Genetic variation in CYP3A43 explains racial difference in olanzapine clearance.

The antipsychotic drug, olanzapine, one of the most widely used drugs in clinical medicine, has a high rate of discontinuation due to inefficacy and/or adverse effects. We identified a single nucleotide polymorphism in the drug metabolizing enzyme, cytochrome P450 3A43 (CYP3A43; rs472660), that highly significantly predicted olanzapine clearance in the Clinical Antipsychotic Trials of Intervention Effectiveness trial (P=5.9e(-7)). Moreover, at standard antipsychotic doses, 50% of individuals with the high clearance genotype (AA) have trough blood levels below the therapeutic range. Interestingly, a much higher proportion of African Americans carry the A allele compared with Caucasians (allele frequency 67 vs 14%). After accounting for CYP3A43 genotype, race is no longer a significant predictor of olanzapine clearance. Olanzapine clearance was associated with measures of clinical response. Patients with greater clearance had higher symptom ratings and were more likely to discontinue treatment due to an inadequate response. Our data identify a genetic mechanism for variation in olanzapine response and demonstrate that blood level monitoring of olanzapine treatment is advisable.

Increased pain tolerance as an indicator of return to work in low-back injuries after work hardening.

OBJECTIVE: This study examined retrospective data from a multidisciplinary work-hardening program that compared patients who did and did not return to work after low-back injury. The objective of this study was to identify differences between these groups to better guide work-hardening programs and return-to-work decisions. METHOD: Retrospective data from patients with low-back injuries (n = 115) who participated in a northern California work-hardening program were analyzed. Using two-way analysis of variance, male and female patients who did and did not return to work were compared. RESULTS: No significant differences were found between men and women for any of the variables studied. Patients who did and did not return to work were not significantly different in age, length of injury, and subjective pain at the beginning or end of the work-hardening program or in activity tolerance (p = .08). Patients who returned to work perceived a significantly (p < or = . 05) greater improvement in pain tolerance by the end of the work-hardening program than those who did not return to work. CONCLUSION: The results of this study suggest that rehabilitation emphasis should not be placed on the reduction of subjective pain but, rather, on strategies to cope with existing pain while improving functional ability.

Extensibility and symmetry of actin filaments in contracting muscles.

When isometrically contracting muscles are subjected to a quick release followed by a shortening ramp of appropriate speed (V(o)), tension decays from its value at the isometric plateau (P(o)) to <0. 05 P(o) with the same time course as the quick part of the release; thereafter, tension remains at a negligible level for the duration of the shortening ramp. X-ray diffraction data obtained under these conditions provide evidence that 1) at V(o) very few heads form an actomyosin complex, while the number of heads doing so at P(o) is significant; 2) relative to rest the actin filament at V(o) is approximately 0.12% shorter and more twisted, while it is approximately 0.3% longer and less twisted at P(o); and 3) the myosin heads attaching to actin during force development do so against a thin filament compliance of at least 0.646 +/- 0.046% nm per P(o).

Small segmental rearrangements in the myosin head can explain force generation in muscle.

Poisson-Boltzmann calculations of the distribution of electrostatic potentials around an actin filament in physiological-strength solutions show that negative isopotential surfaces protrude into the solvent. Each protrusion follows the actin two-start helix and is located on the sites implicated in the formation of the actomyosin complex. Molecular dynamic calculations on the S1 portion of the myosin molecule indicate that in the presence of ATP the crystallographically invisible loops (comprising residues 624-649 and 564-579) remain on the surface, whereas in the absence of ATP they can move toward the actin-binding sites and experience electrostatic forces that range from 1 to 10 pN. The molecular dynamics calculations also suggest that during the ATP cycle there exist at least three states of electrostatic interactions between the loops and actin. Every time a new interaction is formed, the strain in the myosin head increases and the energy of the complex decreases by 2kT to 5kT. This can explain muscular contraction in terms of a Huxley-Simmons-type mechanism, while requiring only rearrangements of small mobile S1 segments rather than the large shape changes in the myosin molecule postulated by the conventional tilting head model.

X-ray evidence that in contracting live frog muscles there exist two distinct populations of myosin heads.

Using synchrotron radiation and whole muscles, 2 ms time-resolved x-ray diffraction patterns were recorded at 8 degrees C. The results show that in both isotonic and isometric contractions, as well as in length changes imposed at maximum tension [Po], the meridional third myosin layer line consists of two distinct reflections with different intensities and spacings that measure approximately 14,623 and 14,412 nm at Po. Although the intensity behavior of the two reflections is strikingly different during quick releases, it is very similar during stretches. Study of the time courses indicates that myosin heads diffracting at Po with the approximately 14.623 nm periodicity are actively involved in tension production. Those diffracting at Po with the periodicity of approximately 14.412 nm appear not be associated with tension production during isometric contraction and releases, but the results suggest that they are recruited during stretches and here contribute to tension production. Our most important conclusion is that under all conditions of contraction we have investigated there exist two populations of myosin heads, each with a well defined axial disposition and configuration.

Time-resolved X-ray diffraction studies of myosin head movements in live frog sartorius muscle during isometric and isotonic contractions.

Using the facilities at the Daresbury Synchrotron Radiation Source, meridional diffraction patterns of muscles at ca 8 degrees C were recorded with a time resolution of 2 or 4 ms. In isometric contractions tetanic peak tension (P0) is reached in ca 400 ms. Under such conditions, following stimulation from rest, the timing of changes in the major reflections (the 38.2 nm troponin reflection, and the 21.5 and 14.34/14.58 nm myosin reflections) can be explained in terms of four types of time courses: K1, K2, K3 and K4. The onset of K1 occurs immediately after stimulation, but that of K2, K3 and K4 is delayed by a latent period of ca 16 ms. Relative to the end of their own latent periods the half-times for K1, K2, K3 and K4 are 14-16, 16, 32 and 52 ms, respectively. In half-times, K1, K2, K3 lead tension rise by 52, 36 and 20 ms, respectively. K4 parallels the time course of tension rise. From an analysis of the data we conclude that K1 reflects thin filament activation which involves the troponin system; K2 arises from an order-disorder transition during which the register between the filaments is lost; K3 is due to the formation of an acto-myosin complex which (at P0) causes 70% or more of the heads to diffract with actin-based periodicities; and K4 is caused by a change in the axial orientation of the myosin heads (relative to thin filament axis) which is estimated to be from 65-70 degrees at rest to ca 90 degrees at P0. Isotonic contraction experiments showed that during shortening under a load of ca 0.27 P0, at least 85% of the heads (relative to those forming an acto-myosin complex at P0) diffract with actin-based periodicities, whilst their axial orientation does not change from that at rest. During shortening under a negligible load, at most 5-10% of the heads (relative to those forming an acto-myosin complex at P0) diffract with actin-based periodicities, and their axial orientation also remains the same as that at rest. This suggests that in isometric contractions the change in axial orientation is not the cause of active tension production, but rather the result of it. Analysis of the data reveals that independent of load, the extent of asynchronous axial motions executed by most of the cycling heads is no more than 0.5-0.65 nm greater than at rest.(ABSTRACT TRUNCATED AT 400 WORDS)

Roles of pilin and PilC in adhesion of Neisseria meningitidis to human epithelial and endothelial cells.

Pili and pilin antigenic variation play important roles in adhesion of Neisseria meningitidis (MC) to human epithelial and endothelial cells. We recently identified one pilin variant that confers high adhesiveness of MC to human epithelial cells in culture. However, other factor(s) also play a role in MC adhesiveness, since some nonadhesive variants of MC strain 8013 are piliated and produce the same pilin variant as adhesive derivatives. PilC1 and PilC2, high molecular weight outer membrane proteins in Neisseria gonorrhoeae, are proposed to play roles in pilus assembly. Strain 8013 also contains pilC1 and pilC2; their products function in a similar if not identical manner in pilus biogenesis. PilC1 has an additional function in that it also modulates adhesiveness of strain 8013.

Two-dimensional time-resolved X-ray diffraction studies of live isometrically contracting frog sartorius muscle.

Results were obtained from contracting frog muscles by collecting high quality time-resolved, two-dimensional, X-ray diffraction patterns at the British Synchrotron Radiation Source (SERC, Daresbury, Laboratory). The structural transitions associated with isometric tension generation were recorded under conditions in which the three-dimensional order characteristic of the rest state is either present or absent. In both cases, new layer lines appear during tension generation, subsequent to changes from activation events in the thin filaments. Compared with the 'decorated' actin layer lines of the rigor state, the spacings of the new layer lines are similar whereas their intensities differ substantially. We conclude that in contracting muscle an actomyosin complex is formed whose structure is not like that in rigor, although it is possible that the interacting sites are the same. Transition from rest to plateau of tension is accompanied by approximately 1.6% increase in the axial spacing of the myosin layer lines. This is explained as arising from axial disposition of the interacting myosin heads in the actomyosin complex. Model calculations are presented which support this view. We argue that in a situation where an actomyosin complex is formed during contraction, one cannot describe the diffraction features as being either thick or thin filament based. Accordingly, the layer lines seen during tension generation are referred to as actomyosin layer lines. It is shown that these layer lines can be indexed as submultiples of a minimum axial repeat of approximately 218.7 nm. After lattice disorder effects are taken into account, the intensity increases on the 15th and 21st AM layer lines at spacings of approximately 14.58 and 10.4 nm respectively, show the same time course as tension rise. However, the time course of the intensity increase of the other actomyosin layer lines and of the spacing change (which is the same for both phenomena) shows a substantial lead over tension rise. These findings suggest that the actomyosin complex formed prior to tension rise is a non-tension-generating state and that this is followed by a transition of the complex to a tension-generating state. The intensity increase in the 15th actomyosin layer line, which parallels tension rise, can be accounted for assuming that in the tension-generating state the attached heads adopt (axially) a more perpendicular orientation with respect to the muscle axis than is seen at rest or in the non-tension-generating state. This suggests the existence of at least two structurally distinct interacting myosin head conformations.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells.

Pili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter- and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low- and high-adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low-adhesive derivatives were completely different from the one found in the high-adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low-adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low- and high-adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence.

X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles.

Using x-rays from a laboratory source and an area detector, myosin layer lines and the diffuse scattering between them in the moderate angle region have been recorded. At full overlap, incubation of rigor muscles with S-1 greatly reduces the diffuse scattering. Also, three of the four actin-based layer lines lying close to the meridian (Huxley, H. E., and W. Brown, 1967. J. Mol. Biol. 30:384-434; Haselgrove, J. C. 1975. J. Mol. Biol. 92:113-143) increase, suggesting fuller labeling of the actin filaments. These results are consistent with the idea (Poulsen, F. R., and J. Lowy, 1983. Nature [Lond.]. 303:146-152) that some of the diffuse scattering in rigor muscles is due to a random mixture of actin monomers with and without attached myosin heads (substitution disorder). In relaxed muscles, regardless of overlap, lowering the temperature from 24 to 4 degrees C practically abolishes the myosin layer lines (a result first obtained by Wray, J.S. 1987. J. Muscle Res. Cell Motil. 8:62 (a). Abstr.), whilst the diffuse scattering between these layer lines increases appreciably. Similar changes occur in the passage from rest to peak tetanic tension in live frog muscle (Lowy, J., and F.R. Poulsen. 1990. Biophys. J. 57:977-985). Cooling the psoas demonstrates that the intensity relation between the layer lines and the diffuse scattering is of an inverse nature, and that the transition occurs over a narrow temperature range (12-14 degrees C) with a sigmoidal function. From these results it would appear that the helical arrangement of the myosin heads is very temperature sensitive, and that the disordering effect does not depend on the presence of actin. Measurements along the meridian reveal that the intensity of the diffuse scattering increases relatively little and does so in a nearly linear manner: evidently the axial order of the myosin heads is much less temperature sensitive. The combined data support the view (Poulsen, F. R., and J. Lowy. 1983. Nature [Lond.]. 303:146-152) that in relaxed muscles a significant part of the diffuse scattering originates from disordered myosin heads. The observation that the extent of the diffuse scattering is greater in the equatorial than in the meridional direction suggests that the disordered myosin heads have an orientation which is on average more parallel to the filament axis.

Studies of the diffuse x-ray scattering from contracting frog skeletal muscles.

Using x-rays from synchrotron radiation, we studied diffuse scattering, sometimes together with the myosin layer lines. With an area detector, sartorius muscles and a time resolution of 150 ms, earlier results from semitendinosus muscles contracting isometrically at 6 degrees C (Lowy, J., and F. R. Poulsen. 1987. J. Mol. Biol. 194:595-600) were confirmed and extended. Evidence from intensity changes both in the diffuse scattering and in the myosin layer lines showed that the majority of the heads become disordered at peak tetanic tension. With a linear detector and a time resolution of 5 ms, it was found that during tension rise the intensity increase of the diffuse scattering (which amounted maximally to 12% recorded near the meridian) runs approximately 20 ms ahead of the mechanical change, comparing half-completion times. This suggests that an appreciable number of heads change orientation before peak tension is reached. In quick release experiments the diffuse scattering intensity showed very little change. Recorded near the meridian during rapid shortening, however, it decreased progressively with a half-time of approximately 40 ms. This change amounted to approximately 35% of that observed during the initial tension rise. We interpret this to indicate that during rapid shortening a certain number of heads assume an orientation characteristic of the relaxed state. Viewed in the context of the behavior of the first myosin layer line and the (1, 1) equatorial reflection in similar experiments (Huxley, H. E., M. Kress, A. R. Faruqi, and R. M. Simmons. 1988. Molecular Mechanism of Muscle Contraction), the present results provide further support for the view that the diffuse scattering is mostly due to disordered myosin heads; whilst ordered heads produce the myosin layer lines (Poulsen, F. R., and J. Lowy.1983. Nature lLond.l. 303:146-152).

Diffuse X-ray scatter from myosin heads in oriented synthetic filaments.

X-ray results are presented concerning the structural state of myosin heads of synthetic filaments in threads. These were made from purified rabbit skeletal muscle myosin and studied by x-ray diffraction and electron microscopy by Cooke et al. (Cooke, P. H., E. M. Bartels, G. F. Elliott, and R. A. Hughes, 1987, Biophys. J., 51:947-957). X-ray patterns show a meridional peak at a spacing of 14.4 nm. We concentrate here on the only other feature of the axial pattern: this is a central region of diffuse scatter, which we find to be similar to that obtained from myosin heads in solution (Mendelson, R. A., K. M. Kretzschmar, 1980, Biochemistry, 19:4103-4108). This means that the myosin heads have very large random displacements in all directions from their average positions, and that they are practically randomly oriented. The myosin heads do not contribute to the 14.4-nm peak, which must come entirely from the backbone. Comparison with x-ray data from the unstriated Taenia coli muscle of the guinea pig indicates that in this muscle at least 75% of the diffuse scatter comes from disordered myosin heads. The results confirm that the diffuse scatter in x-ray patterns from specimens that contain myosin filaments can yield information about the structural behavior of the myosin heads.

X-ray study of myosin heads in contracting frog skeletal muscle.

Using synchrotron radiation, the behaviour of the diffuse X-ray scatter was investigated in the relaxed and active phases of auxotonic and isometric contractions. Muscles were stimulated tetanically for 0.75 of a second, leaving intervals of three minutes between successive contractions. In isometric contractions the scatter is very asymmetric, which means that the myosin heads have a strongly preferred orientation. During tension rise the scatter expands in the meridional direction and contracts in the equatorial direction, the maximal local intensity change being about 20%. The shape change indicates that on average the myosin heads become oriented more perpendicularly to the fibre axis. The distribution of orientations at peak tension is quite different from that we found previously in X-ray scattering data from rigor muscles. In auxotonic contractions where muscles shorten against an increasing tension the scatter is practically circularly symmetrical. This suggests that during shortening the myosin heads go evenly through a wide range of orientations. It is concluded that the results from both the auxotonic and isometric experiments provide strong support for the rotating myosin head model. In isometric contractions the transition between the relaxed phase and peak tension is accompanied by an overall increase in scattering intensity of about 10%: this corresponds to a relative increase in the fraction of disordered myosin heads by almost 30%.

Application of potential energy calculations to the determination of muscle structure from X-ray data with special reference to the configuration of myosin heads.

A method that relates molecular structure to the forces that maintain it and to its X-ray diffraction pattern is described and applied to muscle. In a computer model, the potential energy of the movable components (here the myosin heads) is minimized by letting them move down the steepest gradient in three dimensions from a variety of starting positions. Initial values are assumed for the parameters that determine the forces, and for those that define the structure and arrangement of the fixed components. The X-ray pattern expected from the resulting structures can be calculated in a straightforward manner and compared with relevant observed data. Discrepancies can then be minimized by varying the values initially assumed for the parameters, as in the conventional "trial and error" method. This first application of the present method is concerned with the effects of the hexagonal lattice on the myosin head configuration in thick filaments of the type found in vertebrate skeletal muscle. For that purpose, a very simple model was used with the following main features: smooth cylinders for the thin filaments and for the thick filament backbones, two spherical heads attached by Hookean springs to each point of a 9/3 helix on the surface of the backbone, and repulsive forces of the electrostatic double-layer type acting between each head and all other surfaces. The myosin head configuration was calculated for an isolated thick filament and a study was made of the effects of packing such filaments into a hexagonal lattice of various side spacings in the presence or absence of thin filaments. For the isolated filament, it was found that the 9/3 helical symmetry is maintained in the myosin head configuration and that the two heads of each molecule are splayed azimuthally. When such filaments are packed into the hexagonal lattice with thin filaments present, the 9/3 helical symmetry of the myosin head configuration is lost. As the lattice side spacing is reduced, the myosin heads become increasingly displaced not only in the radial and azimuthal directions but also in the axial direction, although they interact primarily with smooth cylinders. The axial separation of the two heads in each molecule becomes different in one level from that in the other two in the 43 nm axial repeat, thus increasing the repeat in projection onto the axis from 14.3 to 43 nm. This effect may contribute to the "forbidden meridionals" described by Huxley & Brown (1967).(ABSTRACT TRUNCATED AT 400 WORDS)

Small-angle X-ray scattering from myosin heads in relaxed and rigor frog skeletal muscles.

Low-angle X-ray diffraction patterns from relaxed and non-overlap rigor muscles show a central region of diffuse scattering (disk) which is circularly symmetrical, behaves as solution scattering and comes predominantly from myosin heads. In full-overlap rigor the disk is compressed in the diagonal direction, indicating that the myosin heads have a bent shape and a preferred orientation consistent with a 45 degree angle of attachment to actin.

Time-resolved X-ray diffraction studies of the structural behaviour of myosin heads in a living contracting unstriated muscle.

The intensities of three regions of the low-angle X-ray diffraction pattern from a molluscan unstriated muscle have been followed during tension generation at a time resolution of 0.5-1 s using synchrotron radiation. The observed intensity changes can be reasonably interpreted in terms of myosin cross-bridge movements during the contractile cycle. A model that accounts for the intensity changes suggests that myosin heads move out from the thick filament during activation and attach to actin sites to produce tension with a small delay. During relaxation from both phasic and tonic contractions the heads remain attached to actin sites longer than it takes for tension to decay.

Solubilization of an alpha-bungarotoxin-binding component from rat brain.

Binding of [125I]-alpha-bungarotoxin to rat brain was investigated. Picomole quantities of specific toxin binding sites per gram of fresh tissue were found in particulate preparations as well as detergent extracts of whole brain. The toxin-binding macromolecules can be solubilized in low concentrations of Triton X-100. Specific binding occurs to a single class of sites with a dissociation constant of 5.6 X 10(-11) M. The association rate constant in 10 mM sodium phosphate, pH 7.4, was determined to be 6.8 X 10(5) M-1 s-1; the half-life of the complex was found to be 5.1 h, corresponding to a dissociation rate constant of 3.8 X 10(-5) s-1. The binding macromolecules resemble peripheral nicotinic acetylcholine receptors in toxin binding kinetics, solubility, isoelectric point, and hydrodynamic properties.