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.

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.

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.

Evidence from cassette mutagenesis for a structure-function motif in a protein of unknown structure.

The three-dimensional structure of most enzymes is unknown; however, many enzymes may have structural motifs similar to those in the known structures of functionally related enzymes. Evidence is presented that an enzyme of unknown structure [Ile-transfer RNA (tRNA) synthetase] may share a functionally important structural motif with an enzyme of related function (Tyr-tRNA synthetase). This approach involves (i) identifying segments of Ile-tRNA synthetase that have been unusually conserved during evolution, (ii) predicting the function of one such segment by assuming a structural relation between Ile-tRNA synthetase and Tyr-tRNA synthetase, and (iii) testing the predicted function by mutagenesis and subsequent biochemical analysis. Random mutations were introduced by cassette mutagenesis into a ten-amino-acid segment of Ile-tRNA synthetase that was predicted to be involved in the formation of the binding site for isoleucine. Few amino acid substitutions appear to be tolerated in this region. However, one substitution (independently isolated twice) increased the Michaelis constant Km for isoleucine in the adenylate synthesis reaction by greater than 6000-fold, but had little effect on the Km for adenosine triphosphate, the apparent Km for tRNA, or the rate constant kcat.

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.

The DNA binding arm of lambda repressor: critical contacts from a flexible region.

Segments of protein that do not adopt a well-ordered conformation in the absence of DNA can still contribute to site-specific recognition of DNA. The first six residues (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6-) of phage lambda repressor are flexible but are important for site-specific binding. Low-temperature x-ray crystallography and codondirected saturation mutagenesis were used to study the role of this segment. All of the functional sequences have the form [X]1-[X]2-[Lys or Arg]3-[Lys]4-[Lys or Arg]5-[X]6. A high-resolution (1.8 angstrom) crystal structure shows that Lys3 and Lys4 each make multiple hydrogen bonds with guanines and that Lys5 interacts with the phosphate backbone. The symmetry of the complex breaks down near the center of the site, and these results suggest a revision in the traditional alignment of the six lambda operator sites.

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.

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.

Regulation of newly evolved enzymes. III Evolution of the ebg repressor during selection for enhanced lactase activity.

The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.

Sequence 'minimization': exploring the sequence landscape with simplified sequences.

The challenges of protein engineering arise, in part, from the enormous number of possible sequences and the almost unimaginably small fraction of such sequences that can be studied experimentally or computationally. Fortunately, not all possibilities need to be considered because many different sequences can adopt the same structure. Of the vast number of sequences that fold into a given conformation, some are 'simpler' than the sequences of typical proteins. Studying protein sequences that are simpler helps focus attention on the principal determinants of structure. Recent examples of this strategy are the simplification of protein surfaces and cores, the use of a binary 'code' for protein design and the structural analysis of random simple sequences.

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.

Covariation of residues in the homeodomain sequence family.

Homeodomains are 60 amino acid DNA binding domains found in numerous eukaryotic transcription factors. The homeodomain family is a useful system for studying sequence-structure relationships because several hundred sequences are known and the structures of several homeodomains have been determined. Covariation of amino acid residues in the homeodomain family has been investigated to see whether strongly covariant residue pairs can be understood in terms of the structure and function of these domains. Among 16 strongly covariant pairs examined, 2 are explained by the ability to form salt bridges, and 9 appear related to the DNA binding function of the proteins. For the remaining 5 pairs, the rationale for covariance remains unclear and the likelihood of artifactual correlations is discussed in the context of experimental and evolutionary biases in the selection of sequences. No significant correlation was found between covariance and structural proximity in the hydrophobic core.

Structural studies of the engrailed homeodomain.

The structure of the Drosophila engrailed homeodomain has been solved by molecular replacement and refined to an R-factor of 19.7% at a resolution of 2.1 A. This structure offers a high-resolution view of an important family of DNA-binding proteins and allows comparison to the structure of the same protein bound to DNA. The most significant difference between the current structure and that of the 2.8-A engrailed-DNA complex is the close packing of an extended strand against the rest of the protein in the unbound protein. Structural features of the protein not previously noted include a "herringbone" packing of 4 aromatic residues in the core of the protein and an extensive network of salt bridges that covers much of the helix 1-helix 2 surface. Other features that may play a role in stabilizing the native state include the interaction of buried carbonyl oxygen atoms with the edge of Phe 49 and a bias toward statistically preferred side-chain dihedral angles. There is substantial disorder at both ends of the 61 amino acid protein. A 51-amino acid variant of engrailed (residues 6-56) was synthesized and shown by CD and thermal denaturation studies to be structurally and thermodynamically similar to the full-length domain.

Innervation of the chick cornea analyzed in vitro.

PURPOSE: During the early stages (embryonic day 3 [E3]) of avian corneal development, nerve fibers extend from the trigeminal ganglion to the corneal limbus. On E11, these nerve fibers enter the cornea and extend through the secondary stroma to begin innervation of the epithelium on E13. This process of innervation is concomitant with the cornea's dehydration and transition from opacity to transparency; thus, suggesting a link between innervation and the attainment of corneal function. This investigation attempts to ascertain whether the developing cornea can support its innervation in vitro and whether there is a possible developmental interrelationship between corneal innervation and dehydration, with the associated transition from opacity to transparency. METHODS: Isolated corneas from either E8 or E14 chicks were co-cultured with E8 dorsal root ganglia. After 4 days of culture, innervation was visualized by silver staining and immunohistochemistry. Changes in corneal composition and organization associated with this innervation in vitro were analyzed by measuring changes in specific hydration, thickness and compaction, and incorporation of [35S]sulfate into glycosaminoglycans during co-culture. RESULTS: The E8 and E14 corneas support extensive innervation in vitro. Developing nerve fibers extend through the secondary stroma to innervate the epithelium. In vitro innervation of E8, but not E14, corneas was associated with a decrease in corneal specific hydration, whereas control corneas (without dorsal root ganglia) failed to show any such changes. E8 corneas also showed a significant increase in compaction when innervated in vitro. Corneal innervation in vitro did not significantly change the overall incorporation of [35S]sulfate into glycosaminoglycans. Furthermore, incorporation of [35S]sulfate into corneal sulfated glycosaminoglycans (sGAG) is not influenced by either the number of nerve fibers innervating the cornea or nerve growth factor (NGF). In addition, the distribution of staining of the corneal glycosaminoglycans, chondroitin sulfate and keratan sulfate, and peanut agglutinin-binding epitopes, suggests that these molecules are not associated with inhibition of axonal development. CONCLUSIONS: The in vitro system described here is a useful model to understand the process of corneal development. Co-culture has shown that corneal innervation promotes the process of dehydration, which is dependent on the age of the cornea. However, other functionally related refinements necessary for transparency-notably proteoglycan synthesis-may not be linked to innervation or NGF production. The authors conclude that the development of transparency is dependent on corneal innervation, though not exclusively, and that other controlling factors also are required.

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.

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.

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.