An apparatus to measure in vivo biomechanical behavior of dorsi- and plantarflexors of mouse ankle.

We developed an apparatus to quantify the biomechanical behavior of the dorsi- and plantarflexor muscles of the ankle of an anesthetized mouse. When the dorsi- or plantarflexor muscle group is activated by electrical stimulation of either the peroneal or tibial nerve, the apparatus measures the moment developed about the ankle during isometric, isovelocity shortening, or isovelocity lengthening contractions. Displacements may be performed over the full 105 degrees range of ankle motion with an angular resolution of 0.09 degrees. Bidirectional isovelocity ramps in ankle angle up to 1,100 degrees/s are possible and are equivalent to maximum velocities of 2.3 fiber lengths/s (Lf/s) for the fibers in tibialis anterior muscle and 11.9 Lf/s for those in gastrocnemius muscle. During single contractions of the dorsi- and plantarflexor muscle groups at 37 degrees C and with both knee and ankle joint at 90 degrees neutral position, the isometric tetanic force developed was 1.4 and 3.3 N, power output at 2.2 Lf/s was 3.1 and 5.9 mW, and power absorption at 0.5 Lf/s was 4.9 and 9.0 mW, respectively. These values are in reasonable agreement with data from the same muscle groups tested in situ. We conclude that the apparatus provides valid measurements of force and power of the dorsi- and plantarflexor muscle groups.     

Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.     

Obligatory amino acids in primitive proteins

Conformational similarity among amino acid residues, a property derived by analysing (φ, ψ)-probability distributions of 20 proteinous amino acids from 38 different globular proteins, is used to arrive at a set of six ‘obligatory’ amino acids of primitive proteins. The amino acids Ser, Val, Leu, Asp, Gly and Pro have been argued to be ‘obligatory’ and to represent, conformationally, the remaining amino acids. The reasons for consideration of these six residues as ‘obligatory’ are discussed. Methods to check the validity of our proposition are suggested.     

Relation between D-glucose and L- and D-alanine in the initiation of germination of Bacillus subtilis spore

The rate of L-alanine-initiated germination of Bacillus subtilis spore was measured by both loss of heat resistance and loss of turbidity, and the effect of glucose on the germination response to a wide range of concentrations of the germinant was analyzed in the presence and absence of D-alanine, an inhibitor. Glucose stimulated L-alanine germination by means of a cooperative effect: glucose increased the affinity of L-alanine by about 3-fold and the rate of germination by about 1.3-fold. However, glucose had little effect on the binding affinity of D-alanine. The apparent binding constant of L-alanine to the spore, which was determined by the next measurable event in the trigger reaction, was 1.2 X 10(-5), that of D-alanine was 6 X 10(-6), and that of glucose was 5 X 10(-5). The relation between the binding site for glucose and those for L- and D-alanine on the spore is discussed. Effect of glucose analogs was also examined.     

Acetone-regulated synthesis and degradation of cytochrome P450E1 and cytochrome P4502B1 in rat liver [corrected

The regulation of CYP2E1 and 2B1 was studied by following mRNA levels, catalytic activities and the subcellular distribution of the apoproteins in rat liver 0, 6, 12, 24, 48 and 96 h after a single intragastric dose of acetone. No changes were observed in hepatic CYP2E1 mRNA levels at any time after acetone treatment, whereas rapid rises were observed in the microsomal amount of CYP2E1 protein and CYP2E1-catalyzed 4-nitrophenol hydroxylase and carbon-tetrachloride-initiated lipid-peroxidation activities. However, CYP2E1-dependent catalytic activities declined much faster than the immunodetectable CYP2E1 protein, suggesting that this cytochrome P-450 is inactivated prior to degradation. Similar results were seen in primary hepatocyte cultures. By contrast, concomitant changes in levels of CYP2B1 and CYP2B1-dependent O-depentylation of pentoxyresorufin were observed in the same microsomal preparations. Investigation of the degradative mechanism of both CYP2E1 and CYP2B1 by immunoquantitation of the proteins in lysosomes and by immunohistochemistry indicated their degradation via an autophagic-lysosomal pathway. The data suggest that CYP2E1 is acutely inactivated in the endoplasmic reticulum and that degradation of this isozyme occurs, at least in part, by the lysosomal route. By contrast, CYP2B1 is principally controlled at the level of synthesis.