The present study was designed to explore how the interaction between the fascicles and tendinous tissues is involved in storage and utilization of elastic energy during human walking. Eight male subjects walked with a natural cadence (1.4 +/- 0.1 m/s) on a 10-m-long force plate system. In vivo techniques were employed to record the Achilles tendon force and to scan real-time fascicle lengths for two muscles (medial gastrocnemius and soleus). The results showed that tendinous tissues of both medial gastrocnemius and soleus muscles lengthened slowly throughout the single-stance phase and then recoiled rapidly close to the end of the ground contact. However, the fascicle length changes demonstrated different patterns and amplitudes between two muscles. The medial gastrocnemius fascicles were stretched during the early single-stance phase and then remained isometrically during the late-stance phase. In contrast, the soleus fascicles were lengthened until the end of the single-stance phase. These findings suggest that the elastic recoil takes place not as a spring-like bouncing but as a catapult action in natural human walking. The interaction between the muscle fascicles and tendinous tissues plays an important role in the process of release of elastic energy, although the leg muscles, which are commonly accepted as synergists, do not have similar mechanical behavior of fascicles in this catapult action.

The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two ␣-helical turns, in contrast to association throughout the membrane over five ␣-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.Fundamental to cellular physiology is the ability to transmit extracellular signals across the cell membrane to trigger intracellular responses. Although the extracellular and intracellular portions of cell surface receptors are responsible for detecting ligands and initiating signal cascades, respectively, transmembrane (TM) domains are thought to play critical roles by specifically associating and propagating signals across the phospholipid bilayer. However, the mechanisms by which single-pass TM domains associate and conduct signals are poorly understood.The epidermal growth factor receptor (EGFR) is the prototypical type I TM receptor tyrosine kinase. EGFR and related members of the ErbB family-ErbB2, ErbB3, and ErbB4-contain a glycosylated extracellular ligand binding domain; a singlepass TM domain; and intracellular juxtamembrane, tyrosine kinase, and autophosphorylation domains. The extracellular domain of EGFR binds polypeptide growth factor ligands, such as epidermal growth factor (EGF), to stimulate an array of intracellular signaling cascades that regulate normal and oncogenic cellular growth and proliferation (3,17,36). In one model of growth factor-dependent EGFR activation, ligand binding promotes receptor dimerization and activation of intracellular protein tyrosine kinase activity (35); other models suggest that receptors are predimerized on the cell surface and ligand binding alters the equilibrium between inactive and active dimeric (or higher-order oligomeric) configurations (9, 29).Structural mechanisms of growth factor-mediated receptor dimerization and allosteric kinase domain activation have been proposed from recent crystal structures of isolated extracellular ligand binding domains (7) and intracellular tyrosine kinase domains (37). The orientation between the four extracellular domains is dramatically altered upon ligand binding, which frees interfaces that are masked in ...

Conformational changes occurring on the microsecond-millisecond time scale in basic pancreatic trypsin inhibitor (BPTI) are investigated using nuclear magnetic resonance spectroscopy. The rczz CPMG experiment (Wang, C.; Grey, M. J.; Palmer, A. G. J. Biomol. NMR 2001, 21, 361-366) is used to record (15)N spin relaxation dispersion data, R(ex)(1/tau(cp)), in which 1/tau(cp) is the pulsing rate in the CPMG sequence, at two static magnetic fields, 11.7 and 14.1 T, and three temperatures, 280, 290, and 300 K. These data are used to characterize the kinetics and mechanism of chemical exchange line broadening of the backbone (15)N spins of Cys 14, Lys 15, Cys 38, and Arg 39 in BPTI. Line broadening is found to result from two processes: the previously identified isomerization of the Cys 38 side chain between chi(1) rotamers (Otting, G.; Liepinsh, E.; Wüthrich, K. Biochemistry 1993, 32, 3571-3582) and a previously uncharacterized process on a faster time scale. At 300 K, both processes contribute significantly to the relaxation dispersion for Cys 14 and an analytical expression for a linear three-site exchange model is used to analyze the data. At 280 K, isomerization of the Cys 38 side chain is negligibly slow and the faster process dominates the relaxation dispersion for all four spins. Global analysis of the temperature and static field dependence of R(ex)(1/tau(cp)) for Cys 14 and Lys 15 is used to determine the activation parameters and chemical shift changes for the previously uncharacterized chemical exchange process. Through an analysis of a database of chemical shifts, (15)N chemical shift changes for Cys 14 and Lys 15 are interpreted to result from a chi(1) rotamer transition of Cys 14 that converts the Cys 14-Cys 38 disulfide bond between right- and left-handed conformations. At 290 K, isomerization of Cys 14 occurs with a forward and reverse rate constant of 35 s(-1) and 2500 s(-1), respectively, a time scale more than 30-fold faster than the Cys 38 chi(1) isomerization. A comparison of the kinetics and thermodynamics for the transitions between the two alternative Cys 14-Cys 38 conformations highlights the factors that affect the contribution of disulfide bonds to protein stability.

NMR spin relaxation experiments are used to characterize the dynamics of the backbone of ubiquitin. Chemical exchange processes affecting residues Ile 23, Asn 25, Thr 55, and Val 70 are characterized using on-and off-resonance rotating-frame 15 N R 1 relaxation experiments to have a kinetic exchange rate constant of 25,000 sec −1 at 280 K. The exchange process affecting residues 23, 25, and 55 appears to result from disruption of N-cap hydrogen bonds of the ␣-helix and possibly from repacking of the side chain of Ile 23. Chemical exchange processes affecting other residues on the surface of ubiquitin are identified using 1 H-15 N multiple quantum relaxation experiments. These residues are located near or at the regions known to interact with various enzymes of the ubiquitin-dependent protein degradation pathway.

The central nervous system (CNS) takes advantage of a network of complex neural pathways and mechanisms in the control of normal human gait. One such mechanism is the use of afferent feedback from muscle, cutaneous and joint receptors. Our knowledge of the contribution of afferent information in human gait is still limited, although this has been an area of active research for many years (e.g. Dietz et al. 1985;Yang et al. 1991;Sinkjaer et al. 1996). Yang et al. (1991) and Sinkjaer et al. (1996) have shown that afferent-mediated feedback is used by the CNS in the control of gait when an unexpected stretch of the ankle extensors is imposed. More recently, Sinkjaer et al. (2000) provided evidence that during walking, up to 50 % of the background EMG from the soleus muscle can be attributed to afferent feedback. However, the relative importance of the separate afferent pathways may differ for the background locomotor EMG and the EMG that results from an imposed stretch.When the human soleus muscle is stretched in a seated subject, two distinct bursts, with average peak latencies of 59 and 86 ms are evident in the EMG (Toft et al. 1989). These bursts are often referred to as the short (SLR) and medium (MLR) reflex responses, respectively, and have also been labelled the M1 and M2 stretch reflex responses, respectively. The short latency response has an onset latency of approximately 40 ms and is attributed to monosynaptic excitation of spinal motoneurones from the large diameter group Ia afferent fibres (Taylor et al.Group II muscle afferents probably contribute to the medium latency soleus stretch reflex during walking in humans 1. The objective of this study was to determine which afferents contribute to the medium latency response of the soleus stretch reflex resulting from an unexpected perturbation during human walking.2. Fourteen healthy subjects walked on a treadmill at approximately 3.5 km h _1 with the left ankle attached to a portable stretching device. The soleus stretch reflex was elicited by applying small amplitude (~8 deg) dorsiflexion perturbations 200 ms after heel contact.3. Short and medium latency responses were observed with latencies of 55 ± 5 and 78 ± 6 ms, respectively. The short latency response was velocity sensitive (P < 0.001), while the medium latency response was not (P = 0.725).4. Nerve cooling increased the delay of the medium latency component to a greater extent than that of the short latency component (P < 0.005).5. Ischaemia strongly decreased the short latency component (P = 0.004), whereas the medium latency component was unchanged (P = 0.437).6. Two hours after the ingestion of tizanidine, an a 2 -adrenergic receptor agonist known to selectively depress the transmission in the group II afferent pathway, the medium latency reflex was strongly depressed (P = 0.007), whereas the short latency component was unchanged (P = 0.653).7. An ankle block with lidocaine hydrochloride was performed to suppress the cutaneous afferents of the foot and ankle. Neither the short (P = 0.453) nor m...