabridin.

Transport of cryptotanshinone, a major active triterpenoid in Salvia miltiorrhiza Bunge widely used in the treatment of stroke and Alzheimers disease, across the blood-brain

Cryptotanshinone (CTS), a major constituent from the roots of Salvia miltiorrhiza (Danshen), is widely used in the treatment of coronary heart disease, stroke and less commonly Alzheimer's disease. Our recent study indicates that CTS is a substrate for Pglycoprotein (PgP/MDR1/ABCB1). This study has investigated the nature of the brain distribution of CTS across the brain-blood barrier (BBB) using several in vitro and in vivo rodent models. A polarized transport of CTS was found in rat primary microvascular endothelial cell (RBMVEC) monolayers, with facilitated efflux from the abluminal side to luminal side. Addition of a PgP (e.g. verapamil and quinidine) or multi-drug resistance protein 1/2 (MRP1/2) inhibitor (e.g. probenecid and MK-571) in both luminal and abluminal sides attenuated the polarized transport. In a bilateral in situ brain perfusion model, the uptake of CTS into the cerebrum increased from 0.52 ± 0.1% at 1 min to 11.13 ± 2.36 ml/100 g tissue at 30 min and was significantly greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g. verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly increased the brain distribution of CTS by 35.1-163.6%. The brain levels of CTS were only about 21% of those in plasma, and were significantly increased when coadministered with verapamil or probenecid in rats. The brain levels of CTS in rats subjected to middle cerebral artery occlusion and rats treated with quinolinic acid (a neurotoxin) were about 2- to 2.5-fold higher than the control rats. Moreover, the brain levels in mdr1a(-/-) and mrp1(-/-) mice were 10.9- and 1.5-fold higher than those in the wild-type mice, respectively. Taken collectively, these findings indicate that PgP and Mrp1 limit the brain penetration of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further studies are needed to explore the role of other drug transporters in restricting the brain penetration of CTS and the clinical relevance.

Carbohydrate ingestion reduces skeletal muscle acetylcarnitine availability but has no effect on substrate phosphorylation at the onset of exercise in man

This study investigated the effect of reduced acetylcarnitine availability on oxidative metabolism during the transition from rest to steady-state exercise. Eight male subjects completed two randomised exercise trials at 68 % of the peak rate of O2 uptake (V̇O2,peak). On one occasion subjects ingested 1 g (kg body mass)−1 glucose 75 min prior to exercise (CHO), whereas the other trial acted as a control (CON). Muscle samples were obtained pre- and 75 min post-ingestion, and following 1 and 10 min of exercise. Plasma glucose and insulin were elevated (P < 0.05), and plasma free fatty acids (FFA) were lower at the onset of exercise in CHO. Acetylcarnitine (CON, 4.8 ± 1.8; CHO, 1.5 ± 0.9 mmol (kg dry mass (d.m.))−1, P < 0.05) and acetyl CoA (CON, 13.2 ± 2.3; CHO, 6.3 ± 0.6 μmol (kg d.m.)−1, P < 0.05) were lower at rest, whereas pyruvate dehydrogenase activation (PDHa) was greater in CHO compared with CON (CON, 0.78 ± 0.07; CHO, 1.44 ± 0.19 mmol min−1 (kg wet mass (w.m.))−1). Respiratory exchange ratio (RER) was significantly elevated during exercise in CHO. The acetyl groups increased at similar rates at the onset of exercise (1 min) and there was no difference in substrate phosphorylation as determined from lactate accumulation and phosphocreatine degradation between trials. Subsequently, oxidative metabolism during the transition from rest to steady-state exercise was not affected by prior carbohydrate ingestion. Although exercise resulted in the rapid activation of PDH in both trials, PDHa was greater at 1 min in CHO (CON, 2.36 ± 0.22; CHO, 2.91 ± 0.18 mmol min−1 (kg w.m.)−1). No differences in muscle metabolite levels and PDHa were observed after 10 min of moderate exercise between trials. In summary, at rest, carbohydrate ingestion induced multiple metabolic changes which included decreased acetylcarnitine availability and small increases in PDHa. The prior changes in PDHa and acetylcarnitine availability had no effect on substrate phosphorylation and oxidative metabolism at the onset of exercise. These data suggest that acetylcarnitine availability is unlikely to be the site of metabolic inertia during the transition from rest to steady-state moderate intensity exercise.

µ- Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

µ-calpain and calpain-3 are Ca²⁺-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that µ-calpain becomes proteolytically active in the presence of 2–200 µM Ca²⁺. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca²⁺ concentration levels ([Ca²⁺]i; ~1 µM) than the levels at which µ-calpain activation occurs. We have demonstrated the Ca²⁺-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca²⁺] levels (0, 2.5, 10, and 25 µM). Autolysis of calpain-3 was found to occur across a [Ca²⁺] range similar to that for µ-calpain, and both calpains displayed a seemingly higher Ca²⁺ sensitivity in human than in rat muscle homogenates, with ~15% autolysis observed after 1-min exposure to 2.5 µM Ca²⁺ in human muscle and almost none after 1- to 2-min exposure to the same [Ca²⁺]i level in rat muscle. During muscle activity, [Ca²⁺]i may transiently peak in the range found to autolyze µ-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed µ-calpain or calpain-3 in human muscle. No significant autolysis of µ-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca²⁺]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.

Effect of posture on high-intensity constant-load cycling performance in men and women

The time sustained during a graded cycle exercise is ~10% longer in an upright compared with a supine posture. However, during constant-load cycling this effect is unknown. Therefore, we tested the postural effect on the performance of high-intensity constant-load cycling. Twenty-two active subjects (11 men, 11 women) performed two graded tests (one upright, one supine), and of those 22, 10 subjects (5 men, 5 women) performed three high-intensity constant-load tests (one upright, two supine). To test the postural effect on performance at the same absolute intensity, during the upright and one of the supine constant-load tests subjects cycled at 80% of the peak power output achieved during the upright graded test. To test the postural effect on performance at the same relative intensities, during the second supine test subjects cycled at 80% of the peak power output achieved during the supine graded test. Exercise time on the graded and absolute intensity constant-load tests for all subjects was greater (P<0.05) in the upright compared with supine posture (17.9±3.5 vs. 16.1±3.1 min for graded; 13.2±8.7 vs. 5.2±1.9 min for constant-load). This postural effect at the same absolute intensity was larger in men (19.4±8.5 upright vs. 6.6±1.6 supine, P<0.001) than women (7.1±2 upright vs. 3.9±1.4 supine, P>0.05) and it was correlated (P<0.05) with both the difference in VO₂ between positions during the first minute of exercise (r=0.67) and the height of the subjects (r=0.72). In conclusion, there is a very large postural effect on performance during constant-load cycling exercise and this effect is significantly larger in men than women.

Intravenous AICAR administration reduces hepatic glucose output and inhibits whole body lipolysis in type 2 diabetic patients

Aims/hypothesis: The 5′-AMP-activated protein kinase (AMPK) pathway is intact in type 2 diabetic patients and is seen as a target for diabetes treatment. In this study, we aimed to assess the impact of the AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) on both glucose and fatty acid metabolism in vivo in type 2 diabetic patients.

Methods: Stable isotope methodology and blood and muscle biopsy sampling were applied to assess blood glucose and fatty acid kinetics following continuous i.v. infusion of AICAR (0.75 mg kg−1 min−1) and/or NaCl (0.9%) in ten male type 2 diabetic patients (age 64 ± 2 years; BMI 28 ± 1 kg/m2).
Results Plasma glucose rate of appearance (R a) was reduced following AICAR administration, while plasma glucose rate of disappearance (R d) was similar in the AICAR and control test. Consequently, blood glucose disposal (R d expressed as a percentage of R a) was increased following AICAR infusion (p < 0.001). Accordingly, a greater decline in plasma glucose concentration was observed following AICAR infusion (p < 0.001). Plasma NEFA R a and R d were both significantly reduced in response to AICAR infusion, and were accompanied by a significant decline in plasma NEFA concentration. Although AMPK phosphorylation in skeletal muscle was not increased, we observed a significant increase in acetyl-CoA carboxylase phosphorylation (p < 0.001).

Conclusions/interpretation: The i.v. administration of AICAR reduces hepatic glucose output, thereby lowering blood glucose concentrations in vivo in type 2 diabetic patients. Furthermore, AICAR administration stimulates hepatic fatty acid oxidation and/or inhibits whole body lipolysis, thereby reducing plasma NEFA concentration.

Acute signalling responses to intense endurance training commenced with low or normal muscle glycogen

We have previously demonstrated that well-trained subjects who completed a 3 week training programme in which selected high-intensity interval training (HIT) sessions were commenced with low muscle glycogen content increased the maximal activities of several oxidative enzymes that promote endurance adaptations to a greater extent than subjects who began all training sessions with normal glycogen levels. The aim of the present study was to investigate acute skeletal muscle signalling responses to a single bout of HIT commenced with low or normal muscle glycogen stores in an attempt to elucidate potential mechanism(s) that might underlie our previous observations. Six endurance-trained cyclists/triathletes performed a 100 min ride at ∼70% peak O₂ uptake (AT) on day 1 and HIT (8 × 5 min work bouts at maximal self-selected effort with 1 min rest) 24 h later (HIGH). Another six subjects, matched for fitness and training history, performed AT on day 1 then 1–2 h later, HIT (LOW). Muscle biopsies were taken before and after HIT. Muscle glycogen concentration was higher in HIGH versus LOW before the HIT (390 ± 28 versus 256 ± 67 μmol (g dry wt)⁻¹). After HIT, glycogen levels were reduced in both groups (P < 0.05) but HIGH was elevated compared with LOW (229 ± 29 versus 124 ± 41 μmol (g dry wt)−1; P < 0.05). Phosphorylation of 5'AMP-activated protein kinase (AMPK) increased after HIT, but the magnitude of increase was greater in LOW (P < 0.05). Despite the augmented AMPK response in LOW after HIT, selected downstream AMPK substrates were similar between groups. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was unchanged for both groups before and after the HIT training sessions. We conclude that despite a greater activation AMPK phosphorylation when HIT was commenced with low compared with normal muscle glycogen availability, the localization and phosphorylation state of selected downstream targets of AMPK were similar in response to the two interventions.

The contribution of upper limb and total body movement to adolescents’ energy expenditure whilst playing Nintendo Wii

Little research documents the contribution of upper limb and total body movement to energy expenditure (EE) during active video gaming. To address this, EE, heart rate (HR), and, upper limb and total body movement were assessed in 11- to 17-year-old adolescents whilst playing three active (Nintendo Wii) and one sedentary (XBOX 360) video games. Non-dominant upper limb activity, EE and HR were significantly greater during Wii Sports boxing [mean 267.2 (SD 115.8) J kg−1 min−1; 136.7 (24.5) beats min−1] than tennis or bowling (P ≤ 0.044). For all active games hip activity best predicted EE (R 2 ≥ 0.53), with two-measure models of HR and single-site activity data, and multi-site activity data, similarly explaining the variance in EE (R 2 ≥ 0.64). The physiological cost of upper-body orientated active video games increased when movement of both upper limbs was encouraged. Improvements in EE explanatory power provide support for multi-site activity monitoring during unique, non-ambulatory activities.

Exercise performance and V0₂ kinetics during upright and recumbent high-intensity cycling exercise

This study investigated cycling performance and oxygen uptake (VO₂) kinetics between upright and two commonly used recumbent (R) postures, 65ºR and 30ºR. On three occasions, ten young active males performed three bouts of high-intensity constant-load (85% peak workload achieved during a graded test) cycling in one of the three randomly assigned postures (upright, 65ºR or 30ºR). The first bout was performed to fatigue and second and third bouts were limited to 7 min. A subset of seven subjects performed a final constant-load test to failure in the supine posture. Exercise time to failure was not altered when the body inclination was lowered from the upright (13.1 ± 4.5 min) to 65ºR (10.5 ± 2.7 min) and 30ºR (11.5 ± 4.6 min) postures; but it was significantly shorter in the supine posture (5.8 ± 2.1 min) when compared with the three inclined postures. Resulting kinetic parameters from a tri-exponential analysis of breath-by-breath VO₂ data during the first 7 min of exercise were also not different between the three inclined postures. However, inert gas rebreathing analysis of cardiac output revealed a greater cardiac output and stroke volume in both recumbent postures compared with the upright posture at 30 s into the exercise. These data suggest that increased cardiac function may counteract the reduction of hydrostatic pressure from upright ~25 mmHg; to 65ºR ~22 mmHg; and 30ºR ~18 mmHg such that perfusion of active muscle presumably remains largely unchanged, and also therefore, VO₂ kinetics and performance during high-intensity cycling.

Sprint cycling performance is maintained with short-term contrast water immersion

Purpose: Given the widespread use of water immersion during recovery from exercise, we aimed to investigate the effect of contrast water immersion on recovery of sprint cycling performance, HR and, blood lactate.

Methods: Two groups completed high-intensity sprint exercise before and after a 30-min randomized recovery. The Wingate group (n = 8) performed 3 x 30-s Wingate tests (4-min rest periods). The repeated intermittent sprint group (n = 8) cycled for alternating 30-s periods at 40% of predetermined maximum power and 120% maximum power, until exhaustion. Both groups completed three trials using a different recovery treatment for each trial (balanced randomized application). Recovery treatments were passive rest, 1:1 contrast water immersion (2.5 min of cold (8-C) to 2.5 min of hot (40-C)), and 1:4 contrast water immersion (1 min of cold to 4 min of hot). Blood lactate and HR were recorded throughout, and peak power and total work for pre- and postrecovery Wingate performance and exercise time and total work for repeated sprinting were recorded.

Results: Recovery of Wingate peak power was 8% greater after 1:4 contrast water immersion than after passive rest, whereas both contrast water immersion ratios provided a greater recovery of exercise time (È10%) and total work (È14%) for repeated sprinting than for passive rest. Blood lactate was similar between trials. Compared with passive rest, HR initially declined more slowly during contrast water immersion but increased with each transition to a cold immersion phase.

Conclusions: These data support contrast water immersion being effective in maintaining performance during a short-term recovery from sprint exercise. This effect needs further investigation but is likely explained by cardiovascular mechanisms, shown here by an elevation in HR upon each cold immersion.

Lack of glucose and hsp70 responses in haddock Melanogrammus aeglefinus (L.) subjected to handling and heat shock

Juvenile haddock Melanogrammus aeglefinus (c. 39 g) were exposed to either a handling stressor (1 min out of water) or heat shock (increase from 10 to 15° C for 1 h), and plasma cortisol, plasma glucose and gill hsp70 levels were determined before, and at 1, 3, 6, 12, 24 and 48 h post-stress. The pattern of cortisol increase was similar following both stressors, with levels increasing by 25-fold at 1 h post-stress, but returning to pre-stress levels (2–5 ng ml⁻¹) by 3 h. In contrast, neither handling nor heat shock caused an increase in plasma glucose levels. Although gill hsp70 was detected, presumably constitutive levels, in both control and heat shocked groups, there were not significant changes in gill hsp70 levels after exposure to heat shock. The lack of glucose and hsp70 responses to these typical stressors is consistent with previous studies on Atlantic cod Gadus morhua, and suggests that the stress physiology of Gadidae differs from the ‘typical’ teleost.

Changes in behaviour during the inter-nesting period and post-nesting migration for Ascension Island green turtles

Satellite transmitters were attached to green turtles Chelonia mydas while they were nesting on Ascension Island in the South Atlantic (7°57'S, 14°22'W) and individuals were subsequently monitored during the inter-nesting period and the post-nesting migration to Brazil. During the inter-nesting period, data from the transmitters suggested that turtles generally stayed within 5 km of the nesting beach on which they had originally been observed. During both the inter-nesting period and migration, turtles were submerged the vast majority (>95%) of the time, suggesting that they neither basked at the surface nor drifted passively during migration to any great extent. There was a clear dichotomy in submergence behaviour, with submergences tending to be of short duration during post-nesting migration (mean = 7.3 min, 3318 h of data from 5 individuals) and of longer duration during the inter-nesting period (mean = 22.1 min, 714 h of data from 5 different individuals). As submergence duration is generally linked to activity levels in sea turtles, this pattern suggests that turtles were comparatively inactive during the inter-nesting period and comparatively active during migration. During both the inter-nesting period and the post-nesting migration, diel submergence patterns were detected with dive duration tending to be longer at night. As the turtles migrated WSW from Ascension Island, there was a reduction in their speed of travel. A numerical model of the near-surface currents suggested that this reduction was associated with the weakening of the WSW flow of the prevailing South Atlantic Equatorial Current.

High activity and Levy searches : jellyfish can search the water column like fish

Over-fishing may lead to a decrease in fish abundance and a proliferation of jellyfish. Active movements and prey search might be thought to provide a competitive advantage for fish, but here we use data-loggers to show that the frequently occurring coastal jellyfish (Rhizostoma octopus) does not simply passively drift to encounter prey. Jellyfish (327 days of data from 25 jellyfish with depth collected every 1 min) showed very dynamic vertical movements, with their integrated vertical movement averaging 619.2 m d−1, more than 60 times the water depth where they were tagged. The majority of movement patterns were best approximated by exponential models describing normal random walks. However, jellyfish also showed switching behaviour from exponential patterns to patterns best fitted by a truncated Lévy distribution with exponents (mean μ = 1.96, range 1.2–2.9) close to the theoretical optimum for searching for sparse prey (μopt ≈ 2.0). Complex movements in these ‘simple’ animals may help jellyfish to compete effectively with fish for plankton prey, which may enhance their ability to increase in dominance in perturbed ocean systems.