Effects of isokinetic training of the knee extensors on high-intensity exercise performance and skeletal muscle buffering

Twenty-three subjects isokinetically trained the right and left quadriceps femoris, three times per week for 16 weeks; one group (n=13) trained at an angular velocity of 4.19 rad · s^–1 and a second group (n=10), at 1.05 rad · s^–1. A control group (n=10) performed no training. Isometric endurance time at 60% quadriceps maximum voluntary contraction (MVC), mean power output and work done (W) during all-out cycling, and the muscle buffer value (B) and carnosine concentration of biopsy samples from the vastus lateralis, were all assessed before and after training. The two training groups did not differ significantly from each other in their training response to any of these variables (P < 0.05). No significant difference in either 60% MVC endurance time or impulse [(endurance time × force) at 60% MVC] was observed for any group after the 16 week period (P > 0.05). However, the post-training increase (9%) in W during high-intensity cycling was greater in the training group than in the control group (P=0.04). NeitherB nor carnosine concentration showed any significant change following training (P=0.56 andP=0.37, respectively). It is concluded that 16 weeks of isokinetic training of the knee extensors enables subjects to do more work during high-intensity cycling. Although the precise adaptations responsible for the improved performance have yet to be identified, they are unlikely to include an increase inB.     

Muscle activation and the isokinetic torque-velocity relationship of the human triceps surae

The influence of muscle activation and the time allowed for torque generation on the angle-specific torque-velocity relationship of the triceps surae was studied during plantar flexion using supramaximal electrical stimulation and a release technique on six male subjects [mean (SD) age 25 (4) years]. Torque-velocity data were obtained under different levels of constant muscle activation by varying the stimulus frequency and the time allowed for isometric torque generation prior to release and isokinetic shortening. To eliminate the effects of the frequency response on absolute torque the isokinetic data were normalized to the maximum isometric torque values at 0.44 rad. There were no significant differences in the normalized torques generated at any angular velocity using stimulus frequencies of 20, 50 or 80 Hz. When the muscle was stimulated at 50 Hz the torques obtained after a 400 ms and 1 s pre-release isometric contraction did not differ significantly. However, with no pre-release contraction significantly less torque was generated at all angular velocities beyond 1.05 rad · s^–1 when compared with either the 200, 400 ms or 1 s condition. With a 200 ms pre-release contraction significantly less torque was generated at angular velocities beyond 1.05 rad · s^–1 when compared with the 400 ms or 1 s conditions. It would seem that the major factor governing the shape of the torque-velocity curve at a constant level of muscle activation is the time allowed for torque generation.     

Effects of isokinetic training of the knee extensors on isometric strength and peak power output during cycling

Isokinetic training of right and left quadriceps femoris was undertaken three times per week for 16 weeks. One group of subjects (n = 13) trained at an angular velocity of 4.19 rad.s-1 and a second group (n = 10) at 1.05 rad.s-1. A control group (n = 10) performed no training. Maximal voluntary contraction (MVC) of the quadriceps, and peak pedal velocity nu p,peak) and peak power output (Wpeak) during all-out cycling (against loads equivalent to 9, 10, 11, 12, 13 and 14% MVC) were assessed before and after training. The two training groups did not differ significantly from each other in their training response to any of the performance variables (P > 0.05). No significant difference in MVC was observed for any group after the 16-week period (P = 0.167). The post-training increases in average Wpeak (7%) and nu p,peak (6%) during the cycle tests were each significantly different from the control group response (P = 0.018 and P = 0.008, respectively). It is concluded that 16 weeks of isokinetic strength training of the knee extensors is able to significantly improve nu p, peak and Wpeak during spring cycling, an activity which demands considerable involvement of the trained muscle group but with its own distinct pattern of coordination.     

Prolonged intermittent high intensity exercise impairs neuromuscular performance of the knee flexors

This study investigated the effect of prolonged intermittent high intensity exercise upon the isokinetic leg strength and electromechanical delay of the knee flexors. Seven male collegiate soccer players were exposed to: (i) a prolonged intermittent high intensity exercise task (PIHIET) which required subjects to complete a single-leg pedalling task, with the preferred limb, (75 rpm for all constant-load portions of the task) consisting of 48 × 1.8 minute cycles of exercise, and (ii) a control task consisting of no exercise. Pre-, mid- and post-PIHIET gravity corrected indices of knee flexion angle-specific torque (0.44 rad knee flexion (AST); 0 rad = full knee extension; [1.05 rad · s⁻¹]) were made for both intervention and control limbs. Electromechanical delay (EMD) of the m. biceps femoris during supine knee flexion movements was evaluated in the preferred leg on both intervention and control days. Repeated measures ANOVAs revealed significant condition (intervention; control) by time (pre; mid; post) interactions for both knee flexor AST (F_[2,12] = 4.8; p<0.03) and EMD (F_[2,12] = 4.1; p<0.05). AST was observed to decrease by 16% and EMD increase by 30% pre to post intervention. These observations suggest an impairment of neuromuscular control and the ability to maintain force generation in the knee flexors, near the extremes of the range of motion during prolonged intermittent high-intensity exercise activities. Changes of this magnitude may pose a threat to the integrity of the knee joint. Accepted: 6 January 1998     

Human skeletal muscle fibre types and force: velocity properties

It has been reported that there is a relationship between power output and fibre type distribution in mixed muscle. The strength of this relationship is greater in the range of 3–8 rad · s^–1 during knee extension compared to slower or faster angular knee extensor speeds. A mathematical model of the force: velocity properties of muscle with various combinations of fast- and slow-twitch fibres may provide insight into why specific velocities may give better predictions of fibre type distribution. In this paper, a mathematical model of the force: velocity relationship for mixed muscle is presented. This model demonstrates that peak power and optimal velocity should be predictive of fibre distribution and that the greatest fibre type discrimination in human knee extensor muscles should occur with measurement of power output at an angular velocity just greater than 7 rad · s^–1. Measurements of torque: angular velocity relationships for knee extension on an isokinetic dynamometer and fibre type distribution in biopsies of vastus lateralis muscles were made on 31 subjects. Peak power and optimal velocity were determined in three ways: (1) direct measurement, (2) linear regression, and (3) fitting to the Hill equation. Estimation of peak power and optimal velocity using the Hill equation gave the best correlation with fibre type distribution (r > 0.5 for peak power or optimal velocity and percentage of fast-twitch fibres). The results of this study confirm that prediction of fibre type distribution is facilitated by measurement of peak power at optimal velocity and that fitting of the data to the Hill equation is a suitable method for evaluation of these parameters.     

The reliability of electromechanical delay and torque during isometric and concentric isokinetic contractions

The purpose of this investigation was to examine the intra-subject reliability of electromechanical delay (EMD) and torque of the dominant and non-dominant elbow flexors during isometric and isokinetic muscle contractions repeated over five consecutive days. Eleven volunteers that were unfamiliar with isokinetic dynamometry participated in this study and were asked to attend the laboratory on five consecutive days. An isokinetic dynamometer was used to exercise the elbow flexors under isometric, slow (60° s⁻¹) and fast (210° s⁻¹) isokinetic conditions; surface electromyography was recorded from the belly of biceps brachii and the signal was synchronised with the dynamometer to determine EMD. Intra-subject reliability for all measures was good (CV range, 3.1–6.5%) with no discernable difference between the dominant and non-dominant arms during isometric and isokinetic conditions. In addition, there was little difference in EMD and torque variability between the dominant and non-dominant arms which may have applications for clinicians and future research design when monitoring and investigating human muscle function. These data provide researchers and clinicians with an indication of the magnitude of change that is required to elucidate the presence of a meaningful change to muscle function in the elbow flexors.     

Reproducibility of isokinetic leg strength and endurance characteristics of adult men and women

Day-to-day variability and single-measurement reliability of selected isokinetic knee extension-flexion strength and endurance indices were assessed in 10 adult men and 8 adult women. On three occasions separated by at least 5 days, the subjects completed 4 reciprocal maximal voluntary contractions (MVC) at different angular velocities (1.05 rad.s-1 and 3.14 rad.s-1). The men also completed a muscular endurance test consisting of 30 reciprocal, MVC at 3.14 rad.s-1. Coefficient of variation, intra-class correlation coefficient and standard error of single-measurement scores support the continued use of gravity corrected peak torque (PT) and average peak torque (APT) as indices of isokinetic leg strength. Similarly, gravity corrected APT and total work should be the recommended indices of isokinetic leg muscular endurance in men. The results suggest that these isokinetic indices must be assessed using multiple day-to-day trial protocols adequately to describe performance capacity. Composite indices such as the ratio of Knee flexion to extension PT and fatigue measurements offer considerably reduced reliability and a greater potential for misinterpretation. The reliability of knee extension indices generally exceeds that of flexion indices. Similar variability and reproducibility of responses were observed between men and women and between reciprocal contractions performed at angular velocities of 1.05 rad.s-1 and 3.14 rad.s-1.     

Validity and reliability of isometric,isokinetic and isoinertial modalities for the assessment of quadriceps muscle strength in patients with total knee arthroplasty

Reliability of isometric, isokinetic and isoinertial modalities for quadriceps strength evaluation, and the relation between quadriceps strength and physical function was investigated in 29 total knee arthroplasty (TKA) patients, with an average age of 63 years. Isometric maximal voluntary contraction torque, isokinetic peak torque, and isoinertial one-repetition maximum load of the involved and uninvolved quadriceps were evaluated as well as objective (walking parameters) and subjective physical function (WOMAC). Reliability was good and comparable for the isometric, isokinetic, and isoinertial strength outcomes on both sides (intraclass correlation coefficient range: 0.947–0.966; standard error of measurement range: 5.1–9.3%). Involved quadriceps strength was significantly correlated to walking speed (r range: 0.641–0.710), step length (r range: 0.685–0.820) and WOMAC function (r range: 0.575–0.663), independent from the modality (P < 0.05). Uninvolved quadriceps strength was also significantly correlated to walking speed (r range: 0.413–0.539), step length (r range: 0.514–0.608) and WOMAC function (r range: 0.374–0.554) (P < 0.05), except for WOMAC function/isokinetic peak torque (P > 0.05). In conclusion, isometric, isokinetic, and isoinertial modalities ensure valid and reliable assessment of quadriceps muscle strength in TKA patients.     

Frequency content asymmetry of the isokinetic curve between ACL deficient and healthy knee

The torque-time curve patterns of concentric isokinetic knee extension in anterior cruciate ligament (ACL) deficient patients usually present mid-range irregularities associated with the level of anterior tibial translation. The purpose of this study was to compare the smoothness in isokinetic torque production between the ACL deficient and the healthy knee. Thirty ACL deficient soccer players performed bilaterally five trials of maximum concentric knee extension-flexion at 60 degrees /s on a Biodex dynamometer. The three middle trials (a total of six curves) were retained and submitted to further data processing. Maximum frequency values contained within the 90%, 95% and 99% level of the signal power were calculated for each extension and flexion curve. The frequency content of the ACL deficient side proved to be statistically higher compared to the intact side at all levels of the power spectrum. The percentage differences in the frequency content were 18.8%, 10.6% and 40.0% for knee extension, and 49.5%, 24.5% and 16.3% for knee flexion, for the respective power levels. This indicated higher oscillations and, therefore, more unstable mechanical output of the injured knee. An overall biological interpretation of the present results is based on the notion that disturbed motion is generally connected to poor level of joint functionality.     

The distribution of capillaries in the somatic musculature of two vertebrate types with particular reference to teleost fish

The distribution of capillaries in teleost and rat striated muscles was investigated using a number of different methods. A new method for directly viewing capillaries was developed. Teleost white muscle has a capillary: fibre (C:F) ratio of between 0.2 and 0.3; and 0.6 to 1.0 peripheral capillaries per muscle fibre. 26-49% of fibres had no peripheral capillaries. Values for the rat gastrocnemius were 1.2, 2.6 and 4.8% respectively which compares well with literature values. Flathead red muscle had a C:F ratio of between 1.9 and 2.5; and between 5.3 and 6.6 peripheral capillaries per muscle fibre depending on the method used. Values for rat soleus were 1.8 and 4.1 respectively. Teleost pink fibres had an intermediate number of capillaries. Rat striated muscle, particularly the gastrocnemius, was found to be heterogeneous with respect to the distribution of capillaries. Flathead red muscle was homogeneous whilst teleost white muscle was only slightly variable. Flathead red muscle fibres are well suppled with subsarcolemmal mitochondria. These show a clumped distribution corresponding to the position of capillaries. In contrast teleost white fibres are almost totally devoid of these and all other mitochondria. No differences were observed in the vascularisation of either muscle type along the length of the fish. The results are discussed in relation to the division of labour between fibre types during swimming.     

The relationship between cell and population growth

A steady-state model of cell volume frequency distribution using the method of Williams (1971) is derived. Results are compared to a Monte Carlo simulation of cell growth and division. It is suggested that the Monte Carlo method might be of value for investigating cell and population properties for which analytic methods are not currently available.     

Maximal mechanical power output and capacity of cyclists and young adults

The maximal average power output (Wmax) has been examined in 10 male students, 22 pursuit and 12 sprint cyclists. In 24 of these subjects (8 students, 10 pursuit and 6 sprint cyclists), estimates of the maximal capacity (Wcap) of the short-term anaerobic energy yielding processes were made. The results show that the sprinters had a higher absolute Wmax (1241 +/- 266 W) and Wcap (16.7 +/- 4.9 kJ) than either the students (1019 +/- 183 W, 14.7 +/- 2.8 kJ) or the pursuit cyclists (962 +/- 206 W, 14.0 +/- 2.9 kJ). However, the differences were removed when the values were standardised for muscle size. In the sprinters the Wmax was attained at an optimal pedal frequency Vopt of 132 +/- 3 min-1 and the estimated maximal velocity of pedalling (V0) was 262 +/- 8 min-1. The comparable figures in the students and pursuit cyclists were 118 +/- 8 min-1, 235 +/- 17 min-1 and 122 +/- 6 min-1, 242 +/- 12 min-1 respectively. The coefficient of variation of duplicate measurements of Wcap was found to be +/- 9%. Using data of Wilkie (1968) for muscle phosphagen and glycolytic stores (27 mmol.kg-1), it was estimated that the probable efficiency of the anaerobic processes during maximal cycling was 0.22. It was concluded that Wmax and Wcap are largely determined by body size and muscularity. The efficiency of anaerobiosis appears to be of the same order of magnitude as found for oxidative work.     

Testing ecological interactions between Gnomoniopsis castaneae and Dryocosmus kuriphilus

An emerging nut rot of chestnut caused by the fungus Gnomoniopsis castaneae was reported soon after the invasion of the exotic gall wasp Dryocosmus kuriphilus in Italy. The goal of this work was to assess the association between the spread of the fungal pathogen and the infestation of the pest by testing if:I) viable inoculum of G. castaneae can be carried by adults of D. kuriphilus;II) the fungal colonization is related to the number of adults inhabiting the galls;III) the fungal colonization of chestnut buds and the oviposition are associated.Fungal isolations and PCR-based molecular assays were performed on 323 chestnut galls and on their emerging D. kuriphilus adults, whose number was compared between galls colonized and not colonized by G. castaneae. To test the association between fungal colonization and oviposition, Monte Carlo simulations assuming different scenarios of ecological interactions were carried out and validated through isolation trials performed on 597 and 688 chestnut buds collected before and after oviposition, respectively.Although DNA of G. castaneae was detected in a sample of 40% of the adults developed in colonized galls, the fungus could never be isolated from insects, suggesting that the pest is an unlikely vector of viable inoculum.On average, the emerging adults were significantly more abundant from galls colonized by G. castaneae than from not colonized ones (3.76 vs. 2.54, P < 0.05), indicating a possible fungus/pest synergy.The simulations implying no interaction between G. castaneae and D. kuriphilus after fungal colonization were confirmed as the most likely. In fact, G. castaneae was present in 33.8% of the buds before oviposition, while no association was detected between fungal colonization and oviposition (odds ratio 0.98, 0.71–1.33 95% CI). These findings suggest that the fungus/pest synergy is asymmetrically favorable to the pest and occurs after oviposition.     

Body composition and isokinetic strength of professional Sumo wrestlers

The purpose of this study was to investigate the profiles of body composition and force generation capability in professional Sumo wrestlers. The subjects were 23 professional Sumo wrestlers [mean age 22.0 (SEM 1.2) years] including those ranked in the lower- (Jonokuchi, n = 10), middle- (Sandanme, n = 8) and higher-division (Makuuchi, n = 5), 22 weight-classified athletes [5 judo athletes, 5 wrestlers, and 12 weight lifters, mean age 20.7 (SEM 0.7) years], and 21 untrained men [mean age 20.1 (SEM 0.2) years]. In the Sumo wrestlers, body mass ranged between 77.0 and 150.0 kg, body mass index between 25.9 and 44.5 kg · m⁻², relative fat mass (%FM) between 11.9 and 37.0%, and fat-free mass (FFM) between 59.1 and 107.6 kg. The Sumo wrestlers showed significantly higher %FM and smaller elbow and knee extensor cross-sectional areas (CSA) than the weight-classified athletes who weighed from 90.4 kg to 133.2 kg. Moreover, isokinetic forces in the flexion and extension of elbow and knee joints, respectively, at three constant velocities of 1.05, 3.14 and 5.24 rad · s⁻¹ were significantly lower in the Sumo wrestlers than in the weight-classified athletes and untrained subjects when expressed per unit of body mass. However, the median value of FFM relative to body height in the higher-division Sumo wrestlers was ranked high in the range of magnitude among those reported previously in the literature for heavyweight athletes. Moreover, the results on the comparisons within the Sumo wrestlers showed that not only FFM but also force generation capability, expressed both as an absolute term and as a value relative to both body mass and muscle CSA, might be factors contributing to the performance of Sumo wrestlers. Accepted: 18 August 1997     

Genome analyses and modelling the relationships between coding density, recombination rate and chromosome length

In the human genomes, recombination frequency between homologous chromosomes during meiosis is highly correlated with their physical length while it differs significantly when their coding density is considered. Furthermore, it has been observed that the recombination events are distributed unevenly along the chromosomes. We have found that many of such recombination properties can be predicted by computer simulations of population evolution based on the Monte Carlo methods. For example, these simulations have shown that the probability of acceptance of the recombination events by selection is higher at the ends of chromosomes and lower in their middle parts. The regions of high coding density are more prone to enter the strategy of haplotype complementation and to form clusters of genes, which are “recombination deserts”. The phenomenon of switching in-between the purifying selection and haplotype complementation has a phase transition character, and many relations between the effective population size, coding density, chromosome size and recombination frequency are those of the power law type.     

The measurement of force/velocity relationships of fresh and fatigued human adductor pollicis muscle

The purpose of the study was to obtain force/velocity relationships for electrically stimulated (80 Hz) human adductor pollicis muscle (n = 6) and to quantify the effects of fatigue. There are two major problems of studying human muscle in situ; the first is the contribution of the series elastic component, and the second is a loss of force consequent upon the extent of loaded shortening. These problems were tackled in two ways. Records obtained from isokinetic releases from maximal isometric tetani showed a late linear phase of force decline, and this was extrapolated back to the time of release to obtain measures of instantaneous force. This method gave usable data up to velocities of shortening equivalent to approximately one-third of maximal velocity. An alternative procedure (short activation, SA) allowed the muscle to begin shortening when isometric force reached a value that could be sustained during shortening (essentially an isotonic protocol). At low velocities both protocols gave very similar data (r2 = 0.96), but for high velocities only the SA procedure could be used. Results obtained using the SA protocol in fresh muscle were compared to those for muscle that had been fatigued by 25 s of ischaemic isometric contractions, induced by electrical stimulation at the ulnar nerve. Fatigue resulted in a decrease of isometric force [to 69 (3)%], an increase in half-relaxation time [to 431 (10)%], and decreases in maximal shortening velocity [to 77 (8)%] and power [to 42 (5)%]. These are the first data for human skeletal muscle to show convincingly that during acute fatigue, power is reduced as a consequence of both the loss of force and slowing of the contractile speed.     

Atomistic simulations of the effects of polyglutamine chain length and solvent quality on conformational equilibria and spontaneous homodimerization

Aggregation of expanded polyglutamine tracts is associated with nine different neurodegenerative diseases, including Huntington's disease. Experiments and computer simulations have demonstrated that monomeric forms of polyglutamine molecules sample heterogeneous sets of collapsed structures in water. The current work focuses on a mechanistic characterization of polyglutamine homodimerization as a function of chain length and temperature. These studies were carried out using molecular simulations based on a recently developed continuum solvation model that was designed for studying conformational and binding equilibria of intrinsically disordered molecules such as polyglutamine systems. The main results are as follows: Polyglutamine molecules form disordered, collapsed globules in aqueous solution. These molecules spontaneously associate at conditions approaching those of typical in vitro experiments for chains of length N ≥ 15. The spontaneity of these homotypic associations increases with increasing chain length and decreases with increasing temperature. Similar and generic driving forces govern both collapse and spontaneous homodimerization of polyglutamine in aqueous milieus. Collapse and dimerization maximize self-interactions and reduce the interface between polyglutamine molecules and the surrounding solvent. Other than these generic considerations, there do not appear to be any specific structural requirements for either chain collapse or chain dimerization; that is, both collapse and dimerization are nonspecific in that disordered globules form disordered dimers. In fact, it is shown that the driving force for intermolecular associations is governed by spontaneous conformational fluctuations within monomeric polyglutamine. These results suggest that polyglutamine aggregation is unlikely to follow a homogeneous nucleation mechanism with the monomer as the critical nucleus. Instead, the results support the formation of disordered, non-β-sheet-like soluble molten oligomers as early intermediates—a proposal that is congruent with recent experimental data.     

Specific effects of eccentric and concentric training on muscle strength and morphology in humans

The purpose of this study was to compare pure eccentric and concentric isokinetic training with respect to their possible specificity in the adaptation of strength and morphology of the knee extensor muscles. Ten moderately trained male physical education students were divided into groups undertaking eccentric (ETG) and concentric (CTG) training. They performed 10 weeks of maximal isokinetic (90 degrees x s(-1)) training of the left leg, 4x10 repetitions - three times a week, followed by a second 10-week period of similar training of the right-leg. Mean eccentric and concentric peak torques increased by 18% and 2% for ETG and by 10% and 14% for CTG, respectively. The highest increase in peak torque occurred in the eccentric 90 degrees x s(-1) test for ETG (35%) whereas in CTG strength gains ranged 8%-15% at velocities equal or lower than the training velocity. Significant increases in strength were observed in the untrained contra-lateral leg only at the velocity and mode used in ipsilateral training. Cross-sectional area of the quadriceps muscle increased 3%-4% with training in both groups, reaching statistical significance only in ETG. No major changes in muscle fibre composition or areas were detected in biopsies from the vastus lateralis muscle for either leg or training group. In conclusion, effects of eccentric training on muscle strength appeared to be more mode and speed specific than corresponding concentric training. Only minor adaptations in gross muscle morphology indicated that other factors, such as changes in neural activation patterns, were causing the specific training-induced gains in muscle strength.     

Muscle metabolic profile and oxygen transport capacity as determinants of aerobic and anaerobic thresholds

Aerobic and anaerobic thresholds determined by different methods in repeated exercise tests were correlated with cardiorespiratory variables and variables of muscle metabolic profile in 33 men aged 20-50 years. Aerobic threshold was determined from blood lactate, ventilation, and respiratory gas exchange by two methods (AerT1 and AerT2) and anaerobic threshold from venous lactate (AnTLa), from ventilation and gas exchange (AnTr) and by using the criterion of 4 mmol.1(-1) of venous lactate (AnT4mmol). In addition to ordinary correlative analyses, applications of LISREL models were used. The 8 explanatory variables chosen for the regression analyses were height, relative heart volume, relative diffusing capacity of the lung, muscle fiber composition, citrate synthase (CS) and succinate dehydrogenase activities, the lactate dehydrogenase--CS ratio, and age. They explained 58% of the variation in AerT1, 73.5% that of AerT2, 71% that of AnTr, 74.5% that of AnTLa, and 67.5% that of AnT4mmol.AerT and AnT alone explained 77% of the variation in each other. Both AerT and AnT were determined mainly by a muscle metabolic profile, with the CS activity of vastus lateralis as the strongest determinant. The factor 'submaximal endurance' which was measured with AerT and AnT seemed to be slightly more closely connected to 'muscle metabolic profile' than was 'maximal aerobic power' (= VO2max), but both also correlated strongly with each other (r = 0.92).     

Two types of linkage between codon usage and gene-expression levels

The relation between codon usage and gene-expression levels is an intensively investigated and discussed topic in the field of molecular evolution. We statistically analyzed 25 Escherichia coli gene sequences by a new classification of synonymous codons and found that (i) there are two distinct types of linkage between codon usage and gene-expression levels in E. coli, and (ii) one of the two kinds of codon preferences (the codon preference concerned with interaction of GC/AT choice at three codon positions) is observed significantly in weakly expressed genes.