Influence of muscle activity on the forces in the femur: An in vivo study

Experiments were performed on two patients with custom-made instrumented massive proximal femoral prostheses implanted after tumour resection. In vivo axial forces transmitted along the prostheses were telemetered during level walking, single- and double-leg stance, and isometric exercises of the hip muscles. These activities varied the lever arms available to the external loads: minimum for double-leg stance and maximum for hip isometric exercises. Kinematic, force plate, EMG and telemetered force data were recorded simultaneously. The force magnification ration (FMR; the ratio of the telemetered axial force to the external force) was calculated. The FMRs ranged from 1.3 (during double-leg stance) to 29.8 (during abductors test), indicating that a major part of the axial force in the long bones is a response to muscle activity, the strength of which depends on the lever arms available to the external loads. From these results, it was shown that the bulk of the bending moment along limbs is transmitted by a combination of tensile forces in muscles and compressive forces in bones, so moments transmitted by the bones are smaller than the limb moments. It was concluded that appropriate simulation of muscle forces is important in experimental or theoretical studies of load transmission along bones.     

Self-organization of an oscillatory neural system

Hebbian dynamics is used to derive the differential equations for the synaptic strengths in the neural circuitry of the locomotive oscillator. Initially, neural connection are random. Under a specified arborization hypothesis relating to the density of neural connections, the differential equations are shown to model the self-organization and the stability of the oscillator.     

Molecular mapping of wheat. Homoeologous group 2.

A molecular-marker map of bread wheat having many markers in common with other grasses in the Gramineae family is a prerequisite for molecular level genetic studies and breeding in this crop species. We have constructed restriction fragment length polymorphism maps of the A-, B-, and D-genome chromosomes of homoeologous group 2 of hexaploid wheat (Triticum aestivum L. em. Thell) using 114 F7 lines from a synthetic x bread wheat cross and clones from 11 libraries. Chromosomes 2A, 2B, and 2D comprise 57, 60, and 56 markers and each spans about 200 cM. Comparisons between chromosomes are facilitated by 26 sets of homoeoloci. Genes mapped include a heterologous abscisic acid responsive locus cloned as pBS128, the epidermal waxiness inhibitor W21, and two presumed leaf rust and stem rust resistance genes. Anomalies suggesting ancestral rearrangements in chromosome 2B are pointed out and features of wheat group 2 chromosomes that are common to barley (Hordeum vulgare L.), rice (Oryza spp.), and T. tauschii are discussed.     

Purification and characterization of two essential cytochromes of the thiosulphate-oxidizing multi-enzyme system from Thiobacillus A2 (Thiobacillus versutus)

Four c-type cytochromes were purified by several procedures including chromatography on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Sephadex G-75, G-100 and G-200 and chromatofocusing. Cytochrome c-551 had a pI value of 5.2 and an M_r of 260 000 consisting of six non-covalently bound polypeptides each with an M_r of 43 000, and contained four to five haems. Cytochrome c-552.5 had a pI value of 4.8 and an M_r of 56 000 consisting of two polypeptides with the same M_r 29 000, and contained two haems. Cytochromes c-551 and c-552.5 were reduced by ascorbate to about 70 and 60% of the fully dithionite-reduced values, respectively, and both were essential components in the thiosulphate-oxidizing multi-enzyme system (other components of the system were ‘enzyme A’, ‘enzyme B’ and sulphite: cytochrome c oxidoreductase). These two cytochromes functioned as electron carriers and effectors in the oxidation of thiosulphate. Some evidence suggested that cytochrome c-551 might be a specialized electron transfer component for sulphonate-sulphur oxidation. Both cytochromes could be reduced by thiosulphate in the presence of enzymes A and B. Cytochrome c-550 (basic) and cytochrome c-550 (acidic) were small proteins with M_r 15 000 and 14 000 and pI values of over 8 and 5, respectively. Their physiological role is uncertain.     

Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.     

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.     

Adenine affects the structure and stability of telomeric sequences.

Adenine occurs in the strand containing repeated G clusters in the telomeric DNA of a variety of organisms, including that of humans. The role of adenine has been investigated by constructing two sets of oligonucleotides each with one, two, or four copies of the telomeric sequence dTTTAGGG together with a control sequence in which T replaces the A residue, dTTTTGGG. Comparison of the stability and spectral properties of these two sequences in the presence of Na+ or K+ affords a basis for defining the role of adenine in these structures. In Na+, the A residue stabilizes the structure formed by each oligomer significantly, presumably by a base-pairing interaction with T. In K+, by contrast, there is little difference in stability. In two- and four-copy oligomers, the A sequence has a different structure from its T analog, as detected by CD spectroscopy. In the presence of either Na+ or K+, the tetraplexes of A and T interact with intercalators.     

Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites.

Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor.     

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.