SUMMARY1. Five subjects exercised with the knee extensor of one limb at work loads ranging from 10 to 60 W. Measurements of pulmonary oxygen uptake, heart rate, leg blood flow, blood pressure and femoral arterial-venous differences for oxygen and lactate were made between 5 and 10 min of the exercise.2. Flow in the femoral vein was measured using constant infusion of saline near 0 'C. Since a cuff was inflated just below the knee during the measurements and because the hamstrings were inactive, the measured flow represented primarily the perfusion of the knee extensors.3. Blood flow increased linearly with work load right up to an average value of 57 1 min-. Mean arterial pressure was unchanged up to a work load of 30 W, but increased thereafter from 100 to 130 mmHg. The femoral arterial-venous oxygen difference at maximum work averaged 14-6 % (v/v), resulting in an oxygen uptake of 0-80 1 min-. With a mean estimated weight of the knee extensors of 2-30 kg the perfusion of maximally exercising skeletal muscle of man is thus in the order of 2-5 1 kg-' min', and the oxygen uptake 0-35 1 kg-' min-. 4. Limitations in the methods used previously to determine flow and/or the characteristics ofthe exercise model used may explain why earlier studies in man have failed to demonstrate the high perfusion of muscle reported here.5. It is concluded that muscle blood flow is closely related to the oxygen demand of the exercising muscles. The hyperaemia at low work intensities is due to vasodilatation, and an elevated mean arterial blood pressure only contributes to the linear increase in flow at high work rates. The magnitude of perfusion observed during intense exercise indicates that the vascular bed of skeletal muscle is not a limiting factor for oxygen transport.

Discovery of discontinuous B-cell epitopes is a major challenge in vaccine design. Previous epitope prediction methods have mostly been based on protein sequences and are not very effective. Here, we present DiscoTope, a novel method for discontinuous epitope prediction that uses protein threedimensional structural data. The method is based on amino acid statistics, spatial information, and surface accessibility in a compiled data set of discontinuous epitopes determined by X-ray crystallography of antibody/antigen protein complexes. DiscoTope is the first method to focus explicitly on discontinuous epitopes. We show that the new structure-based method has a better performance for predicting residues of discontinuous epitopes than methods based solely on sequence information, and that it can successfully predict epitope residues that have been identified by different techniques. DiscoTope detects 15.5% of residues located in discontinuous epitopes with a specificity of 95%. At this level of specificity, the conventional Parker hydrophilicity scale for predicting linear B-cell epitopes identifies only 11.0% of residues located in discontinuous epitopes. Predictions by the DiscoTope method can guide experimental epitope mapping in both rational vaccine design and development of diagnostic tools, and may lead to more efficient epitope identification.Keywords: discontinuous epitopes; B-cell epitope; antibody; vaccine design; protein structure; antigen; accessibility; hydrophilicity A major task in vaccine design is to select and design proteins containing antibody-binding epitopes (B-cell epitopes) able to induce an efficient immune response. The selection can be aided by epitope prediction in relevant proteins or regions of proteins. In addition, prediction of B-cell epitopes may help to identify epitopes in proteins that have been analyzed using experimental techniques based on antibody affinity binding, e.g., Western blotting, immunohistochemistry, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA).Most existing methods for prediction of B-cell epitopes exclusively use protein sequences as input, and are best suited to predict epitopes composed of a continuous stretch of amino acids (linear epitopes) (Hopp and Woods 1981;Parker et al. 1986;Jameson and Wolf 1988;Debelle et al. 1992;Maksyutov and Zagrebelnaya 1993;Alix 1999;Odorico and Pellequer 2003). In general, these methods are based on prediction of hydrophilicity, flexibility, b-turns, and surface accessibility using a number of amino acid propensity scales. A large amount of data exists on linear epitopes (Leitner et al. 2003;Saha et al. 2005;Toseland et al. 2005), since the annotation can be done by measuring the binding of antigen peptide fragments to antibodies. However, this method of annotation may lead to annotation errors, because a peptide can specifically bind an antibody even if some residues of the peptide are not interacting with the antibody. Predicting linear epitopes is still a nontrivial task, and the obtainable pre...

BackgroundEstimation of the reliability of specific real value predictions is nontrivial and the efficacy of this is often questionable. It is important to know if you can trust a given prediction and therefore the best methods associate a prediction with a reliability score or index. For discrete qualitative predictions, the reliability is conventionally estimated as the difference between output scores of selected classes. Such an approach is not feasible for methods that predict a biological feature as a single real value rather than a classification. As a solution to this challenge, we have implemented a method that predicts the relative surface accessibility of an amino acid and simultaneously predicts the reliability for each prediction, in the form of a Z-score.ResultsAn ensemble of artificial neural networks has been trained on a set of experimentally solved protein structures to predict the relative exposure of the amino acids. The method assigns a reliability score to each surface accessibility prediction as an inherent part of the training process. This is in contrast to the most commonly used procedures where reliabilities are obtained by post-processing the output.ConclusionThe performance of the neural networks was evaluated on a commonly used set of sequences known as the CB513 set. An overall Pearson's correlation coefficient of 0.72 was obtained, which is comparable to the performance of the currently best public available method, Real-SPINE. Both methods associate a reliability score with the individual predictions. However, our implementation of reliability scores in the form of a Z-score is shown to be the more informative measure for discriminating good predictions from bad ones in the entire range from completely buried to fully exposed amino acids. This is evident when comparing the Pearson's correlation coefficient for the upper 20% of predictions sorted according to reliability. For this subset, values of 0.79 and 0.74 are obtained using our and the compared method, respectively. This tendency is true for any selected subset.

SUMMARY1. Five subjects trained for 8 weeks on a bicycle ergometer for an average of 40 min/day, four times a week at a work load requiring 80 % of the maximal oxygen uptake (02maX.)-1o2 max. determinations were performed, and muscle biopsies from the quadriceps femoris muscle (vastus lateralis) were taken before, as well as repeatedly during, the training period. The muscle biopsies were histochemically stained for fibre-types (myofibrillar ATPase) and capillaries (amylase-PAS method), and analysed biochemically for succinate dehydrogenase and cytochrome oxidase activities.2. The training programme resulted in a 16% increase in l2max., a 20 % increase in capillary density, a 20 % increase in mean fibre area, and an approximately 40 % increase in the activities of succinate dehydrogenase and cytochrome oxidase.3. The capillary supply to type I, IIA and IIB fibres, expressed as the mean number of capillaries in contact with each fibre-type, relative to fibre-type area, increased equally.4. The present study shows that endurance training constitutes a powerful stimulus for capillary proliferation in human skeletal muscle.