The metabolic and mechanical costs of step time asymmetry in walking

Animals use both pendular and elastic mechanisms to minimize energy expenditure during terrestrial locomotion. Elastic gaits can be either bilaterally symmetric (e.g. run and trot) or asymmetric (e.g. skip, canter and gallop), yet only symmetric pendular gaits (e.g. walk) are observed in nature. Does minimizing metabolic and mechanical power constrain pendular gaits to temporal symmetry? We measured rates of metabolic energy expenditure and calculated mechanical power production while healthy humans walked symmetrically and asymmetrically at a range of step and stride times. We found that walking with a 42 per cent step time asymmetry required 80 per cent (2.5 W kg⁻¹) more metabolic power than preferred symmetric gait. Positive mechanical power production increased by 64 per cent (approx. 0.24 W kg⁻¹), paralleling the increases we observed in metabolic power. We found that when walking asymmetrically, subjects absorbed more power during double support than during symmetric walking and compensated by increasing power production during single support. Overall, we identify inherent metabolic and mechanical costs to gait asymmetry and find that symmetry is optimal in healthy human walking.     

Total synthesis,purification, and characterization of human [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20–58, [Tyr52, Nle32,53,56,Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58

The synthesis of [Phe(p-CH₂SO₃Na)⁵², Nle^32,53,56 Nal⁵⁵]-CCK_20–58, [Tyr⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58 and of [Phe(p-CH₂SO₃Na)⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58 using the (9-fluorenylmethyloxy)-carbonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude peptide preparations were extremely complex when analyzed by RP-HPLC, capillary zone electrophoresis (CZE), and ion-exchange chromatography (IE-FPLC). We found that the most effective strategy for purification included cation-exchange chromatography followed by a RP-HPLC desalting step. The highly purified peptides (purity greater than 90%) were characterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spectrometry, amino acid analysis, and Edman sequence analysis {for [Tyr⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58}. The results demonstrate the applicability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of long peptides amides (58 residues in length in this case) as well as the efficacy of an FPLC/RP-HPLC approach for the purification of very long, heterogeneous crude peptides, allowing a true assessment of the biological properties of these analogs to be carried out. [Phe(p-CH₂SO₃Na)⁵², Nle^32,53,56, Nal⁵⁵]-CCK_20–58 was less than 1% as potent as CCK-8 while [Tyr⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58 and [Phe(p-CH₂SO₃Na)⁵², Nle^32,53,56, Nal⁵⁵]-CCK-58 were inactive at the doses tested (<0.01%).     

Identification of critical contact residues in the NC41 epitope of a subtype N9 influenza virus neuraminidase

We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab¹ shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment. © 1993 Wiley-Liss, Inc.     

Molecular-genetic evaluation of fungal molecules for roles in pathogenesis to plants

Fungus-plant interactions involve complex developmental processes in which a variety of fungal and plant molecules are required to determine whether the outcome is a susceptible reaction (successful fungal colonization of plant tissues) or a resistant reaction (the plant mounts a defence that aborts fungal invasion). To understand the molecular basis of fungal disease, it is necessary to identify the fungal molecules that areessential for pathogenic processes, and to distinguish them from molecules that may be present during infection but not critical to its outcome. Molecular-genetic technology has been developed for fungal pathogens and used to evaluate the roles of fungal molecules in fungal infection processes. Although the field is in its infancy, several molecules have already been proven as essential components of fungal pathogenesis. Some are clearly involved in the adhesion and penetration phases of infection, i.e. hydrophobins, melanin, glycerol, cutinase, and components of signal transduction pathways (which mediate colonization as well), whereas others are required for colonization of plant tissues after penetration, i.e. toxins that induce susceptibility, toxins that induce resistance, and enzymes that inactivate plant defence mechanisms. Molecular-genetic manipulation has also been used to show that certain candidates for roles in pathogenesis are in fact not involved in any detectable way.     

In situ determination of different dehydrogenase activity profiles in the linings of odontogenic keratocysts and radicular cysts

Summary The levels of succinate, lactate, glutamate, glycerophosphate and glucose-6-phosphate dehydrogenases within the linings of keratinizing and non-keratinizing odontogenic cysts were investigated using static end-point and continuously monitored Nitroblue Tetrazolium-based histochemical methods. The use of TV image analysis for quantification of formazan final reaction products was validated by demonstrating significant relationships between the integrated absorbance at 585 nm and the amount of formazan in, and thickness of, gelatin films containing reduced tetrazolium salt (r=1.0,p<0.001). Absorbance readings of stained sections gave mean coefficients of variation of 1.8±0.9% between day of measurement, and of 5.65±1.32% between serial sections. End-point assays indicated that the linings of odontogenic keratocysts contained higher levels of glucose-6-phosphate dehydrogenases (p<0.0002) and lower levels of lactate dehydrogenase (p<0.002) than those of radicular cysts. Succinate, glutamate and glycerophosphate dehydrogenase activities were similar in both cyst types. Results from continously monitored assays, performed for glucose-6-phosphate and succinate dehydrogenases, demonstrated linear reaction rates over the first 2.75 min of reaction. The calculated enzyme activities from continuous assays were between 1.49 and 3.49 times higher than those determined from end-point assays and confirmed that levels of glucose-6-phosphate dehydrogenase were significantly higher in the linings of odontogenic keratocysts than those of radicular cysts (p<0.004). By contrast, succinate dehydrogenase activity was significantly higher in radicular cyst linings (p<0.03). These results highlight the benefits of an approach toin situ determination of enzyme activity using image analysis and continous monitoring methodologies. Overall, the high level of glucose-6-phosphate dehydrogenase found in keratocyst linings is consistent with their clinical behaviour and higher level of proliferation and synthetic activity whereas the level of lactate dehydrogenase in radicular cysts probably reflects the presence of local tissue damage within these inflammatory lesions.     

Microscale purification of proteins by line immunoelectrophoresis: Application of the technique in protein biogenesis studies

A small-scale version of line immunoelectrophoresis in combination with immunoprecipitate excision is describeb as a rapid convenient technique to purify proteins on a micro scale in biogenesis studies. In the purification of pig small intestinal microvillar enzymes the method was found to be capable of a quantitative purification and to result in a higher state of purity than an isolation procedure using protein A-Sepharose. Since the method furthermore allows a simultaneous purification of several different protein antigens from the sample, it may be of interest as an alternative method to other procedures in the purification of proteins on a micro scale.     

The CD of ligand-DNA systems. I. Poly(dG-dC) B-DNA.

A systematic theoretical study of the CD of double-stranded poly(dG-dC) and its complexes with small molecules is presented. The intrinsic CD of the polymer and the induced CD of a transition belonging to a molecule bound to DNA are calculated using the matrix method. The calculations show considerable differences between pyrimidine-purine and purine-pyrimidine binding sites, and we find that the induced CD of a groove bound molecule is one order of magnitude stronger than that of an intercalated molecule. The results form a sound basis for interpreting the CD of ligand-DNA systems in terms of molecular geometry, interactions, and spectroscopy.