Energy expenditure of bipedal walking is higher than that of quadrupedal walking in Japanese macaques.

The authors previously compared energetic costs of bipedal and quadrupedal walking in bipedally trained macaques used for traditional Japanese monkey performances (Nakatsukasa et al. 2004 Am. J. Phys. Anthropol. 124:248-256). These macaques used inverted pendulum mechanics during bipedal walking, which resulted in an efficient exchange of potential and kinetic energy. Nonetheless, energy expenditure during bipedal walking was significantly higher than that of quadrupedal walking. In Nakatsukasa et al. (2004 Am. J. Phys. Anthropol. 124:248-256), locomotor costs were measured before subjects reached a steady state due to technical limitations. The present investigation reports sequential changes of energy consumption during 15 min of walking in two trained macaques, using carbon dioxide production as a proxy of energy consumption, as in Nakatsukasa et al. (2004 Am. J. Phys. Anthropol. 124:248-256). Although a limited number of sessions were conducted, carbon dioxide production was consistently greater during bipedal walking, with the exception of some irregularity during the first minute. Carbon dioxide production gradually decreased after 1 min, and both subjects reached a steady state within 10 min. Energy expenditure during bipedalism relative to quadrupedalism differed between the two subjects. It was considerably higher (140% of the quadrupedal walking cost) in one subject who walked with more bent-knee, bent-hip gaits. This high cost strongly suggests that ordinary macaques, who adopt further bent-knee, bent-hip gaits, consume a far greater magnitude of energy during bipedal walking.

Energetic costs of bipedal and quadrupedal walking in Japanese macaques.

We investigated the energetic costs of quadrupedal and bipedal walking in two Japanese macaques. The subjects were engaged in traditional bipedal performance for years, and are extremely adept bipeds. The experiment was conducted in an airtight chamber with a gas analyzer. The subjects walked quadrupedally and bipedally at fixed velocities (<5 km/hr) on a treadmill in the chamber for 2.5-6 min. We estimated energy consumption from carbon dioxide (CO2) production. While walking bipedally, energetic expenditure increased by 30% relative to quadrupedalism in one subject, and by 20% in another younger subject. Energetic costs increased linearly with velocity in quadrupedalism and bipedalism, with bipedal/quadrupedal ratios remaining almost constant. Our experiments were relatively short in duration, and thus the observed locomotor costs may include presteady-state high values. However, there was no difference in experimental duration between bipedal and quadrupedal trials. Thus, the issue of steady state cannot cancel the difference in energetic costs. Furthermore, we observed that switching of locomotor mode (quadrupedalism to bipedalism) during a session resulted in a significant increase of CO2 production. Taylor and Rowntree ([1973] Science 179:186-187) noted that the energetic costs for bipedal and quadrupedal walking were the same in chimpanzees and capuchin monkeys. Although the reason for this inconsistency is not clear, species-specific differences should be considered regarding bipedal locomotor energetics among nonhuman primates. Extra costs for bipedalism may not be great in these macaques. Indeed, it is known that suspensory locomotion in Ateles consumes 1.3-1.4 times as much energy relative to quadrupedal progression. This excess ratio surpasses the bipedal/quadrupedal energetic ratios in these macaques.

Induction of eotaxin production by interleukin-4, interleukin-13 and lipopolysaccharide by nasal fibroblasts.

BACKGROUND: There is growing evidence that eotaxin is a key mediator in the development of tissue eosinophilia. Fibroblasts are a major source of eotaxin. The severity of diseases with eosinophilic inflammation like nasal polyposis, atopic dermatitis and asthma, where Th2-type cytokines (IL-4 and IL-13) and TGF-beta are expressed locally, was shown to correlate with bacterial factors such as lipopolysaccharide (LPS) rather than allergen. OBJECTIVE: We examined eotaxin production by nasal fibroblasts stimulated with IL-4 or IL-13 alone or in combination with LPS, and the effect of TGF-beta(1) on it. Moreover, we compared the magnitude of eotaxin produced by nasal fibroblasts with that produced by lung or skin fibroblasts. METHODS: Fibroblast lines were established from human biopsy tissue. The expression of eotaxin mRNA was evaluated by RT-PCR. The amount of eotaxin in the supernatants was measured by ELISA. RESULTS: IL-4, but not IL-13, synergized with LPS to produce eotaxin in a dose- and time-dependent manner. Sequential treatment of nasal fibroblasts with IL-4 and LPS did not have any effect. But when IL-4 and LPS were added together, synergy for eotaxin production was observed. Moreover, this synergy was observed in nasal and skin fibroblasts, but not in lung fibroblasts. The production of eotaxin by IL-4 and LPS was modulated by TGF-beta(1). CONCLUSION: These results suggest that a co-stimulus like LPS is necessary for IL-4 to make a strong induction of eotaxin in eosinophilic inflammations such as nasal polyposis. Modulation by TGF-beta(1) may have important implications for the development of eosinophilic inflammation.

Characterization of the portal signal during 24-h glucose delivery in unrestrained, conscious rats.

To characterize the "portal signal" during physiological glucose delivery, liver glycogen was measured in unrestrained rats during portal (Po) and peripheral (Pe) constant-rate infusion, with minimal differences in hepatic glucose load (HGL) and portal insulin between the delivery routes. Hepatic blood flows were measured by Doppler flowmetry during open surgery. Changes in hepatic glucose, portal insulin, glucagon, lactate, and free fatty acid concentrations were generally similar in either delivery except for glucagon at 4 h. Hepatic glycogen, however, increased continuously in Po and was higher than Pe at 8 and 24 h, although it decreased to the level of Pe upon the removal of Po at 8 h. There was a near-linear relationship between hepatic glycogen and HGL in either delivery, with the slope being twice as high in Po and the intercepts converging to basal HGL. The hepatic response to Po did not alter upon 80% replacement by Pe. These results suggest that negative arterial-portal glucose gradients increase the rate of hepatic glycogen synthesis against the incremental HGL in an all-or-nothing mode.

Characterization of the portal signal in a nonsteady hyperglycemic state in conscious dogs.

To characterize the "portal signal" in a nonsteady hyperglycemic state, the kinetic relationship between net hepatic glucose balance (NHGB) and either hepatic glucose load (HGL) or plasma insulin level was determined during glucose infusion using a catheter technique in 36 conscious dogs. Glucose was infused intraportally (Po group) and peripherally (Pe group) at 39, 56, and 83 micromol x kg(-1) x min(-1) over 2 h. There was a linear relationship between mean NHGB and either mean HGL or plasma insulin levels at each rate in either delivery (HGL: Po r = 0.99, Pe r = 0.95; insulin: Po r = 99, Pe r = 0.79). The threshold levels for net hepatic glucose uptake were 3.8 and 11.7 mmol/l for plasma glucose and 65 and 392 pmol/l for plasma insulin, respectively. The slope of the regression line against the abscissa was four times larger in portal than in peripheral delivery (HGL: Po 0.20 vs. Pe 0.05, P < 0.05; insulin: Po 0.19 vs. Pe 0.04, P < 0.05). These results suggest that the portal signal overrules the threshold of glucose for hepatic uptake by increasing hepatic extraction rate in a nonsteady hyperglycemic state.

Comparison of insulinotrophic actions of nateglinide with glibenclamide dissociated from absorption in conscious dogs.

Nateglinide is more rapidly absorbed than glibenclamide. Therefore, the different absorption kinetics of both drugs were eliminated by intraportal administration in conscious fasted dogs. The plasma insulin profiles were compared under similar kinetic changes in plasma drug concentrations. After a priming dose of nateglinide (1 mg/kg. 5 min) or glibenclamide (40 microg/kg. 5 min), plasma drug concentrations reached a peak at 4 minutes (nateglinide, 80 +/- 5 micromol/L, n = 6 and glibenclamide, 263 +/- 60 nmol/L, n = 6) followed by a sustained level at approximately 30% of the peak concentration at 30 minutes. Nateglinide led to a rapid and constant reduction in arterial glucose of approximately 30% basal, while glibenclamide promoted a gradual decrease to approximately 50% basal at 120 minutes. An increase in plasma insulin level by nateglinide of 4 times basal (218 +/- 58 pmol/L v 47 +/- 3 pmol/L, P <.05, n = 6) occurred at 6 to 10 minutes followed by sustained release of 1.4 times basal (67 +/- 15 pmol/L, n = 6). The insulin surge was more than doubled (484 +/- 209 pmol/L, n = 6) under a euglycemic clamp. Insulin release by glibenclamide increased gradually reaching 10-fold basal (449 +/- 166 pmol/L, n = 6) at 60 minutes. This was not enhanced during a euglycemic clamp. Lowering the primed doses of nateglinide resulted in a diminished peak plasma insulin concentration. In contrast, glibenclamide caused only a slower increase, but eventually reaching a similar peak. By increasing the continuous infusion of nateglinide, the sustained insulin release was not altered. Glibenclamide, but not nateglinide, evoked prompt and sustained insulin release in the continuing presence of the other. These results are consistent with the concept that nateglinide produces a quick, but very short-lived, interaction with sulfonylurea (SU)-receptors on plasma membrane by free access of the drug from the cell exterior. In contrast, glibenclamide promotes a slow and longer interaction with the receptor by distribution of the drug into the cell inferior. We conclude, therefore, that not only the different kinetics of gastrointestinal (GI) absorption, but also the inherent difference in the interaction with beta cells is attributed to the different insulin release characteristics between nateglinide and glibenclamide in vivo.

Generation of human bipedal locomotion by a bio-mimetic neuro-musculo-skeletal model.

To emulate the actual neuro-control mechanism of human bipedal locomotion, an anatomically and physiologically based neuro-musculo-skeletal model is developed. The human musculo-skeletal system is constructed as seven rigid links in a sagittal plane, with a total of nine principal muscles. The nervous system consists of an alpha motoneuron and proprioceptors such as a muscle spindle and a Golgi tendon organ for each muscle. At the motoneurons, feedback signals from the proprioceptors are integrated with the signal induced by foot-ground contact and input from the rhythm pattern generator; a muscle activation signal is produced accordingly. Weights of connection in the neural network are optimized using a genetic algorithm, thus maximizing walking distance and minimizing energy consumption. The generated walking pattern is in remarkably good agreement with that of actual human walking, indicating that the locomotory pattern could be generated automatically, according to the musculoskeletal structures and the connections of the peripheral nervous system, particularly due to the reciprocal innervation in the muscle spindles. Using the proposed model, the flow of sensory-motor information during locomotion is estimated and a possible neuro-control mechanism is discussed.

Design of three-dimensional domain-swapped dimers and fibrous oligomers.

Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 A resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.

A structure-based mechanism for copper-zinc superoxide dismutase.

A reaction cycle is proposed for the mechanism of copper-zinc superoxide dismutase (CuZnSOD) that involves inner sphere electron transfer from superoxide to Cu(II) in one portion of the cycle and outer sphere electron transfer from Cu(I) to superoxide in the other portion of the cycle. This mechanism is based on three yeast CuZnSOD structures determined by X-ray crystallography together with many other observations. The new structures reported here are (1) wild type under 15 atm of oxygen pressure, (2) wild type in the presence of azide, and (3) the His48Cys mutant. Final R-values for the three structures are respectively 20.0%, 17.3%, and 20.9%. Comparison of these three new structures to the wild-type yeast Cu(I)ZnSOD model, which has a broken imidazolate bridge, reveals the following: (i) The protein backbones (the "SOD rack") remain essentially unchanged. (ii) A pressure of 15 atm of oxygen causes a displacement of the copper ion 0.37 A from its Cu(I) position in the trigonal plane formed by His46, His48, and His120. The displacement is perpendicular to this plane and toward the NE2 atom of His63 and is accompanied by elongated copper electron density in the direction of the displacement suggestive of two copper positions in the crystal. The copper geometry remains three coordinate, but the His48-Cu bond distance increases by 0.18 A. (iii) Azide binding also causes a displacement of the copper toward His63 such that it moves 1.28 A from the wild-type Cu(I) position, but unlike the effect of 15 atm of oxygen, there is no two-state character. The geometry becomes five-coordinate square pyramidal, and the His63 imidazolate bridge re-forms. The His48-Cu distance increases by 0.70 A, suggesting that His48 becomes an axial ligand. (iv) The His63 imidazole ring tilts upon 15 atm of oxygen treatment and azide binding. Its NE2 atom moves toward the trigonal plane by 0.28 and 0.66 A, respectively, in these structures. (v) The replacement of His48 by Cys, which does not bind copper, results in a five-coordinate square pyramidal, bridge-intact copper geometry with a novel chloride ligand. Combining results from these and other CuZnSOD crystal structures, we offer the outlines of a structure-based cyclic mechanism.

The crystal structure of the designed trimeric coiled coil coil-VaLd: implications for engineering crystals and supramolecular assemblies.

The three-dimensional structure of the 29-residue designed coiled coil having the amino acid sequence acetyl-E VEALEKK VAALESK VQALEKK VEALEHG-amide has been determined and refined to a crystallographic R-factor of 21.4% for all data from 10-A to 2.1-A resolution. This molecule is called coil-VaLd because it contains valine in the a heptad positions and leucine in the d heptad positions. In the trigonal crystal, three molecules, related by a crystallographic threefold axis, form a parallel three-helix bundle. The bundles are stacked head-to-tail to form a continuous coiled coil along the c-direction of the crystal. The contacts among the three helices within the coiled coil are mainly hydrophobic: four layers of valine residues alternate with four layers of leucine residues to form the core of the bundle. In contrast, mostly hydrophilic contacts mediate the interaction between trimers: here a total of two direct protein--protein hydrogen bonds are found. Based on the structure, we propose a scheme for designing crystals of peptides containing continuous two-, three-, and four-stranded coiled coils.

Unusual trigonal-planar copper configuration revealed in the atomic structure of yeast copper-zinc superoxide dismutase.

The three-dimensional structure of yeast copper-zinc superoxide dismutase (CuZnSOD) has been determined in a new crystal form in space group R32 and refined against X-ray diffraction data using difference Fourier and restrained crystallographic refinement techniques. The unexpected result is that the copper ion has moved approximately 1 angstrom from its position in previously reported CuZnSOD models, the copper-imidazolate bridge is broken, and a roughly trigonal planar ligand geometry characteristic of Cu(I) rather than Cu(II) is revealed. Final R values for the two nearly identical room temperature structures are 18.6% for all 19 149 reflections in the 10.0-1.7 angstrom resolution range and 18. 2% for 17 682 reflections (F > 2 sigma) in the 10.0-1.73 angstrom resolution range. A third structure has been determined using X-ray data collected at -180 degrees C. The final R value for this structure is 19.0% (R(free) = 22.9%) for all 24 356 reflections in the 10.0-1.55 angstrom resolution range. Virtually no change in the positions of the ligands to the zinc center is observed in these models. The origin of the broken bridge and altered Cu-ligand geometry is discussed.

Biomechanical analysis of the development of human bipedal walking by a neuro-musculo-skeletal model.

A new computer simulation method, using a neuro-musculo-skeletal model, is used to clarify the process of acquisition of erect bipedal walking during human ontogeny. Walking was autonomously generated as a dynamic interaction called 'mutual entrainment' between the neural oscillation and the pendular movement of differently proportioned bodies. Walking patterns of humans with 8 different sets of alternative body proportions, varying from those of 8-month-old children to those of 22 years old adults, were simulated. The development of bipedal walking is characterized as the change from a forced oscillation controlled by the nervous system to the natural oscillation of pendular motion, determined by body proportions. Body proportions are the fundamental factor in the development of bipedal walking.

Crystallization of a designed peptide from a molten globule ensemble.

BACKGROUND: The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles. RESULTS & CONCLUSIONS: Crystals diffracting to 3.5 A resolution have been grown of a 16-residue peptide (alpha 1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha 1, has previously been shown to yield crystals that diffract to 1.2 A resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins.

Crystallization of a designed peptide from a molten globule ensemble.

Backgound. The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles. Results. Crystals diffracting to 3.5Å resolution have been grown of a 16-residue peptide (alpha1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha1, has previously been shown to yield crystals that diffract to 1.2Å resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins.

Inhibitory effect of 15-oxygenated sterols on cholesterol synthesis from 24,25-dihydrolanosterol.

Several 15-oxygenated sterols were examined as to their inhibitory activity toward cholesterol synthesis from [24,25-3H]-24,25-dihydrolanosterol in the 10,000 X g supernatant fraction of a rat liver homogenate. At 40 microM, three 15 alpha-hydroxylated compounds, 14 alpha-ethylcholest-7-ene-3 beta,15 alpha-diol, 14 alpha-methylcholest-7-ene-3 beta,15 alpha-diol, and lanost-7-ene-3 beta,15 alpha-diol, were found to be extremely potent inhibitors (more than 90% inhibition) of dihydrolanosterol metabolism. The inhibitory effect of the C-15 substituents appeared to be in the order of: 15 alpha-hydroxyl greater than 15-ketone greater than 15 beta-hydroxyl.