Etiology of musculoskeletal injuries in amateur breakdancers.

AIM: The aim of this study was to assess the incidence of musculoskeletal injuries in breakdancers and investigate the association with training habits. METHODS: Forty-six males and sixteen females completed a questionnaire regarding their training and competition habits (frequency, warm-up and stretching, strength training, protective equipment, move types and supervision) and the musculoskeletal injuries sustained as a result of breakdancing in the previous 12 months. The effects of training habits and sex on injury rates were analyzed by a Mann-Whitney Test and a Kruskal-Wallis Test, while a stepwise linear regression analysis assessed the link between injury rates and quantitative risk factors. RESULTS: The injury rate was 4.02 injuries per 1000 h, with no significant difference between males and females (P>0.05). The main injuries affected were the knee (23.4%) and wrist (15.3%), and females were characterized by a significantly greater number of finger injuries and a lower number of shoulder injuries that males (P<0.05). In addition, of all the factors evaluated, only the amount of time spent performing breakdance training showed a significant association with injury rate (P<0.05). CONCLUSION: These results suggest that interventions should focus on protecting specific body parts and improving training quality and recovery.

The effect of caffeine ingestion on functional performance in older adults.

UNLABELLED: Caffeine is a widely used nutritional supplement which has been shown to enhance both physical and cognitive performance in younger adults. However, few studies have assessed the effect of caffeine ingestion on performance, particularly functional performance in older adults. The present study aims to assess the effect of acute caffeine ingestion on functional performance, manual dexterity and readiness to invest effort in older adults. METHODS: 19 apparently healthy, volunteers (10 females and 9 males aged 61-79; 66 ± 2 years) performed tests of functional fitness and manual dexterity post ingestion of caffeine (3mg*kg-1) or placebo in a randomised order. Pre and 60 minutes post ingestion, participants also completed measures of readiness to invest physical (RTIPE) and mental (RTIME) effort. RESULTS: A series of repeated measures ANOVAS indicated enhanced performance in the following functional fitness tests; arm curls (P = .04), 8 foot up and go (P = .007), six minute walk (P = .016). Manual dexterity was also improved in the presence of caffeine (P = .001). RTIME increased (P = .015) pre to post ingestion in the caffeine condition but not in the placebo condition. There were no significant main effects or interactions for RTIPE or gender in any analysis (all P > .05). CONCLUSIONS: The results of this study suggest that acute caffeine ingestion positively enhances functional performance, manual dexterity and readiness to invest effort in apparently healthy older adults.

Effect of a simulated soccer match on the functional hamstrings-to-quadriceps ratio in amateur female players.

The purpose of this study was to investigate the effect of a simulated soccer test on the functional hamstrings-to-quadriceps ratio (H(ecc) : Q(con)) in female soccer players. Fourteen amateur players (age, 26.1 ± 4.6 years; height, 168 ± 12 cm; body mass, 62.7 ± 5.5 kg; body fat, 23.7 ± 2.2%) performed the modified Loughborough Intermittent Shuttle Test (modified LIST). Isokinetic strength assessments of the hamstrings and quadriceps on the dominant and non-dominant legs at 120°/s were performed before and immediately after the modified LIST. H(ecc) : Q(con) was calculated as the ratio of the peak eccentric torque of the hamstrings to the peak concentric torque of the quadriceps. A two-way univariate analysis of variance was used to assess the effect of time and leg dominance on H(ecc) : Q(con). The main results showed that the modified LIST led to a significant decrease in H(ecc) : Q(con) in the dominant (-14.1%) and non-dominant legs (-8.0%) (P = 0.02). However, this decrease was not significantly different between dominant and non-dominant legs (P = 0.42). These results reflect a greater risk of hamstrings tears and ACL sprains at the end of soccer matches.

The ingestion of combined carbohydrates does not alter metabolic responses or performance capacity during soccer-specific exercise in the heat compared to ingestion of a single carbohydrate.

This study was designed to investigate the effect of ingesting a glucose plus fructose solution on the metabolic responses to soccer-specific exercise in the heat and the impact on subsequent exercise capacity. Eleven male soccer players performed a 90 min soccer-specific protocol on three occasions. Either 3 ml · kg(-1) body mass of a solution containing glucose (1 g · min(-1) glucose) (GLU), or glucose (0.66 g · min(-1)) plus fructose (0.33 g · min(-1)) (MIX) or placebo (PLA) was consumed every 15 minutes. Respiratory measures were undertaken at 15-min intervals, blood samples were drawn at rest, half-time and on completion of the protocol, and muscle glycogen concentration was assessed pre- and post-exercise. Following the soccer-specific protocol the Cunningham and Faulkner test was performed. No significant differences in post-exercise muscle glycogen concentration (PLA, 62.99 ± 8.39 mmol · kg wet weight(-1); GLU 68.62 ± 2.70; mmol · kg wet weight(-1) and MIX 76.63 ± 6.92 mmol · kg wet weight(-1)) or exercise capacity (PLA, 73.62 ± 8.61 s; GLU, 77.11 ± 7.17 s; MIX, 83.04 ± 9.65 s) were observed between treatments (P > 0.05). However, total carbohydrate oxidation was significantly increased during MIX compared with PLA (P < 0.05). These results suggest that when ingested in moderate amounts, the type of carbohydrate does not influence metabolism during soccer-specific intermittent exercise or affect performance capacity after exercise in the heat.

Effects of carbohydrate beverage ingestion on the salivary IgA response to intermittent exercise in the heat.

The purpose of this study was to establish if provision of CHO altered the mucosal immune and salivary cortisol responses to intermittent exercise in the heat. In a double-blind design, 10 males undertook soccer-specific intermittent exercise on a motorized treadmill on 2 occasions, each over 90 min and separated by 1 week. During CHO and placebo trials, subjects were given either a carbohydrate solution (3 ml · kg (-1) body weight) or placebo drink, 5 min before the commencement of exercise, at 15, 30 min, at half time, 60 and 75 min into exercise. Salivary flow rate increased throughout the placebo trial and decreased throughout the CHO treatment; the difference between conditions neared statistical significance (P=0.055). Neither s-IgA concentration nor s-IgA to osmolality ratio was affected by 2 conditions or differed at any time-point post-exercise (P>0.05). The s-IgA secretion rate increased, s-IgA to protein ratio decreased post-exercise and salivary cortisol decreased 24 h post-exercise (P<0.05) compared to pre-exercise. Carbohydrate supplementation whilst exercising in the heat, does not influence rating of perceived exertion, thermal sensation, salivary flow rate, s-IgA concentration, s-IgA secretion rate, s-IgA to osmolality ratio or s-IgA to protein ratio and salivary cortisol but heart rate was increased.

Carbohydrate ingestion and pre-cooling improves exercise capacity following soccer-specific intermittent exercise performed in the heat.

Ingestion of carbohydrate and reducing core body temperature pre-exercise, either separately or combined, may have ergogenic effects during prolonged intermittent exercise in hot conditions. The aim of this investigation was to examine the effect of carbohydrate ingestion and pre-cooling on the physiological responses to soccer-specific intermittent exercise and the impact on subsequent high-intensity exercise performance in the heat. Twelve male soccer players performed a soccer-specific intermittent protocol for 90 min in the heat (30.5°C and 42.2% r.h.) on four occasions. On two occasions, the participants underwent a pre-cooling manoeuvre. During these sessions either a carbohydrate-electrolyte solution (CHOc) or a placebo was consumed at (PLAc). During the remaining sessions either the carbohydrate-electrolyte solution (CHO) or placebo (PLA) was consumed. At 15-min intervals throughout the protocol participants performed a mental concentration test. Following the soccer-specific protocol participants performed a self-chosen pace test and a test of high-intensity exercise capacity. The period of pre-cooling significantly reduced core temperature, muscle temperature and thermal sensation (P < 0.05). Self-chosen pace was greater with CHOc (12.5 ± 0.5 km h(-1)) compared with CHO (11.3 ± 0.4 km h(-1)), PLA (11.3 ± 0.4 km h(-1)) and PLAc (11.6 ± 0.5 km h(-1)) (P < 0.05). High-intensity exercise capacity was improved with CHOc and CHO when compared with PLA (CHOc; 79.8 ± 7 s, CHO; 72.1 ± 5 s, PLAc; 70.1 ± 8 s, PLA; 57.1 ± 5 s; P < 0.05). Mental concentration during the protocol was also enhanced during CHOc compared with PLA (P < 0.05). These results suggest pre-cooling in conjunction with the ingestion of carbohydrate during exercise enhances exercise capacity and helps maintain mental performance during intermittent exercise in hot conditions.

Fluid provision and metabolic responses to soccer-specific exercise.

The present study aimed to investigate the impact on metabolism of altering the timing and volume of ingested carbohydrate during soccer-specific exercise. Twelve soccer players performed a soccer-specific protocol on three occasions. On two, 7 ml kg(-1) carbohydrate-electrolyte or placebo were ingested at 0 and 45 min. On a third, the same total volume of carbohydrate-electrolyte was consumed but at 0, 15, 30, 45, 60 and 75 min. Carbohydrate-electrolyte ingestion increased blood glucose, insulin and carbohydrate oxidation, whilst suppressing NEFA, glycerol and fat oxidation (P < 0.05) although manipulating the schedule of carbohydrate ingestion elicited similar metabolic responses (P > 0.05). However, consuming fluid in small volumes reduced the sensation of gut fullness (P < 0.05). The results demonstrated that when the total volume of carbohydrate consumed is equal, manipulating the timing and volume of ingestion elicits similar metabolic responses. Furthermore, consuming a small volume of fluid at regular intervals reduces the sensation of gut fullness.

Strategies for hydration and energy provision during soccer-specific exercise.

The aim of the present study was to investigate the effect of manipulating the provision of sports drink during soccer-specific exercise on metabolism and performance. Soccer players (N = 12) performed a soccer-specific protocol on three occasions. On two, 7 mL/kg carbohydrate-electrolyte (CHOv) or placebo (PLA) solutions were ingested at 0 and 45 min. On a third, the same total volume of carbohydrate-electrolyte was consumed (CHOf) in smaller volumes at 0, 15, 30, 45, 60, and 75 min. Plasma glucose, glycerol, non-esterified free fatty acids (NEFA), cortisol, and CHO oxidation were not significantly different between CHOv and CHOf (P > 0.05). Sprint power was not significantly affected (P > 0.05) by the experimental trials. This study demonstrates when the total volume of carbohydrate consumed is equal, manipulating the timing and volume of ingestion elicits similar metabolic responses without affecting exercise performance.

Covalent capture of a human O(6)-alkylguanine alkyltransferase-DNA complex using N(1),O(6)-ethanoxanthosine, a mechanism-based crosslinker.

The DNA repair protein O(6)-alkylguanine alkyltransferase (AGT) is responsible for removing promutagenic alkyl lesions from exocyclic oxygens located in the major groove of DNA, i.e. the O(6) and O(4) positions of guanine and thymine. The protein carries out this repair reaction by transferring the alkyl group to an active site cysteine and in doing so directly repairs the premutagenic lesion in a reaction that inactivates the protein. In order to trap a covalent AGT-DNA complex, oligodeoxyribonucleotides containing the novel nucleoside N(1),O(6)-ethanoxanthosine ((e)X) have been prepared. The (e)X nucleoside was prepared by deamination of 3',5'-protected O(6)-hydroxyethyl-2'-deoxyguanosine followed by cyclization to produce 3',5'-protected N(1),O(6)-ethano-2'-deoxyxanthosine, which was converted to the nucleoside phosphoramidite and used in the preparation of oligodeoxyribonucleotides. Incubation of human AGT with a DNA duplex containing (e)X resulted in the formation of a covalent protein-DNA complex. Formation of this complex was dependent on both active human AGT and (e)X and could be prevented by chemical inactivation of the AGT with O(6)-benzylguanine. The crosslinking of AGT to DNA using (e)X occurs with high yield and the resulting complex appears to be well suited for further biochemical and biophysical characterization.

Assignment of enzyme substrate specificity by principal component analysis of aligned protein sequences: an experimental test using DNA glycosylase homologs.

We have studied the relationship between amino acid sequence and substrate specificity in a DNA glycosylase family by characterizing experimentally the specificity of four new members of the family. We show that principal component analysis (PCA) of the sequence family correctly predicts the substrate specificity of one of the novel homologs even though conventional sequence analysis methods fail to group this homolog with other sequences of the same specificity. PCA also suggested, correctly, that another homolog characterized previously differs in its specificity from those sequences with which it clusters by conventional criteria. These results suggest that principal component analysis of sequence families can be a useful tool in annotating genome sequences when there is ambiguity concerning which subfamily a new homolog belongs to. Published 2000 Wiley-Liss, Inc.

Dominant sensitization variants of human O(6)-methylguanine-DNA-methyltransferase obtained by a mutational screen of surface residues.

A scanning mutagenesis experiment was performed on human O(6)-methylguanine methyltransferase (hMGMT), directed largely at non-conserved surface residues that have not previously been studied. Variants typically contained two or more substitutions. Two of the 16 variants characterized in detail are inactive for methyltransfer, but increase the cytotoxicity and mutagenic effects of methylating agents. This phenotype is reminiscent of a variant (C145A) that has a mutation in the methyl-accepting cysteine. C145A is inactive, but reportedly binds methylated DNA and confers sensitivity to methylating agents. The sensitization phenotype of the two new variants is more striking in strains that are wild-type for DNA repair than in strains that are deficient for repair, suggesting that these proteins inhibit functional DNA repair proteins by competitively binding to methylated DNA. Both variants have multiple substitutions in the last helix of the protein. These results suggest that the C-terminal helix is necessary for methyltransfer activity, but not for methylguanine-specific binding.

Characterization of an 8-oxoguanine DNA glycosylase from Methanococcus jannaschii.

A thermostable 8-oxoguanine (oxoG) DNA glycosylase from Methanococcus jannaschii has been expressed in Escherichia coli, purified, and characterized. The enzyme, which has been named mjOgg, belongs to the same diverse DNA glycosylase superfamily as the 8-oxoguanine DNA glycosylases from yeast (yOgg1) and human (hOgg1) but is substantially different in sequence. In addition, unlike its eukaryotic counterparts, which have a strong preference for oxoG.C base pairs, mjOgg has little specificity for the base opposite oxoG. mjOgg has both DNA glycosylase and DNA lyase (beta-elimination) activity, and the combined glycosylase/lyase activity occurs at a rate comparable with the glycosylase activity alone. Mutation of Lys-129, analogous to Lys-241 of yOgg1, abolishes glycosylase activity.

The C-terminal domain of the adenine-DNA glycosylase MutY confers specificity for 8-oxoguanine.adenine mispairs and may have evolved from MutT, an 8-oxo-dGTPase.

MutY is an adenine-DNA glycosylase with specificity for mismatches involving 8-oxoguanine (oG.A) or guanine (G.A). In addition to a 25 kDa catalytic domain common to all members of its DNA glycosylase superfamily, MutY has a 14 kDa C-terminal domain. Sequence analyses suggest that this C-terminal domain is distantly related to MutT, a pyrophosphohydrolase specific for 2'-deoxy-8-oxoguanosine triphosphate (doGTP). Here we present biochemical evidence that the MutT-like domain of MutY is the principal determinant of oG specificity. First, MutY dissociates approximately 1500-fold more slowly from oG-containing product DNA than from G-containing product, but a truncated protein lacking the C-terminal domain dissociates as rapidly from oG-DNA as the full-length protein dissociates from G-DNA. Second, MutY removes adenine from oG.A mismatches almost 30-fold faster than from G.A mismatches in a pre-steady-state assay, but deletion of the C-terminal domain reduces this specificity for oG.A to less than 4-fold. The kinetic data are consistent with a model in which binding of oG to the C-terminal domain of MutY accelerates the pre-steady-state glycosylase reaction by facilitating adenine base flipping. The observation that oG specificity derives almost exclusively from the C-terminal domain of MutY adds credence to the sequence analyses and suggests that specificity for oG.A mismatches was acquired by fusion of a MutT-like protein onto the core catalytic domain of an adenine-DNA glycosylase.

Zinc fingers in Caenorhabditis elegans: finding families and probing pathways.

More than 3 percent of the protein sequences inferred from the Caenorhabditis elegans genome contain sequence motifs characteristic of zinc-binding structural domains, and of these more than half are believed to be sequence-specific DNA-binding proteins. The distribution of these zinc-binding domains among the genomes of various organisms offers insights into the role of zinc-binding proteins in evolution. In addition, the complete genome sequence of C. elegans provides an opportunity to analyze, and perhaps predict, pathways of transcriptional regulation.

Computer search algorithms in protein modification and design.

The computer-aided design of protein sequences requires efficient search algorithms to handle the enormous combinatorial complexity involved. A variety of different algorithms have now been applied with some success. The choice of algorithm can influence the representation of the problem in several important ways--the discreteness of the configuration, the types of energy terms that can be used and the ability to find the global minimum energy configuration. The use of dead end elimination to design the complete sequence for a small protein motif and the use of genetic and mean-field algorithms to design hydrophobic cores for proteins represent the major themes of the past year.

A hybrid sequence approach to the paracelsus challenge.

Inspired by the Paracelsus Challenge of Rose and Creamer (Proteins 19: 1-3, 1994), we have designed a protein sequence that is 50% identical to an all-helical protein but is intended to fold into a largely beta-sheet structure. Rather than attempt a de novo design, our strategy was to construct a hybrid sequence based on a helical "parent" protein (434 Cro) and a "target" protein with the desired fold (the B1 domain of protein G). The hybrid sequence (Crotein-G) is 50% identical to 434 Cro but is also 62% identical to the B1 domain of protein G. We also created a variant of Crotein-G (ZCrotein-G) that contains a potential His3Cys1 zinc binding site. At low protein concentrations and in the presence of 20% 2,2,2-trifluoroethanol (TFE) (v/v), the circular dichroism spectra of the designed proteins are distinct from that of 434 Cro and similar to that of the B1 domain of protein G. However, the proteins fail to denature in a cooperative manner. Furthermore, aggregation occurs at moderate protein concentrations or in the absence of TFE. Addition of zinc to ZCrotein-G does not promote structure formation. In summary, 434 Cro has been altered to something that may resemble the B1 domain of protein G, but the protein does not adopt a native structure.

Specificity of DNA repair methyltransferases determined by competitive inactivation with oligonucleotide substrates: evidence that Escherichia coli Ada repairs O6-methylguanine and O4-methylthymine with similar efficiency.

DNA repair methyltransferases (MTases) are stoichiometric acceptor molecules that are irreversibly inactivated in the course of removing a methyl group from O6-methylguanine (meG)-DNA or O4-methylthymine (meT)-DNA. A new assay has been developed to determine the relative efficiency of repair of meG and meT. The assay is based on the deprotection of methylated restriction sites in synthetic oligonucleotides and can be used to measure meG repair or meT repair directly. More importantly, relative repair efficiencies can be measured in competition experiments, using each of the methylated oligomers in turn as an inhibitor of repair for the other. Relative repair rates are determined by numerical solution of the coupled rate equations that describe this competition to the experimental data. We find that the human MTase repairs meT about 35-fold less well than meG, qualitatively similar to earlier studies. Contrary to previous reports, however, we find that Escherichia coli Ada repairs meG and meT with nearly equal efficiency. This finding, in conjunction with other recent reports, may indicate that low meT repair is a relatively unusual characteristic of the human homolog.

Formation of a covalent complex between methylguanine methyltransferase and DNA via disulfide bond formation between the active site cysteine and a thiol-containing analog of guanine.

DNA repair methyltransferases (MTases) remove methyl or other alkyl groups from the O6 position of guanine or the O4 position of thymine by transfering the group to an active site cysteine. In order to trap an MTase-DNA complex via a disulfide bond, 2'-deoxy-6-(cystamine)-2-aminopurine (d6Cys2AP) was synthesized and incorporated into oligonucleotides. d6Cys2AP has a disulfide bond within an alkyl chain linked to the 6 position of 2,6-diaminopurine, which disulfide can be reduced to form a free thiol. Addition of human MTase to reduced oligonucleotide resulted in a protein-DNA complex that was insensitive to denaturation by SDS and high salt, but which readily dissociated in the presence of dithiothreitol. Formation of this complex was prevented by methylation of the active site cysteine. Evidence that the active site cysteine is directly involved in disulfide bond formation was obtained by N-terminal sequencing of peptides that remained associated with DNA after proteolysis of the complex.

Construction and overexpression of a synthetic gene for human DNA methylguanine methyltransferase: renaturation and rapid purification of the protein.

A synthetic gene was constructed that encodes human DNA methylguanine methyltransferase (hMGMT). The synthetic gene was designed with a number of unique restriction sites to facilitate cassette mutagenesis and to reflect the preferences found among genes in Escherichia coli. Both the full-length gene and a gene for a functional variant (hMGMT delta C) that lacks the C-terminal 28 codons were constructed, and the genes were overexpressed using a T7 RNA polymerase promoter. The proteins are made in the form of insoluble aggregates but the truncated form of the protein (hMGMT delta C) has been successfully denatured, renatured, and purified to near homogeneity by ion exchange. Methyltransferase activity assays of hMGMT delta C demonstrate that the reconstituted protein has substantial DNA repair activity, though somewhat less than full-length hMGMT that had been expressed and purified in a soluble form. Mass spectrometry of a mixture of proteolytic fragments confirmed the protein sequence and indicated no detectable oxidation of the active site cysteine. The protein was determined to be monomeric by gel filtration chromatography, and circular dichroism spectra for renatured hMGMT delta C and fully soluble hMGMT are consistent with the renatured protein preparation being fully folded. Refolded hMGMT delta C had a curious propensity to form large aggregates in a time-dependent manner when injected into a dynamic light scattering instrument; this aggregation behavior was not observed for hMGMT purified in a soluble form. Differences in susceptibility to aggregation may account for differences in methyltransfer activity. Yields of purified protein were approximately 5 mg/liter of culture.

Specific recognition of A/G and A/7,8-dihydro-8-oxoguanine (8-oxoG) mismatches by Escherichia coli MutY: removal of the C-terminal domain preferentially affects A/8-oxoG recognition.

Escherichia coli MutY is a 39 kDa adenine DNA glycosylase and 3' apurinic/apyrimidinic (AP) lyase that is active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. 8-oxoG (7,8-dihydro-8-oxoguanine or GO) is a major stable product of oxidative damage, and A/GO mismatches may be particularly important biological substrates for MutY. Proteolytic digestion of MutY using thermolysin was found to produce two relatively stable fragments of 25 and 12 kDa. The 25 kDa fragment begins at the N terminus of MutY and spans the region homologous with E. coli endonuclease III, a DNA glycosylase/AP lyase that repairs oxidatively damaged pyrimidines. The 12 kDa fragment, which consists of much of the rest of MutY, had no detectable activity. The purified 25 kDa fragment (M25) had nearly wild-type binding and cleavage activities with A/G-mismatched substrates. Binding to A/GO-mismatched DNA, however, was dramatically reduced in M25 compared to that in intact protein. Borohydride-dependent enzyme-DNA cross-linking, which is a hallmark of the reaction of several DNA glycosylases that possess concomitant AP lyase activity, was also substantially reduced when M25 was allowed to react with A/GO-mismatched DNA. The significant differences in M25 recognition and reactivity with A/G and A/GO mismatches suggest that the C-terminal region of MutY, a region with no homologous counterpart in E. coli endonuclease III, plays an important role in the repair of mismatched DNA arising from oxidation damage.