Carlene Starck

Molecular analysis of survivin isoforms : Evidence that alternatively spliced variants do not play a role in mitosis

Survivin is a protein with proposed roles in cell division and apoptosis. Transcripts encoding splice variants of human survivin have been described and their expression correlated with cancer progression. As survivin forms homodimers in vitro, it has been suggested that these isoforms could interfere with wild type function by forming heterodimers. Here we show that survivin-2β and survivin-δEx3 can interact with wild type survivin but have reduced affinity for the partner protein of survivin, borealin, and thus do not localize with the chromosomal passenger complex in vivo. Furthermore, we demonstrate that overexpression of survivin-2β-green fluorescent protein (GFP) or survivin-δEx3-GFP does not impede cell cycle progression. We also report that wild type survivin, but not survivin-2β-GFP or survivin-δEx3-GFP, can rescue cell proliferation inhibited by small interfering RNA-mediated survivin depletion. These data suggest that, despite their ability to interact with wild type survivin, neither of these isoforms acts as its competitor during mitosis nor has an essential function.

Cytotoxic Aggregation and Amyloid Formation by the Myostatin Precursor Protein

Myostatin, a negative regulator of muscle growth, has been implicated in sporadic inclusion body myositis (sIBM). sIBM is the most common age-related muscle-wastage disease with a pathogenesis similar to that of amyloid disorders such as Alzheimers and Parkinsons diseases. Myostatin precursor protein (MstnPP) has been shown to associate with large molecular weight filamentous inclusions containing the Alzheimers amyloid beta peptide in sIBM tissue, and MstnPP is upregulated following ER stress. The mechanism for how MstnPP contributes to disease pathogenesis is unknown. Here, we show for the first time that MstnPP is capable of forming amyloid fibrils in vitro. When MstnPP-containing Escherichia coli inclusion bodies are refolded and purified, a proportion of MstnPP spontaneously misfolds into amyloid-like aggregates as characterised by electron microscopy and binding of the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the aggregates form straight and unbranched amyloid fibrils 15 nm in diameter and also exhibit higher order amyloid structures. Circular dichroism spectroscopy reveals that the amyloid fibrils are dominated by β-sheet and that their formation occurs via a conformational change that occurs at a physiologically relevant temperature. Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts. These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.

The Effect of Caffeine Ingestion during Evening Exercise on Subsequent Sleep Quality in Females.

In a randomised, double-blind, placebo-controlled crossover design, 10 females taking monophasic oral contraceptives completed 90 min intermittent treadmill-running 45 min after ingestion of 6 mg∙kg(-1) body mass anhydrous caffeine or artificial sweetener (placebo). Water (3 mL∙kg(-1)) was provided every 15 min during exercise. Venous blood samples were taken before, during and after exercise, as well as after sleep (~15 h post-ingestion), and levels of caffeine, paraxanthine, theobromine and theophylline were measured using high-performance liquid chromatography. Sleep quality was assessed using the Leeds Sleep Evaluation Questionnaire. Plasma caffeine concentration peaked 100 min after ingestion. Caffeine clearance was 0.95±0.14 mL·min(-1)·kg(-1) while the elimination half-life of caffeine was 17.63±8.06 h. Paraxanthine and theophylline levels were significantly elevated at 15 h with no significant change in theobromine. Sleep latency and subsequent quality of sleep was impaired following caffeine supplementation (P<0.05); there were no differences between trials for how participants were feeling upon awakening. This is the first controlled study to examine caffeine supplementation on sleep quality in female athletes taking a low-dose monophasic oral contraceptive steroid following an intermittent-exercise running protocol. The data shows that female athletes using monophasic oral contraceptive steroids will have impaired sleep quality following evening caffeine ingestion.

Caffeine ingestion enhances perceptual responses during intermittent exercise in female team-game players

Abstract We examined the influence of caffeine supplementation on cognitive performance and perceptual responses in female team-game players taking low-dose monophasic oral contraceptives of the same hormonal composition. Ten females (24 ± 4 years; 59.7 ± 3.5 kg body mass; 2–6 training sessions per week) took part in a randomised, double-blind, placebo-controlled crossover-design trial. A 90-min intermittent treadmill-running protocol was completed 60 min following ingestion of a capsule containing either 6 mg • kg−1 anhydrous caffeine or artificial sweetener (placebo). Perceptual responses (ratings of perceived exertion (RPE), feeling scale (FS), felt arousal scale (FAS)), mood (profile of mood states (POMS)) and cognitive performance (Stroop test, choice reaction time (CRT)) were completed before, during and after the exercise protocol, as well as after ~12 h post exercise. Caffeine ingestion significantly enhanced the ratings of pleasure (P = 0.008) and arousal (P = 0.002) during the exercise protocol, as well as increased vigour (POMS; P = 0.007), while there was a tendency for reduced fatigue (POMS; P = 0.068). Caffeine ingestion showed a tendency to decrease RPE (P = 0.068) and improve reaction times in the Stroop (P = 0.072) and CRT (P = 0.087) tests. Caffeine supplementation showed a positive effect on perceptual parameters by increasing vigour and a tendency to decrease fatigue during intermittent running activity in female games players taking low-dose monophasic oral contraceptive steroids (OCS).

Salivary Diagnostic Markers in Males and Females During Rest and Exercise

BackgroundSaliva is a useful diagnostic tool for analysis in sports, exercise and nutrition research, as collection is easy and non-invasive and it contains a large number of analytes affected by a range of physiological and pathological stressors and conditions. This study examined key salivary electrolytes and stress and immune markers in males and females at rest and during exercise.MethodsUnstimulated whole saliva from 20 healthy, recreationally active participants (8 males and 12 females) was analysed for flow rate, osmolality, sodium (Na+), potassium (K+), chloride (Cl−), secretory immunoglobulin A (SIgA), α-amylase activity and cortisol during both rest and moderate intensity (70% peak power) cycling exercise in a randomised crossover design. Each trial lasted 60 min and sampling was carried out at 15 and 45 min after the start of the trial. Saliva was collected using the gold-standard drool method; participants were required to provide at least 1 mL sample over 2 or 3-min period.ResultsFemales showed a greater response to steady-state exercise stress than males, with significant increases in osmolality (P < 0.001), α-amylase activity (P = 0.001) and secretion rate (P = 0.023) and SIgA secretion rate (P = 0.023), with trends for an increase in K+ (P = 0.053) and decrease in Cl− (P = 0.067). There were no differences between rest and exercise for any salivary analytes in males. In addition, females showed a trend for higher levels of cortisol than males at both rest (P = 0.099) and exercise (P = 0.070), as well as a higher heart rate (P < 0.001) and greater ratings of perceived exertion (P < 0.001) during the exercise trial. The coordination of the two stress response pathways (α-amylase vs cortisol) was positive in males (r = 0.799; P = 0.017) yet negative in females (r = −0.475; P = 0.036).ConclusionsMales and females show a markedly different response to steady-state exercise stress as measured in unstimulated whole saliva.

Dietary thiols in exercise: oxidative stress defence, exercise performance, and adaptation

Endurance athletes are susceptible to cellular damage initiated by excessive levels of aerobic exercise-produced reactive oxygen species (ROS). Whilst ROS can contribute to the onset of fatigue, there is increasing evidence that they play a crucial role in exercise adaptations. The use of antioxidant supplements such as vitamin C and E in athletes is common; however, their ability to enhance performance and facilitate recovery is controversial, with many studies suggesting a blunting of training adaptations with supplementation. The up-regulation of endogenous antioxidant systems brought about by exercise training allows for greater tolerance to subsequent ROS, thus, athletes may benefit from increasing these systems through dietary thiol donors. Recent work has shown supplementation with a cysteine donor (N-acetylcysteine; NAC) improves antioxidant capacity by augmenting glutathione levels and reducing markers of oxidative stress, as well as ergogenic potential through association with delayed fatigue in numerous experimental models. However, the use of this, and other thiol donors may have adverse physiological effects. A recent discovery for the use of a thiol donor food source, keratin, to potentially enhance endogenous antioxidants may have important implications for endurance athletes hoping to enhance performance and recovery without blunting training adaptations.

The C313Y Piedmontese mutation decreases myostatin covalent dimerisation and stability

BackgroundMyostatin is a key negative regulator of muscle growth and development, whose activity has important implications for the treatment of muscle wastage disorders. Piedmontese cattle display a double-muscled phenotype associated with the expression of C313Y mutant myostatin. In vivo, C313Y myostatin is proteolytically processed, exported and circulated extracellularly but fails to correctly regulate muscle growth. The C313Y mutation removes the C313-containing disulphide bond, an integral part of the characteristic TGF-β cystine-knot structural motif.ResultsHere we present in vitro analysis of the structure and stability of the C313Y myostatin protein that reveals significantly decreased covalent dimerisation for C313Y myostatin accompanied by a loss of structural stability compared to wild type. The C313Y myostatin growth factor, processed from full length precursor protein, fails to inhibit C2C12 myoblast proliferation in contrast to wild type myostatin. Although structural modeling shows the substitution of tyrosine causes structural perturbation, biochemical analysis of additional disulphide mutants, C313A and C374A, indicates that an intact cystine-knot motif is a major determinant in myostatin growth factor stability and covalent dimerisation.ConclusionsThis research shows that the cystine-knot structure is important for myostatin dimerisation and stability, and that disruption of this structural motif perturbs myostatin signaling.

Factors contributing to the selection of dietary protein food sources

Protein is the only dietary macronutrient required for life. As such, it is reasonable to consider dietary protein as the centerpiece of a healthy eating pattern. To do so requires consideration of what type of protein should be eaten. Account should be taken of the quality of the protein, the density of the protein in the protein food source, and the non-protein components of protein food source. The quality of protein can be quantified based on the amount and profile of essential amino acids (EAAs), as well as the true ileal digestibility of the EAAs in the protein. The density of protein in a food source can be quantified on the basis of the amount of total calories ingested to achieve intake of the daily requirement of all EAAs. Non-protein components of protein food sources can be considered in terms of the amount and nature of fat, carbohydrate and fiber, as well as the content of micronutrients. Potential beneficial effects of high-quality protein food sources should be balanced against any possible adverse effects. When all of these factors are considered we conclude that animal-based protein foods (e.g., eggs, dairy, meat, fish, poultry) occupy an important place in a healthy eating pattern.

Endogenous Amino Acid Losses from the Gastrointestinal Tract of the Adult Human—A Quantitative Model

Background The loss of endogenous (nondietary) amino acids (AAs) from the gastrointestinal tract (GIT) is an important component underlying the adult human dietary requirement for protein and essential AAs (EAAs). Although data with regard to endogenous AA losses to the end of the small intestine have been published, to our knowledge there are no direct measures of colonic endogenous AA losses. Objective The objective was to derive quantitative estimates for daily endogenous protein and EAAs lost from the colon of the adult human. Methods A factorial model was developed for the prediction of endogenous AA losses across the adult human GIT. Estimates of AAs entering the upper GIT lumen were combined with relative protein synthesis rates in the colon to predict colonic AA losses. The AA composition of human colonic endogenous protein was calculated by estimating the relative contributions of epithelial cell protein and mucin protein on the basis of published data for cell shedding in the pig small intestine, small intestinal protein synthesis rates in pigs and humans, and human upper and lower GIT surface areas. Colonic AA losses were summed with empirical estimates of ileal AA losses in humans to estimate total daily GIT endogenous AA losses. Results Colonic AA loss was estimated to total 3.5 g/d in the adult male human, comprising 33% of total GIT endogenous AA loss (10.2 g/d). GIT essential AA losses accounted for 25-97% of the current recommended daily AA requirement for adult humans. For threonine, colonic losses were 54% of total GIT threonine losses, which were 97% of the current recommended daily threonine requirement. Conclusions Colonic endogenous AA losses represent a significant fraction of total GIT endogenous AA losses. The requirement of the GIT for EAAs to replace AAs lost via the gut lumen comprises a substantial proportion of the Recommended Daily Intake of AAs.