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OBJECTIVE: Many trials of new therapies for cardiovascular disease include economic measures to assess the impact of treatment on healthcare costs, however, it is difficult to compare results between trials due to variation in methods for assigning costs. Therefore we developed a standard library of inpatient hospital costs for major cardiovascular events commonly reported in trials for new cardiovascular therapies. DESIGN: Mean and median hospital charges for each event were calculated from Medicare admissions selected by ICD-9-CM codes from the most recent Healthcare Cost and Utilisation Project (HCUP) Nationwide Inpatient Sample (NIS) database available. Charges were converted to costs using the cost-to-charge ratio from the most recent Medicare cost report data and updated to 1999 using a model derived from the Medicare Payment Advisory Commission (MedPAC) forecast to recommend annual updates to Medicare. RESULTS: Total hospital costs for medical events ranged from $US3654 (1999 values) to $US7833; total hospital costs for surgery and procedures ranged from $US7054 to $US46 317. The distribution of hospital costs is skewed with median costs and lengths of stay lower than mean values. Costs for patients who died in the hospital were generally higher than costs for patients who were discharged. CONCLUSIONS: The library of costs was calculated using a uniform method based on publicly available and easily accessible data and may be updated from year to year. This method provides standardised estimates of hospital costs that can be used in economic analyses of cardiovascular clinical trials.

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The growing problem of resistance to antimicrobial chemotherapy was discussed by participants at the February 1995 workshop at Emory University on population biology, evolution, and control of infectious diseases. They discussed the nature and source of this problem and identified areas of research in which information is lacking for the development of programs to control of the emergence and spread of resistant bacteria. Particular attention was given to theoretical (mathematical modeling) and empirical studies of the within and between-host population biology (epidemiology) and the evolution of microbial resistance to chemotherapeutic agents. Suggestions were made about the kinds of models and data needed, and the procedures that could be employed to stem the ascent and dissemination of resistant bacteria. This article summarizes the observations and recommendations made at the 1995 meeting and in the correspondence between participants that followed. It concludes with an update on the theoretical and empirical research on the between- and within-host population biology and evolution of resistance to antimicrobial chemotherapy most of which has been done since that meeting.

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Previous studies on the motor enzyme kinesin suggesting that the enzyme molecule tightly binds to a microtubule by only one of its two mechanochemical head domains were performed with multiple kinesin molecules on each microtubule, raising the possibility that interactions between adjacent bound molecules may interfere with the binding of the second head. To characterize the microtubule-bound state of isolated single kinesin molecules, we have measured the rates of nucleotide-induced dissociation of the complex between microtubules and bead-labeled single molecules of the dimeric kinesin derivative K448-BIO using novel single-molecule kinetic methods. Complex dissociation by <2 microM ADP displays an apparent second-order rate constant of 1.2 x 10(4) M-1 s-1. The data suggest that only one of the two heads is bound to the microtubule in the absence of ATP, that binding of a single ADP to the complex is sufficient to induce dissociation, and that even lengthy exposure of kinesin to the microtubule fails to produce significant amounts of a two-head-bound state under the conditions used. The inhibitor adenylyl imidodiphosphate (AMP-PNP) induces stochastic pauses in the movement of bead-labeled enzyme molecules in 1 mM ATP. Exit from pauses occurs at 2 s-1 independent of AMP-PNP concentration. The same rate constant is obtained for dissociation of the transient kinesin-microtubule complexes formed in 1 mM ADP, 0.5 mM AMP-PNP, suggesting that release of a single AMP-PNP molecule from the enzyme is the common rate-limiting step of the two processes. The results are consistent with alternating-sites movement mechanisms in which two-head-bound states do not occur in the enzyme catalytic cycle until after ATP binding.

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The oligomeric structure was determined for four recombinant kinesin derivatives containing N-terminal fragments of the kinesin alpha-subunit. Some of the proteins were dimeric (two-headed) molecules with mechanochemical properties similar to those of intact kinesin. Comparison of the primary and quaternary structures of the derivatives with those of intact kinesin suggests that structures distinct from the long alpha-helical coiled-coil rod domain contribute to subunit self-association. Three of the proteins contain a single engineered site for post-translational biotination in vivo; this facilitates analysis of motility in experiments in which the proteins are specifically bound to streptavidin-conjugated microscopic plastic beads. One of the derivatives is monomeric (one-headed); like the two-headed derivatives, it is functional in the motility assay and is a microtubule-dependent ATPase. Unlike intact kinesin and the two-headed derivatives, the one-headed enzyme fails to track microtubule protofilaments. This confirms a prediction of proposed "hand-over-hand" mechanisms of kinesin movement. The ability of molecules with a one-headed solution structure to generate movement is consistent with a translocation-generating conformational change internal to the kinesin head. A simple set of coupling rules can be used to formulate consistent mechano-chemical mechanisms that explain movement by both one- and two-headed kinesin molecules.

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Kinesin, a two-headed motor enzyme molecule, hydrolyses ATP to direct organelle transport along microtubules. As it moves along a microtubule, kinesin remains associated with, or 'tracks', microtubule protofilaments. We have prepared truncated kinesin derivatives that contain either two mechanochemical head domains or only a single head. Unlike intact kinesin and the two-headed derivatives, the one-headed enzyme frequently fails to track protofilaments, suggesting that it detaches from microtubules during movement. In this way, the one-headed kinesin derivative is similar to the motor enzyme myosin, which frequently detaches from the actin filament during movement. For myosin (which has two heads), the consequence of this detachment is that single molecules do not appear to drive continuous movement along the filament. Our observations suggest that the ability of single two-headed kinesin molecules to drive continuous movement results from a 'hand-over-hand' mechanism in which one head remains bound to the microtubule while the other detaches and moves forwards.

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The N-terminal residues of the two heavy chains of the motor enzyme kinesin form two globular "heads"; the heads are attached to a "rod" domain which is a two-stranded alpha-helical coiled-coil. Interaction between the heads is thought to be important to kinesin function. The rod may not be necessary for head-head interactions because a heavy chain N-terminal fragment containing only residues from the head and adjacent region forms dimers (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). However, the nature and stability of the subunit-subunit interactions in such derivatives are unclear. To examine the physical properties of heavy chain interaction in and near the head domains, we characterized the self-association behavior of two dimeric kinesin derivatives predicted (Lupas, A., van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164) to lack the rod. Derivative K448-BIO contains the 448 N-terminal residues of Drosophila kinesin heavy chain fused at the C terminus to a 2-residue linker and a C-terminal fragment from Escherichia coli biotin carboxyl carrier protein; derivative K448-L is the same except that it lacks the biotin carboxyl carrier protein fragment. Both derivatives expressed in insect cells display microtubule-stimulated ATPase activity; K448-BIO also displays microtubule motility. Equilibrium sedimentation and gel filtration indicate that purified K448-BIO and K448-L at 0.02-0.4 mg/ml form homogeneous solutions of homodimers with no detectable formation of monomers or higher order oligomers. Derivative self-association is non-covalent but extremely stable with an association constant > or = 2 x 10(8) M-1. Stable subunit-subunit association induced by structures in and near the kinesin heads may be necessary for full mechanochemical function.

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Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.

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The myocardial MR signal reduction associated with an intravenous bolus of Gd-DTPA and Dy-DTPA was studied in a canine model. Imaging was performed with a high speed echo-planar type imaging system (Instascan, Advanced NMR Systems, Inc.). Gated spin-echo images were obtained with TE of 30 ms, which permits image acquisition in approximately 40 ms. The gated TR was dependent on the heart rate, with an average TR of 2.4 s. After 0.1 mmol/kg of contrast was injected, 70 images were acquired, which showed in an 80-image data set a reduction in myocardial signal with a gradual return to normal. After dipyridamole infusion, the signal loss was significantly more pronounced, and earlier than in the control data set. There was no significant difference between Gd-DTPA and Dy-DTPA in these imaging studies despite the theoretical prediction of better Dy signal reduction, possibly due to physiological variability during the course of a study or between studies. The cause of enhanced contrast effect after dipyridamole infusion is discussed, as is the basis for dipyridamole enhancement, and the possible role of contrast enhanced MR imaging in the detection of cardiac disease.

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The regulatory domain of scallop myosin, consisting of a regulatory light chain (R-LC), an essential light chain (E-LC), and a portion of heavy chain, occupies the neck region of myosin. This domain is directly involved in the regulation of molluscan muscle contraction, which is triggered by direct Ca2+ binding to myosin. We have isolated a soluble functional complex (regulatory complex) comprised of R-LC, E-LC, and a 10-kDa heavy chain fragment in a 1:1:1 stoichiometry by clostripain digestion of the myosin head (papain subfragment 1). N termini of the heavy chain fragments were either leucine-812 or valine-817. The isolated complex retained the specific Ca2(+)-binding site and bound Ca2+ with a similar affinity and selectivity as myosin. The individual components of the regulatory complex were isolated after complete denaturation with guanidine hydrochloride. The regulatory complex was reconstituted from isolated light chains and the heavy chain fragment. The renatured complex regained Ca2+ binding quantitatively. To elucidate the function of the E-LC in Ca2+ binding, we constructed hybrid regulatory complexes. The hybrid complexes reconstituted with molluscan E-LC and R-LC regained the specific Ca2(+)-binding site, whereas the hybrid complex formed with rabbit skeletal E-LC [alkali LC 2 (A2-LC)] and scallop R-LC did not. The results demonstrate that E-LCs from myosins regulated by direct Ca2+ binding are required for the specific Ca2+ binding in the molluscan muscle.

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Hypothalamic suprachiasmatic nucleus (Schn) and Arquate nucleus (AN) were studied in female rats kept under constant illumination (rats with persistent estrus) or under light-dark cyclic illumination (normal estrus cycle). To destroy monoaminergic terminals, some rats from each group were injected with neurotoxic agents into the lateral cerebral ventricle. 6-hydroxydophamine which disturbs catecholaminergic terminals induced activation of the Schn and AN in rats with persistent estrus only. Destruction of serotoninergic terminals with 5,6-hydroxytryptophan induced activation of the Schn in rats with normal sex cycle and in those with a persistent estrus; the AN was also activated but only in rats with normal estrous cycle. As reported earlier, destruction of serotoninergic terminals withstands the decrease of plasma LH under constant illumination although persistent estrus still lasts. It seems there are different parallel ways to disturb normal sex cycle under constant light illumination.

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Contrary to auditory and somatosensory evoked potentials, surface recorded visual evoked potentials which arise in subcortical neural elements have rarely been described. Considerable disagreement exists between the reports in the literature on such visual potentials. In this study, flash stimuli were used to evoke the potentials which were recorded from the skin overlying the infra-orbital ridge, outer canthus, middle of the forehead, vertex, mastoid ipsilateral to the stimulated eye and inion, using a non-cephalic reference. The potentials were amplified in a band which was chosen to omit slow retinal and cortical potentials, and to enhance activity which might include compound neural activity. Potentials were recorded from 9 subjects (13 eyes), and for each one the effects of eye position and stimulus intensity were studied. The results indicate that the series of components recorded within the first 100 msec following photic stimulation were volume-conducted activity generated by a subset of the visual system which is activated by luminosity changes. The generators of the first 4 or 5 components seem to be situated within the retina, the subsequent components seem to be generated in the optic nerve or tracts, and the later components may be thalamo-cortical in origin. These potentials may complement pattern evoked potentials in a more accurate definition of sites of lesions along the visual pathway.

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