Comparison of system identification techniques in the analysis of a phantom for studying shaken-baby syndrome.

This article compares two techniques for estimating the parameters describing the motion of a phantom designed to investigate shaking baby syndrome. Parameters of a simple computational model and an impulse response function for a linear second order system were both fitted using kinematic measurements of the motion of an inverted jointed pendulum. From the two methods respectively, the rotational stiffness of the joint was calculated to be 1.396 kgm(2) s(-2) and 1.355 kgm(2) s(-2) and the damping coefficient was calculated to be 0.0142 kgm(2) s(-1) and 0.0133 kgm(2) s(-1). The parameter estimates were similar demonstrating that the two techniques were comparable. Identifying accurate parameters will allow more complex phantoms to be modeled, and will provide insight into the relationship between the shaking of the torso and the resultant head motion during shaken baby syndrome.

The tether connecting cytosolic (N terminus) and membrane (C terminus) domains of yeast V-ATPase subunit a (Vph1) is required for assembly of V0 subunit d.

V-ATPases are molecular motors that reversibly disassemble in vivo. Anchored in the membrane is subunit a. Subunit a has a movable N terminus that switches positions during disassembly and reassembly. Deletions were made at residues securing the N terminus of subunit a (yeast isoform Vph1) to its membrane-bound C-terminal domain in order to understand the role of this conserved region for V-ATPase function. Shrinking of the tether made cells pH-sensitive (vma phenotype) because assembly of V(0) subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V(1) and V(0)(-d) that failed to form V(1)V(0), and very low levels of V-ATPase subunits were found at the membrane. Although subunit d expression was stable and at wild-type levels, growth defects were rescued by exogenous VMA6 (subunit d). Stable V(1)V(0) assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V(1)V(0) was delivered to the vacuole and active. It retained 63-71% of the wild-type activity and was responsive to glucose. Tether-less V(1)V(0) disassembled and reassembled after brief glucose depletion and readdition. The N terminus retained binding to V(1) subunits and the C terminus to phosphofructokinase. Thus, no major structural change was generated at the N and C termini of subunit a. We concluded that early steps of V(0) assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6.

Identification of a domain in the V0 subunit d that is critical for coupling of the yeast vacuolar proton-translocating ATPase.

Vacuolar proton-translocating ATPase pumps consist of two domains, V(1) and V(o). Subunit d is a component of V(o) located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59-72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V(1)V(o), mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N- and C-terminal deletions of various lengths were inhibitory and gradually destabilized subunit d, limiting V(1)V(o) formation. Both N and C terminus were required for V(o) assembly. The N-terminal truncation 2-19Delta prevented V(1)V(o) formation, although subunit d was available. The C terminus was required for retention of subunits E and d at the membrane. In addition, the C terminus of its bacterial homolog (subunit C from T. thermophilus) stabilized the yeast subunit d mutant 310-345Delta and allowed assembly of the rotor structure with subunits A and B. Structural features conserved between bacterial and eukaryotic subunit d and the significance of domain 3 for vacuolar proton-translocating ATPase function are discussed.