Acute Effects of Low-intensity Isometric Exercise at Long and Short Muscle-tendon Unit Lengths.

Low-intensity training at long muscle-tendon unit lengths with a greater passive force may cause muscle swelling, which may be related to hypertrophy, even if the active force production is lower than that at short muscle-tendon unit lengths. This study compared muscle swelling after low-intensity torque-matched isometric exercises at long and short muscle-tendon unit lengths. Twenty-six volunteers performed isometric knee flexion exercises (30% of maximal voluntary contraction× 5 seconds×10 repetitions×9 sets) either at long or short lengths of the hamstrings (90° hip flexion and 30° knee flexion, or 90° hip and knee flexion, respectively). Active torque was calculated by subtracting passive torque from the total torque generated during exercise. Swelling-induced changes in cross-sectional area was assessed before and after exercise using ultrasonography. There was no between-group difference in the total torque during exercise; however, the active torque was significantly lower in the group trained at long than in the group trained at short muscle-tendon unit lengths. Muscle swelling occurred in both groups. The results suggest that exercise at long muscle-tendon unit lengths can cause similar muscle swelling as exercise at short muscle-tendon unit lengths, even in cases where active torque production is lower than that at short lengths.

Effects of Low-Intensity Torque-Matched Isometric Training at Long and Short Muscle Lengths of the Hamstrings on Muscle Strength and Hypertrophy: A Randomized Controlled Study.

ABSTRACT: Nakao, S, Ikezoe, T, Taniguchi, M, Motomura, Y, Hirono, T, Nojiri, S, Hayashi, R, Tanaka, H, and Ichihashi, N. Effects of low-intensity torque-matched isometric training at long and short muscle lengths of the hamstrings on muscle strength and hypertrophy: A randomized controlled study. J Strength Cond Res 37(10): 1978-1984, 2023-This study investigated the effects of low-intensity torque-matched isometric training on muscle hypertrophy and strengthening at long (LL) and short muscle lengths (SL). Twenty-eight young subjects completed an 8-week hamstring isometric training program (30% of maximal voluntary contraction (MVC) × 5 s × 20 repetitions × 5 sets × 3 times/week) at 30° knee flexion (LL) or 90° knee flexion (SL). The cross-sectional area (CSA) of the hamstrings and MVC were measured before and after the intervention. The active torque because of muscle contraction was calculated by subtracting the passive torque at rest from the total torque (30% MVC). The active torque was significantly lower in the LL training group than in the SL training group (p < 0.01), whereas there was no between-group difference in total torque during training. For CSA and MVC at 30° knee flexion, the split-plot analysis of variance (ANOVA) showed no significant time × group interaction; however, it did show a significant main effect of time (p < 0.05), indicating a significant increase after training intervention. As for MVC at 90° knee flexion, there was a significant time × group interaction (p < 0.05) and a significant simple main effect of time in both the LL (p < 0.01; Cohen's d effect size [ES] = 0.36) and SL (p < 0.01; ES = 0.64) training groups. Therefore, low-intensity isometric training at LL can induce hypertrophy and strengthening, even in cases where the active torque production is lower than that at SL, whereas the training at SL may be more effective for muscle strengthening at SL.

Effects of trunk lean and foot lift exercises in sitting position on abdominal muscle activity and the contribution rate of transversus abdominis.

PURPOSE: Abdominal hollowing exercise has been recommended to improve trunk stability. Trunk lean and foot lift exercises while sitting may easily promote abdominal muscle activity even in people who cannot perform abdominal hollowing consciously. The purpose of the present study was to examine the changes in abdominal muscle activity and contribution rate of the transversus abdominis muscle (TrA) when leaning the trunk and lifting the foot during sitting. METHODS: The muscle stiffnesses (indicators of muscle activity) of the right rectus abdominis, external oblique, internal oblique, and TrA of 14 healthy men were measured during abdominal hollowing and the following nine sitting tasks: reference posture, 15° and maximal posterior trunk lean, 20° and maximal ipsilateral and contralateral trunk lean, and ipsilateral and contralateral foot lift. The TrA contribution rate was calculated by dividing the TrA stiffness by the sum of the abdominal muscles' stiffnesses. RESULTS: The TrA stiffness was significantly higher in abdominal hollowing than in reference posture, posterior and ipsilateral trunk lean, and ipsilateral foot lift, but not higher than in contralateral trunk lean and contralateral foot lift. There was no significant difference in the TrA contribution rates between abdominal hollowing and ipsilateral or contralateral foot lift. CONCLUSION: The contralateral trunk lean or contralateral foot lift could enhance TrA activity for people who cannot perform abdominal hollowing consciously. The contralateral foot lift could particularly be beneficial to obtain selective activity of TrA.

Chronic Effects of a Static Stretching Program on Hamstring Strength.

ABSTRACT: Nakao, S, Ikezoe, T, Nakamura, M, Umegaki, H, Fujita, K, Umehara, J, Kobayashi, T, Ibuki, S, and Ichihashi, N. Chronic effects of a static stretching program on hamstring strength. J Strength Cond Res 35(7): 1924-1929, 2021-This study investigated the effects of a 4-week static stretching (SS) program on isokinetic and isometric knee flexor peak torque and angle of peak torque. Thirty healthy men (age, 22.7 ± 2.2 years) were randomized to receive either of the following: (a) a 4-week stretch intervention for the hamstrings (SS intervention group; n = 15) or (b) no intervention (control group; n = 15). The maximum pain-free knee angle, passive stiffness, which was determined by a slope of torque-angle curve, isometric and isokinetic (at 60°·s-1 and 180°·s-1) peak torque, and angle of peak torque for knee flexors were measured before and after 4 weeks. After 4 weeks, passive stiffness decreased significantly in the intervention group. There were no significant changes in isometric and isokinetic (neither at 60°·s-1 nor at 180°·s-1) peak torque, or angle of peak torque at 180°·s-1. A significantly increased peak extension angle at 60°·s-1 was observed in the intervention group. These results suggest that SS intervention is effective for decreasing musculotendinous unit stiffness of the hamstrings and that an SS program influences the angle of peak torque, whereas no significant changes occur in peak torque. Because a previous study suggests that angle of peak torque is associated with hamstring strain injuries, the results of this study would be helpful when considering the training program for preventing or treating hamstring strain injuries.

Effect of different knee flexion angles with a constant hip and knee torque on the muscle forces and neuromuscular activities of hamstrings and gluteus maximus muscles.

PURPOSE: This study examined the effect of different knee flexion angles with a constant hip and knee torque on the muscle force and neuromuscular activity of the hamstrings and gluteus maximus. METHODS: Twenty healthy males lay in prone position and held their lower limb with hip flexion at 45° and knee flexion at either 10° or 80°. At these angles, the hip and knee torques are identical. Under three load conditions: passive (referred to as Unloaded), active (Loaded), and active with 3-kg weight added to the shank (Loaded + 3 kg), the muscle stiffness (i.e., an indicator of muscle force) and neuromuscular activity of the hamstrings and gluteus maximus were measured using shear wave elastography and surface electromyography. RESULTS: The muscle stiffness and neuromuscular activity of the hamstrings and gluteus maximus increased significantly with the load. Muscle stiffness in the hamstrings was significantly lower at knee flexion of 80° than at 10° for Unloaded, but not for either Loaded or Loaded + 3 kg. The neuromuscular activity of the hamstrings was significantly greater at knee flexion of 80° than at 10° for both Loaded and Loaded + 3 kg. The muscle stiffness or neuromuscular activity of the gluteus maximus showed no significant differences between knee angles. CONCLUSIONS: When the passive force in the hamstrings decreases with knee flexion, sufficient muscle force to maintain the hip and knee torques against an external load is generated by preferentially increasing the neuromuscular activity of the hamstrings, rather than increasing the synergetic muscle force.

The effects of a 4-week static stretching programme on the individual muscles comprising the hamstrings.

The aims of this study were to investigate the effects of a 4-week intervention of static stretching (SS) on muscle hardness of the semitendinosus (ST), semimembranosus (SM) and biceps femoris (BF) muscles. Shear elastic modulus was measured by using ultrasound shear wave elastography as the index of muscle hardness. Thirty healthy men (age 22.7 ± 2.2 years) volunteered for this study and were randomly assigned to the SS intervention group (n = 15) or the control group (n = 15). Participants in the SS intervention group received a 4-week stretch intervention for the hamstrings of their dominant leg. Shear elastic moduli of the hamstrings were measured at initial evaluation and after 4 weeks in both groups at a determined angle. In all muscles, the shear elastic modulus decreased significantly after SS intervention. The percentage change in the shear elastic modulus from the value at initial evaluation to after 4 weeks intervention was greatest in the SM. These results suggest that SS intervention has chronic effects on reducing hardness of the hamstring muscle components, especially the SM muscle.

Effects of abdominal hollowing and bracing on each intervertebral angle during quadruped upper and lower extremity lift: Three-dimensional motion analysis of the spine.

To investigate the effects of intentionally minimizing spinal motion and abdominal muscle contractions on intervertebral angles during quadruped upper and lower extremity lift (QULEL). Fifteen healthy men performed the QULEL under four conditions: without any special instructions (basic), with the intention to minimize spinal motion (intentional), with abdominal bracing (bracing), and with abdominal hollowing (hollowing). Each intervertebral angle was calculated from the local coordinate system using the marker data obtained from a motion capture system. Shear moduli, as indicators of the activities of the right transversus abdominis (TrA), internal and external oblique, and rectus abdominis muscles, were assessed using shear wave elastography during QULEL. One-way repeated-measures analysis of variance and multiple comparisons among conditions were used to compare each shear modulus of the abdominal muscle and the changes in thoracic kyphosis (Th1-12), lumbar lordosis (L1-5), and lumbar intervertebral angles from the quadruped position to QULEL. The significance level was set at P < 0.05. Changes in lumbar lordosis and L2/L3 and L3/L4 extension angles were significantly lower under hollowing than under other conditions (effect size ηG2: lumbar lordosis, 0.068; L2/L3, 0.072; L3/L4, 0.043). The change in the L1/L2 extension angle significantly decreased in bracing and hollowing compared with the basic (ηG2 = 0.070). Only the TrA shear modulus significantly increased in bracing and hollowing compared with the basic (ηG2 = 0.146). Abdominal hollowing during the QULEL increased TrA activity and suppressed lumbar extension, except at L4/L5, and may be more effective as a rehabilitation exercise for controlling spinal motion.

Changes in echo intensity of the gastrocnemius muscle with passive ankle dorsiflexion: can echo intensity be used to assess muscle elongation?

Introduction: While shear modulus has been used as an index of muscle elongation, high costs prevent its general adoption. A more general indicator that can quantify muscle elongation non-invasively is needed to develop effective methods for stretching each muscle. The purpose of this study was to determine whether the echo intensity of the muscle changes with muscle elongation compared with the shear modulus. Methods: Sixteen healthy males (24.1 ± 2.8 years) participated in the study. Shear modulus and echo intensity of the medial gastrocnemius were assessed at 20° and 10° of ankle plantar-flexion, and 0°, 10°, and 20° of ankle dorsiflexion (presented as -20°, -10°, and 0°, +10°, +20°, respectively). Shear modulus was measured using ultrasound shear wave elastography. The echo intensity was quantified using the average grayscale value of a given region of interest (RoI) in longitudinal and transverse B-mode images. Grayscale analysis was performed using two RoIs: one which included as much of the muscle as possible (maximum RoI), and a rectangular one whose size and depth was identical for all images (rectangular RoI). Pearson's correlation coefficients between either the shear modulus or echo intensity and the ankle angle and between the echo intensity and shear modulus were calculated separately for each participant. Results: Average Pearson's correlation coefficient between shear modulus and ankle angle of the participants was 0.904. The average Pearson's correlation coefficients between the echo intensity and ankle angle were estimated to be 0.797 and 0.222 for the maximum RoI and 0.698 and 0.323 for the rectangular RoI in the longitudinal and transverse images, respectively. The average Pearson's correlation coefficients between the echo intensity and shear modulus were 0.684 and 0.514 for the maximum RoI, and 0.611 and 0.409 for rectangular RoI in the longitudinal and transverse images, respectively. Discussion: The results indicate that the echo intensity in the longitudinal image of the gastrocnemius, especially when assessed using the maximum RoI, increased with muscle elongation by passive ankle dorsiflexion. Therefore, assessment of the echo intensity using the maximum RoI in the longitudinal image might be useful for quantifying the muscle elongation.

Investigation of Endotoxin Positivity in Ascites at the Time of Cell-free and Concentrated Ascites Re-infusion Therapy.

Objective The presence of endotoxin (ET) in ascites at the time of cell-free and concentrated ascites reinfusion therapy (CART) is generally assessed in patients with infectious disease status, but the exact rate of ET positivity in ascites for patients treated with CART is unknown. Methods We evaluated ET levels in ascites at the time of CART, regardless of the presence of infectious symptoms. The analysis was performed for 529 cases in 183 patients in whom ET levels in ascites were measured at 2 time points (pre- and post-processing). Results ET in ascites was positive in 8 of 529 cases. In the positive cases, the ET level after CART was significantly decreased. ET-positive patients had a significantly higher white blood cell count, neutrophil count, and serum CRP level before CART than ET-negative patients. Conclusion Collectively, our data suggest that ET may be present in ascites, regardless of the infectious symptoms, especially in patients with a high white blood cell count, neutrophil count, and serum CRP level. Although the ET level in the re-infusion ascites seems to be decreased by CART, the possibility of endotoxemia after CART should be considered for such patients.

Properties of triceps surae and Achilles tendon in forefoot and non-forefoot strike runners.

BACKGROUND: The purpose of this study was to compare the properties of the triceps surae and Achilles tendon of forefoot strike runners and non-forefoot strike runners. METHODS: Fourteen male college forefoot strike runners and 11 non-forefoot strike runners participated in this study. The shear elastic moduli and cross-sectional areas of the medial and lateral gastrocnemius, soleus, and Achilles tendon were measured. Passive ankle joint stiffness, maximum voluntary isometric contraction on plantar flexion and Rebound Jump Index were also measured. RESULTS: Forefoot strike runners had higher shear elastic moduli of the medial and lateral gastrocnemius, higher passive ankle joint stiffness, higher Rebound Jump Index, and greater cross-sectional area of the gastrocnemius and Achilles tendon than non-forefoot strike runners. CONCLUSIONS: These findings suggest that forefoot strike runners have different mechanical properties in the gastrocnemius muscles, and different morphological properties in the Achilles tendon and gastrocnemius muscles, compared with non-forefoot strike runners.

Effective stretching position of the coracobrachialis muscle.

An increase in the stiffness of the coracobrachialis muscle can restrain proper movement of the glenohumeral joint and scapula during arm elevation. Therefore, muscle stiffness should be reduced through stretching. The aim of this study was to determine the effective stretching position of the coracobrachialis muscle using ultrasound shear wave elastography imaging to evaluate the stiffness of individual muscles. Eighteen healthy young men participated in this study. The shear modulus of the coracobrachialis muscle was measured at the following eight shoulder positions: i) 20° abduction (Rest), ii) maximal external rotation at 90° abduction (ER2), iii) maximal internal rotation at 90° abduction (IR2), iv) maximal flexion (Flex), v) maximal extension (Ext), vi) maximal horizontal abduction at 90° abduction (Hab), vii) maximal horizontal abduction and maximal external rotation at 90° abduction (HabER), and viii) maximal horizontal abduction and maximal internal rotation at 90° abduction (HabIR). The shear modulus in each position was compared with that of Rest using the Wilcoxon signed-rank test, and a multiple comparison test was performed among the positions that exhibited significant difference. The shear modulus of all stretching positions was significantly higher than that of Rest, except for Flex. Moreover, the shear moduli of IR2, Ext, Hab, HabER, and HabIR were significantly higher than that of ER2. The shear modulus of Ext was significantly higher than that of HabIR. The coracobrachialis muscle could be stretched effectively at IR2, Ext, Hab, HabER, and HabIR. Among these positions, Ext, Hab, and HabER are recommended for clinical settings.

Epimuscular myofascial force transmission from biarticular rectus femoris elongation increases shear modulus of monoarticular quadriceps muscles.

This study aimed to examine the effect of rectus femoris (RF) elongation with passive hip angle change on the shear moduli of the vastus lateralis (VL) and medialis (VM) to verify whether Epimuscular myofascial force transmission (EMFT) occurs in the human quadriceps. Fourteen healthy men participated in this study. The shear moduli of the RF, VL, and VM were measured in four hip positions: flexion (Flex), extension and abduction (Ext-Abd), extension (Ext), and extension and adduction (Ext-Add). As the behavior of shear moduli may differ depending on the parts of the vasti muscles, we measured the medial and lateral parts of the VL (Medial-VL and Lateral-VL) and VM (Medial-VM and Lateral-VM). The shear moduli at the Ext and Ext-Add positions were higher than at the Flex position in the RF, VL, and VM. The shear moduli during Ext and Ext-Add were higher than at the Ext-Abd in the RF, VL, and Lateral-VM. Moreover, the shear modulus of the Lateral-VM was higher than of the Medial-VM (Flex: 8.5% higher; Ext-Abd: 15.6%; Ext: 30.2%; Ext-Add: 32.6%). The shear moduli of the VL and VM, which are monoarticular muscles of the knee, increased with passive hip extension or adduction with extension, even when the knee angle was kept constant. The results suggest that EMFT occurs in the quadriceps, and EMFT had a great impact in the Lateral-VM, which is anatomically adjacent to the RF, but it had little effect in the Medial-VM, which is further away from the RF.

Age-related changes in gait speeds and asymmetry during circular gait and straight-line gait in older individuals aged 60-79 years.

AIM: The present study aimed to investigate the age-related changes in gait speeds and asymmetry during circular and straight-line gaits among older adults aged 60-79 years. METHODS: The study included 391 community-dwelling older adults aged >60 years, who participated in the Nagahama cohort study. They were assigned to four age groups: 60-64 years (early 60s), 65-69 years (late 60s), 70-74 years (early 70s) and 75-79 years (late 70s). For the circular gait test, the time required to walk twice around a 1-m diameter circle for right and left rotations were measured. The average time of the two trials was measured as the circular gait time, and the side-to-side difference in the circular gait times was calculated as an asymmetry index. Walking speed, asymmetry of step length, and asymmetry of stance duration time during straight-line gait at comfortable and maximal walking pace were measured. RESULTS: Circular gait time in older women in the late 70s group was significantly slower than that in other age groups; however, no age-related change was observed in older men. Maximal gait speeds in the early and late 70s groups were significantly slower than those in the early 60s group. CONCLUSIONS: Age-related decline in circular gait speed was observed in older women aged ≥75 years, but not in older men. Maximal straight-line gait speed decreased significantly in both genders after the age of 70 years. Geriatr Gerontol Int 2021; 21: 404-410.

Regional differential stretching of the pectoralis major muscle: An ultrasound elastography study.

Pectoralis major (PMa) muscle injuries are becoming more prevalent, and their incidence differs among the PMa regions, i.e., the clavicular, sternal, and abdominal regions. Therefore, identifying the position for effectively lengthening each PMa region is critical in preventing PMa injuries. The purpose of this study was to determine the effective stretching position for each PMa region through shear wave elastography, which can indirectly assess individual muscle lengthening. Fifteen men participated in this study. Twelve stretching positions were compounded with shoulder abductions (45°, 90°, and 135°), pelvic rotation (with or without), shoulder external rotation (with or without), and shoulder horizontal abductions. The shear modulus of each PMa region was measured through shear wave elastography in the stretching positions mentioned above. At the clavicular region, the shear modulus was higher for three stretching positions: shoulder horizontal abduction at 45° abduction during pelvic rotation and shoulder external rotation, shoulder horizontal abduction at 90° abduction, and shoulder horizontal abduction at 90° abduction while considering shoulder external rotation. For the sternal region, the shear modulus was higher in two stretching positions: shoulder horizontal abduction at 90° abduction while adding external rotation, and combination of pelvic rotation and external rotation. For the abdominal region, the shear modulus was higher in the shoulder horizontal abduction at 135° abduction with pelvic and external rotation. These results indicated that the effective stretching position was different for each PMa region.

Effects of ankle position during static stretching for the hamstrings on the decrease in passive stiffness.

Static stretching is frequently performed to improve flexibility of the hamstrings, although the ankle position during hamstring stretching has not been fully investigated. We investigated the effects of ankle position during hamstring stretching on the decrease in passive stiffness. Fourteen healthy men performed static stretching for the hamstrings with the ankle dorsiflexed and plantar-flexed in a randomized order on different days. The hip was passively flexed to the maximum angle which could be tolerated without stretch pain with the knee fully extended; this was maintained for 5 min, with 1-min stretching performed in 5 sessions. Final angles and passive stiffness were measured before and after stretching. The final angle was defined as that formed by the tibia and horizontal plane when the knee was passively extended from hip and knee angles at 90° flexion to the maximum extension angle which could be tolerated without stretch pain. Passive stiffness was determined by the slope of torque-angle curve during the measurement of the final angle. The final angle significantly increased after stretching with the ankle dorsiflexed and plantar-flexed, whereas passive stiffness significantly decreased only after stretching with the ankle planter-flexed. The results suggest that passive stiffness decreases after stretching with the ankle planter-flexed but not after stretching with the ankle dorsiflexed, although the range of joint motion increases regardless of the ankle position during 5-min stretching for the hamstrings. These results indicate that static stretching should be performed with the ankle plantar-flexed when aiming to decrease passive stiffness of the hamstrings.

Effect of static stretching with different rest intervals on muscle stiffness.

The aim of the study was to investigate the effect of static stretching (SS) with different rest intervals on muscle stiffness. Fifteen healthy males participated in the study. Four bouts of thirty-second SS for the gastrocnemii were performed at the maximal dorsiflexion using dynamometer with two different rest intervals between stretches, namely 0 s (R0) and 30 s (R30). Each participant underwent both stretching protocols at least 48 h apart in a random order. Between each bout of SS, the ankle was moved to 20°-plantar-flexion in 3 s, held for each rest interval time, and then returned to the stretching position in 3 s. The shear elastic modulus of the medial gastrocnemius was measured before (PRE) and immediately after (POST) four bouts of SS to assess muscle stiffness of the medial gastrocnemius. Two-way repeated measures analysis of variance (protocol × time) indicated a significant interaction effect on the shear elastic modulus. The shear elastic modulus significantly decreased after SS in both protocols [R0, PRE: 11.5 ±â€¯3.3 kPa, POST: 10.0 ±â€¯2.6 kPa, amount of change: 1.6 ±â€¯0.9 kPa (13.0 ±â€¯5.2%); R30, PRE: 11.0 ±â€¯2.8 kPa, POST: 10.2 ±â€¯2.1 kPa, amount of change: 0.8 ±â€¯1.3 kPa (6.0 ±â€¯10.4%)]. Furthermore, the SS with 0-s rest interval induced greater decrease in shear elastic modulus when compared to SS with 30-s rest interval (p = 0.023). Thus, when performing SS to decrease muscle stiffness, rest intervals between stretches should be minimized.

Ankle and toe muscle strength characteristics in runners with a history of medial tibial stress syndrome.

BACKGROUND: A high proportion of flexor digitorum longus attachment is found at the posteromedial border of the tibia, which is the most common location of medial tibial stress syndrome (MTSS). Therefore, plantar flexion strength of the lesser toes could be related to MTSS; however, the relationship between MTSS and muscle strength of the hallux and lesser toes is not yet evaluated due to the lack of quantitative methods. This study investigated the muscle strength characteristics in runners with a history of MTSS by using a newly developed device to measure the muscle strength of the hallux, lesser toes, and ankle. METHODS: This study comprised 27 collegiate male runner participants (20.0 ± 1.6 years, 172.1 ± 5.1 cm, 57.5 ± 4.0 kg). Maximal voluntary isometric contraction (MVIC) torque of the plantar flexion, dorsiflexion, inversion, and eversion of the ankle were measured by using an electric dynamometer. MVIC torque of the 1st metatarsophalangeal joint (MTPJ) and 2nd-5th MTPJ were measured by using a custom-made torque-measuring device. MVIC torques were compared between runners with and without a history of MTSS. RESULTS: MVIC torque of the 1st MTPJ plantar flexion was significantly higher in runners with a history of MTSS than in those without it. In contrast, there were no significant differences in the MVIC torque values of the 2nd-5th MTPJ plantar flexion and each MVIC torque of the ankle between runners with and without a history of MTSS. CONCLUSION: A history of MTSS increased the isometric FHL strength.

Vertical-scanning mutagenesis of amino acids in a model N-myristoylation motif reveals the major amino-terminal sequence requirements for protein N-myristoylation.

In order to determine the amino-terminal sequence requirements for protein N-myristoylation, site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAAAAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation. Sequential vertical-scanning mutagenesis of amino acids at a distinct position in this model N-terminal sequence revealed the major sequence requirements for protein N-myristoylation: the combination of amino acids at position 3 and 6 constitutes a major determinant for the susceptibility to protein N-myristoylation. When Ser was located at position 6, 11 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) were permitted at position 3 to direct efficient protein N-myristoylation. In this case, the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6, only 3 amino acids (Ala, Asn, Gln) were permitted at position 3 to direct efficient protein N-myristoylation. The amino acid requirements found in this study were fully consistent with the N-terminal sequence of 78 N-myristoylated proteins in which N-myristoylation was experimentally verified. These observations strongly indicate that the combination of amino acids at position 3, 6 and 7 is a major determinant for protein N-myristoylation.