Bone mineral density in high-level endurance runners: Part B-genotype-dependent characteristics.

PURPOSE: Inter-individual variability in bone mineral density (BMD) exists within and between endurance runners and non-athletes, probably in part due to differing genetic profiles. Certainty is lacking, however, regarding which genetic variants may contribute to BMD in endurance runners and if specific genotypes are sensitive to environmental factors, such as mechanical loading via training. METHOD: Ten single-nucleotide polymorphisms (SNPs) were identified from previous genome-wide and/or candidate gene association studies that have a functional effect on bone physiology. The aims of this study were to investigate (1) associations between genotype at those 10 SNPs and bone phenotypes in high-level endurance runners, and (2) interactions between genotype and athlete status on bone phenotypes. RESULTS: Female runners with P2RX7 rs3751143 AA genotype had 4% higher total-body BMD and 5% higher leg BMD than AC + CC genotypes. Male runners with WNT16 rs3801387 AA genotype had 14% lower lumbar spine BMD than AA genotype non-athletes, whilst AG + GG genotype runners also had 5% higher leg BMD than AG + GG genotype non-athletes. CONCLUSION: We report novel associations between P2RX7 rs3751143 genotype and BMD in female runners, whilst differences in BMD between male runners and non-athletes with the same WNT16 rs3801387 genotype existed, highlighting a potential genetic interaction with factors common in endurance runners, such as high levels of mechanical loading. These findings contribute to our knowledge of the genetic associations with BMD and improve our understanding of why some runners have lower BMD than others.

Bone mineral density in high-level endurance runners: part A-site-specific characteristics.

PURPOSE: Physical activity, particularly mechanical loading that results in high-peak force and is multi-directional in nature, increases bone mineral density (BMD). In athletes such as endurance runners, this association is more complex due to other factors such as low energy availability and menstrual dysfunction. Moreover, many studies of athletes have used small sample sizes and/or athletes of varying abilities, making it difficult to compare BMD phenotypes between studies. METHOD: The primary aim of this study was to compare dual-energy X-ray absorptiometry (DXA) derived bone phenotypes of high-level endurance runners (58 women and 45 men) to non-athletes (60 women and 52 men). Our secondary aim was to examine the influence of menstrual irregularities and sporting activity completed during childhood on these bone phenotypes. RESULTS: Female runners had higher leg (4%) but not total body or lumbar spine BMD than female non-athletes. Male runners had lower lumbar spine (9%) but similar total and leg BMD compared to male non-athletes, suggesting that high levels of site-specific mechanical loading was advantageous for BMD in females only and a potential presence of reduced energy availability in males. Menstrual status in females and the number of sports completed in childhood in males and females had no influence on bone phenotypes within the runners. CONCLUSION: Given the large variability in BMD in runners and non-athletes, other factors such as variation in genetic make-up alongside mechanical loading probably influence BMD across the adult lifespan.

TTN genotype is associated with fascicle length and marathon running performance.

Titin provides a molecular blueprint for muscle sarcomere assembly, and sarcomere length can vary according to titin isoform expression. If variations in sarcomere length influence muscle fascicle length, this may provide an advantage for running performance. Thus, the aim of this study was to investigate whether the titin (TTN) rs10497520 polymorphism was associated with muscle fascicle length in recreationally active men (RA; n=137) and marathon personal best time in male marathon runners (MR; n=141). Fascicle length of the vastus lateralis was assessed in vivo using B-mode ultrasonography at 50% of muscle length in RA. All participants provided either a whole blood, saliva or buccal cell sample, from which DNA was isolated and genotyped using real-time polymerase chain reaction. Vastus lateralis fascicle length was 10.4% longer in CC homozygotes, those carrying two copies of the C-allele, than CT heterozygotes (P=.003) in RA. In the absence of any TT homozygotes, reflective of the low T-allele frequency within Caucasian populations, it is unclear whether fascicle length for this group would have been smaller still. No differences in genotype frequency between the RA and MR groups were observed (P=.500), although within the MR group, the T-allele carriers demonstrated marathon personal best times 2 minutes 25 seconds faster than CC homozygotes (P=.020). These results suggest that the T-allele at rs10497520 in the TTN gene is associated with shorter skeletal muscle fascicle length and conveys an advantage for marathon running performance in habitually trained men.

Fat mass and obesity associated (FTO) gene influences skeletal muscle phenotypes in non-resistance trained males and elite rugby playing position.

BACKGROUND: FTO gene variants have been associated with obesity phenotypes in sedentary and obese populations, but rarely with skeletal muscle and elite athlete phenotypes. METHODS: In 1089 participants, comprising 530 elite rugby athletes and 559 non-athletes, DNA was collected and genotyped for the FTO rs9939609 variant using real-time PCR. In a subgroup of non-resistance trained individuals (NT; n = 120), we also assessed structural and functional skeletal muscle phenotypes using dual energy x-ray absorptiometry, ultrasound and isokinetic dynamometry. In a subgroup of rugby athletes (n = 77), we assessed muscle power during a countermovement jump. RESULTS: In NT, TT genotype and T allele carriers had greater total body (4.8% and 4.1%) and total appendicular lean mass (LM; 3.0% and 2.1%) compared to AA genotype, with greater arm LM (0.8%) in T allele carriers and leg LM (2.1%) for TT, compared to AA genotype. Furthermore, the T allele was more common (94%) in selected elite rugby union athletes (back three and centre players) who are most reliant on LM rather than total body mass for success, compared to other rugby athletes (82%; P = 0.01, OR = 3.34) and controls (84%; P = 0.03, OR = 2.88). Accordingly, these athletes had greater peak power relative to body mass than other rugby athletes (14%; P = 2 x 10-6). CONCLUSION: Collectively, these results suggest that the T allele is associated with increased LM and elite athletic success. This has implications for athletic populations, as well as conditions characterised by low LM such as sarcopenia and cachexia.

Polymorphisms in PTK2 are associated with skeletal muscle specific force: an independent replication study.

PURPOSE: The aim of the study was to investigate two single nucleotide polymorphisms (SNP) in PTK2 for associations with human muscle strength phenotypes in healthy men. METHODS: Measurement of maximal isometric voluntary knee extension (MVCKE) torque, net MVCKE torque and vastus lateralis (VL) specific force, using established techniques, was completed on 120 Caucasian men (age = 20.6 ± 2.3 year; height = 1.79 ± 0.06 m; mass = 75.0 ± 10.0 kg; mean ± SD). All participants provided either a blood (n = 96) or buccal cell sample, from which DNA was isolated and genotyped for the PTK2 rs7843014 A/C and rs7460 A/T SNPs using real-time polymerase chain reaction. RESULTS: Genotype frequencies for both SNPs were in Hardy-Weinberg equilibrium (X 2 ≤ 1.661, P ≥ 0.436). VL specific force was 8.3% higher in rs7843014 AA homozygotes than C-allele carriers (P = 0.017) and 5.4% higher in rs7460 AA homozygotes than T-allele carriers (P = 0.029). No associations between either SNP and net MVCKE torque (P ≥ 0.094) or peak MVCKE torque (P ≥ 0.107) were observed. CONCLUSIONS: These findings identify a genetic contribution to the inter-individual variability within muscle specific force and provides the first independent replication, in a larger Caucasian cohort, of an association between these PTK2 SNPs and muscle specific force, thus extending our understanding of the influence of genetic variation on the intrinsic strength of muscle.

Association of ACTN3 R577X but not ACE I/D gene variants with elite rugby union player status and playing position.

We aimed to quantify the ACE I/D and ACTN3 R577X (rs1815739) genetic variants in elite rugby athletes (rugby union and league) and compare genotype frequencies to controls and between playing positions. The rugby athlete cohort consisted of 507 Caucasian men, including 431 rugby union athletes that for some analyses were divided into backs and forwards and into specific positional groups: front five, back row, half backs, centers, and back three. Controls were 710 Caucasian men and women. Real-time PCR of genomic DNA was used to determine genotypes using TaqMan probes and groups were compared using χ(2) and odds ratio (OR) statistics. Correction of P values for multiple comparisons was according to Benjamini-Hochberg. There was no difference in ACE I/D genotype between groups. ACTN3 XX genotype tended to be underrepresented in rugby union backs (15.7%) compared with forwards (24.8%, P = 0.06). Interestingly, the 69 back three players (wings and full backs) in rugby union included only six XX genotype individuals (8.7%), with the R allele more common in the back three (68.8%) than controls (58.0%; χ(2) = 6.672, P = 0.04; OR = 1.60) and forwards (47.5%; χ(2) = 11.768, P = 0.01; OR = 2.00). Association of ACTN3 R577X with playing position in elite rugby union athletes suggests inherited fatigue resistance is more prevalent in forwards, while inherited sprint ability is more prevalent in backs, especially wings and full backs. These results also demonstrate the advantage of focusing genetic studies on a large cohort within a single sport, especially when intrasport positional differences exist, instead of combining several sports with varied demands and athlete characteristics.

Stereochemistry and biological activities of constituents from Cynanchum taiwanianum.

The stereochemistry of new acetophenones, cynandione B-D (2-4), isolated from Cynanchum taiwanianum, elucidated by computer modelling calculation and NOESY spectrum. It establishes the absolute configurations of cynandiones B-D (2-4) as 7R; 7"S, 7S; 7"S and 7R; 7"R, respectively. Cynandione B (2) strongly inhibited the release of beta-glucuronidase and lysozyme in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils in a concentration-dependent manner with IC50 values of 1.5 +/- 0.2 and 1.6 +/- 0.2 microM, respectively. 2,5-Dihydroxyacetophenone (6) strongly inhibited the aggregation of washed rabbit platelets induced by arachidonic acid in a concentration-dependent manner with an IC50 value of about 4.8 microM. In human citrated platelet-rich plasma, 2,5-dihydroxyacetophenone (6) inhibited the secondary phase, but not the primary phase, of aggregation induced by adrenaline and ADP. These results suggest that the antiplatelet effect of 2,5-dihydroxyacetophenone (6) is due to inhibition of the formation of thromboxane A2.

A semi-automated method for measuring the potential for protein covalent binding in drug discovery.

INTRODUCTION: Covalent protein binding of metabolically reactive intermediates of drugs has been implicated in drug toxicity including the occurrence of idiosyncratic drug toxicity. Investigators therefore would prefer to avoid developing compounds that produce significant amounts of reactive metabolites. By incubating the radiolabeled drug of interest with liver microsomes it is possible to evaluate the propensity of a drug candidate to covalently bind to proteins. METHODS: Here we present a semi-automated method in which a Brandel cell harvester is used to collect and wash proteins that have been incubated with radiolabeled drug. This method utilizes glass fiber filter paper to capture precipitated protein, rather than the more traditional exhaustive extraction/centrifugation approach. Using model compounds (including [14C]diclofenac, [3H]imipramine, [14C]naphthalene, and [14C]L-746530) we compare the covalent binding results obtained using this method to results generated using the traditional method and we performed cross-laboratory testing of assay reproducibility. RESULTS: It was found that results from new method correlated highly with the traditional method (R2=0.89). The cross-laboratory testing of the method showed an average interlaboratory coefficient of variation of only 18.4%. DISCUSSION: This method provides comparable results to the more traditional centrifugation-based method with considerable time and labor savings.

Molecular flexibility profiling using NMR spectroscopy.

Molecular flexibility is a factor that is not extensively studied in most pharmaceutical research efforts. When it is, the level of effort is high involving the preparation of detailed models supported by either molecular dynamics simulations and/or Nuclear Magnetic Resonance data. While these studies are both powerful and illuminating, they cannot be routinely applied in a drug discovery setting as they are time and expertise intensive. Yet there seems to be little doubt that at least in some cases, molecular flexibility plays a key role in complex formation. A simple, rapid and generally applicable flexibility profiling protocol was applied to two model systems and data describing the internal mobility of carbon atoms were obtained. The protocol utilizes the Model Free approach and NMR data to characterize the internal molecular dynamics of these compounds. The first model system consisted of fluorene and diphenylmethane where the anticipated flexibility trends were observed in the data providing a link between chemical intuition and the experimental results. Data on a second model system, which consisted of two Paclitaxel analogs, showed predictable patterns including dynamical phenyl and methyl groups and a relatively immobile taxane core. Subtle differences in the internal dynamics within the taxane core suggest that it cannot be considered as a rigid structure. Key advantages of using this approach are that no prior knowledge or supposition of dynamical features is required, the protocol can be carried out in most medicinal chemistry laboratories and the data obtained provide a common, empirically derived reference point to discuss the effects of molecular flexibility on activity.

Abnormal acuity development in infantile esotropia.

Monocular and binocular grating acuities were measured using a swept spatial frequency visual evoked potential (VEP) technique in a group of fifteen infants with esotropia and alternating fixation. Both monocular and binocular acuity measures fell significantly below the mean for age-matched normals. Infants with esotropia and alternating fixation did not have significant interocular acuity differences.

Conjunctival nodules associated with onchocerciasis.

A 14-year-old native of Ethiopia with previously treated onchocerciasis moved to California where he was examined for evidence of persisting nematode infestation. Skin and conjunctival biopsy specimens initially disclosed no abnormalities. Subsequently, conjunctival nodules developed, and a biopsy specimen of one of these revealed microfilariae of Onchocerca volvulus lying adjacent to a necrotic eosinophilic granulomatous inflammatory nodule. To our knowledge, nodules of this type have not heretofore been reported to be a notable feature of ocular onchocerciasis. This type of inflammation has a relationship to degenerating microfilaria in onchocerciasis and in other nematode infestation.

Induction of cytochrome-P450 in cryopreserved rat and human hepatocytes.

Our laboratory has been routinely using suspended and cultured human hepatocytes for predicting drug metabolism and enzyme induction by drug candidates to aid drug discovery. Increasing limitation and irregular availability of human tissue has indicated the need for maximizing the use of this valuable resource. Cryopreservation of surplus hepatocytes after isolation would greatly increase the potential of this model. However, cryopreservation of hepatocytes by various methods has resulted in cells with poor metabolic activity and unacceptably low survival rates in culture. Recently, Zaleski et al. (Biochem. Pharmacol. 46 (1993) 111-116) reported that cryopreserved rat hepatocytes retained metabolic capacity similar to fresh hepatocytes when the cells were preincubated for 30 min at 37 degrees C in Krebs Ringer bicarbonate buffer prior to freezing. To further explore this methodology, both the functional capacity of the cells in culture as well as their ability to retain CYP inducibility were investigated with thawed cryopreserved hepatocytes. Although human hepatocytes were used in this study the initial work focused on rat hepatocytes as a cell model. Our results showed that while the preincubation step did not appear to effect the initial viability of cryopreserved hepatocytes, survival of the cells in culture was greatly enhanced. Plating efficiencies for nonpreincubated cryopreserved hepatocytes were decreased to approximately 15% of fresh cells after 48 h in culture. In contrast, cells that had been preincubated prior to freezing had an excellent plating efficiency (approximately 60%) and responded to classical CYP inducers dexamethasone, beta-naphthoflavone and phenobarbital in a manner indistinguishable from that of fresh hepatocytes. Experiments with human hepatocytes have also demonstrated similar results. This is the first time to our knowledge that cryopreserved hepatocytes from both rat and human have been shown to reproducibly respond to CYP inducers in culture.

Clinical experience with the Molteno implant in advanced infantile glaucoma.

The alternatives in the management of infantile glaucoma refractory to conventional treatment are quite limited and unrewarding. We did a retrospective study of 49 patients (53 eyes) under 12 years of age with advanced, uncontrolled glaucoma who each underwent implantation of a single-plate Molteno implant in one stage. Over the follow-up period ranging from 6 months to 3 years (mean 18 months), 36 of the 53 eyes (68%) had an intraocular pressure less than or equal to 21 mm Hg and were considered a success. Fourteen eyes (26%) required further glaucoma surgery, 2 eyes developed severe chronic hypotony, and 1 eye lost light perception following endophthalmitis. The number of anti-glaucoma medications was decreased from a mean of 2 (+/- 1) to 1 (+/- 1). The probability for success of the Molteno implant in controlling glaucoma increased with the age of the patient. Six eyes (11%) were late failures, most probably due to fibrous encapsulation of the bleb. Complications observed were: prolonged hypotony and flat anterior chamber, retinal detachment, migration of the tube, erosion of the tube through the conjunctiva, and an ingrowth of fibrous tissue in the anterior chamber. We consider the Molteno implant to be a reasonable option in the management of difficult cases of infantile glaucoma.

Comparison of on- and off-axis photorefraction with cycloplegic retinoscopy in infants.

We have compared the performance of an off-axis (knife-edge) photorefractor with that of an on-axis (isotropic) system. Normal infants and children between the ages of 8 and 208 weeks were photographed with each camera both with and without cycloplegia. Refractive errors were estimated for each technique based on equations derived from ray-tracing. These refractions were compared to the results of retinoscopy under cycloplegia. Sensitivity and specificity of the two photorefraction systems were evaluated as a function of the magnitude of meridional hyperopia defined by retinoscopy. We also examined the effect of varying the photorefraction screening criterion. Thirteen percent of the infants in the screening sample presented with +3.50 diopters or more of meridional hyperopia. Using this level of ametropia as a referral criterion, the sensitivity and specificity of the off-axis system for infants without cycloplegia were 83% and 72%, respectively. For the on-axis system, sensitivity and specificity values were 85% and 53%. The use of cycloplegics did not significantly improve the performance of either system, but rather their use degraded the specificity of the on-axis system in the presence of moderate refractive errors. The results of the present study indicate that both on- and off-axis systems are effective in identifying highly ametropic infants, but that the off-axis system results in significantly fewer false positives. Moreover, the off-axis system has the advantages of an inherently greater dynamic range for a fixed camera design, and also more easily interpreted photographs.

Ophthalmology's future in the next decade: a historical and comparative perspective.

PURPOSE: To gain a historical and comparative perspective about the future of ophthalmology within the profession of medicine. METHODS: A literature search is made of disciplines other than medicine (history, sociology, philosophy, economics, and ethics) in order to assess factors responsible for survival and healthiness of a profession. The "learned" professions (medicine, law, and theology) are assessed. Other "professional" careers valued by society (sports and classical music) are reviewed. RESULTS: From the perspective of other disciplines, the future of ophthalmology is seen as vulnerable and fragile. Survival of professions, be they classically or economically defined, is linked to societal needs, a profession's unique commitment and ability to provide services to society, and the profession's maintenance of knowledge as well as skill-based services. Historical evidence has shown erosion of a profession's power consequent to capitalist influences, government influences, access of skills by less trained individuals, and elitist posturing by a profession. Comparative evidence has shown societal acceptance of an escalation of salaries for designated superstars, increasing roles and influence of managerial personnel, and trivialization of values other than economic ones. CONCLUSION: Attention to historical and comparative trends by individual ophthalmologists as well as associations representing ophthalmologists is mandatory if ophthalmology as we know it is to survive within the profession of medicine.

Photographic detection of amblyogenic factors.

Refractive errors were determined photographically in a group of infants and children and were compared to conventional cycloplegic retinoscopy. The refractor consisted of a mirror telephoto lens and strobe flash designed to mimic the action of a retinoscope. Significant amblyogenic conditions such as anisometropia and high isoametropia were detectable. Strabismus and media opacities were also recordable. The technique provides a potential mass suited for infants.

Photorefraction with a catadioptric lens. Improvement on the method of Kaakinen.

Kaakinen (1979) presented a method of measuring refractive error and ocular alignment by simultaneous photography of corneal and fundus reflexes. As presented, the technique was unable to detect refractive errors of less than 2-3 diopters (Howland 1980; Kaakinen 1979). We demonstrate that the use of a catadioptric lens and a long working distance can improve sensitivity to less than 1.0 D. Refractions of a model eye, an accommodating eye and ametropic eyes indicate that refractive errors greater than 0.75 D are readily detectable. The improvement in sensitivity achieved by the present system is attributable to a reduction of the angle between the flash source and the entrance pupil of the photorefractor.

Activation of hepatic glutaminase by spermine.

Glutaminase activity in intact mitochondria from rat liver is activated by spermine, as indicated both by increased glutamate production from glutamine and by increased respiration with glutamine as sole substrate. Glutaminase activity assayed in membranes from frozen-thawed mitochondria, is activated by spermine about 6-fold at physiological concentrations of its other effectors (NH4+ at 0.7 mM, Pi 5 mM) and at pH 7.4. Spermine decreased the apparent Km for glutamine from 38 to 15 mM at 5 mM Pi, and increased the sensitivity of the enzyme for phosphate activation so that the concentration required for 50% stimulation decreased from 15 to 4 mM. Half-maximal spermine effects occurred at 0.15 mM, which is in the physiological range. Spermine was effective in the presence of physiological concentrations of Mg2+. We suggest that spermine may be a physiological activator of hepatic glutaminase.