Towards a unified theory of muscle contraction. I: foundations.

Molecular models of contractility in striated muscle require an integrated description of the action of myosin motors, firstly in the filament lattice of the half-sarcomere. Existing models do not adequately reflect the biochemistry of the myosin motor and its sarcomeric environment. The biochemical actin-myosin-ATP cycle is reviewed, and we propose a model cycle with two 4- to 5-nm working strokes, where phosphate is released slowly after the first stroke. A smaller third stroke is associated with ATP-induced detachment from actin. A comprehensive model is defined by applying such a cycle to all myosin-S1 heads in the half-sarcomere, subject to generic constraints as follows: (a) all strain-dependent kinetics required for actin-myosin interactions are derived from reaction-energy landscapes and applied to dimeric myosin, (b) actin-myosin interactions in the half-sarcomere are controlled by matching rules derived from the structure of the filaments, so that each dimer may be associated with a target zone of three actin sites, and (c) the myosin and actin filaments are treated as elastically extensible. Numerical predictions for such a model are presented in the following paper.

Mapping the actin filament with myosin.

Structural studies have shown that the heads of the myosin motor molecule bind preferentially to "target zones" of favorably oriented sites on the helices of the actin filament. We present direct evidence for target zones from the interactions of a single myosin head with an actin filament held between two optically trapped beads. With compliant traps, thermal motions of the filament allow the fixed myosin-S1 to interact with at least two zones, observed as a bi-modal distribution of filament displacements due to myosin binding, whose spacing is near the 36-nm helix repeat distance. The number of binding events and the "apparent working stroke" (mean displacement with myosin bound), vary periodically as the filament is moved past the fixed myosin by displacing the traps; observed periods are close to 36 nm and the apparent stroke varies from 0-10 nm. We also observe a strong modulation at the 5.5-nm actin monomer repeat confirming that myosin interacts with a single strand and that the actin is not free to rotate. Each interaction can be assigned to an actin monomer and each active zone on the helix is made up of three actin monomers.

Hidden-Markov methods for the analysis of single-molecule actomyosin displacement data: the variance-Hidden-Markov method.

In single-molecule experiments on the interaction between myosin and actin, mechanical events are embedded in Brownian noise. Methods of detecting events have progressed from simple manual detection of shifts in the position record to threshold-based selection of intermittent periods of reduction in noise. However, none of these methods provides a "best fit" to the data. We have developed a Hidden-Markov algorithm that assumes a simple kinetic model for the actin-myosin interaction and provides automatic, threshold-free, maximum-likelihood detection of events. The method is developed for the case of a weakly trapped actin-bead dumbbell interacting with a stationary myosin molecule (Finer, J. T., R. M. Simmons, and J. A. Spudich. 1994. Nature. 368:113-119). The algorithm operates on the variance of bead position signals in a running window, and is tested using Monte Carlo simulations to formulate ways of determining the optimum window width. The working stroke is derived and corrected for actin-bead link compliance. With experimental data, we find that modulation of myosin binding by the helical structure of the actin filament complicates the determination of the working stroke; however, under conditions that produce a Gaussian distribution of bound levels (cf. Molloy, J. E., J. E. Burns, J. Kendrick-Jones, R. T. Tregear, and D. C. S. White. 1995. Nature. 378:209-212), four experiments gave working strokes in the range 5.4-6.3 nm for rabbit skeletal muscle myosin S1.

Use of a behavioural programme in the first 3 months to prevent infant crying and sleeping problems.

OBJECTIVE: To assess the effectiveness of a behavioural programme introduced in the first 3 months of age in preventing infant crying and sleeping problems. Two issues were addressed: (i) which elements of the behavioural programme would parents implement; and (ii) whether the behavioural programme was more effective in reducing infant crying and encouraging night-time sleeping than an educational intervention or the routine services. METHODOLOGY: Mothers and newborns were assigned at random to the behavioural programme (n = 205), educational intervention (n = 202), or control (n = 203) group. Behaviour diaries kept before randomization and at 3, 6, 9 and 12 weeks of age were used to measure implementation of the interventions and infant behaviour, including crying and sleeping. Crying and sleeping problems were followed up using questionnaire measures at 9 months of age. RESULTS: The educational intervention did not change parental care behaviour. One element of the behavioural programme, a focal feed between 10 PM and midnight, was not implemented. A second element, stretching of interfeed intervals after 3 weeks of age, was implemented initially, but not maintained at older ages. The third element, which asked parents to emphasise day and night differences in the environment, and to settle their babies in the cot and minimise interaction at night, was carried out by more parents in the behavioural group than in the other groups. This led to an increase of around 10% in the number of babies who slept for 5 or more hours at night (a definition of sleeping through the night) at 12 weeks of age. Fewer behavioural programme parents sought help for crying and sleeping problems between 3 and 9 months of age. CONCLUSION: The behavioural programme produced a modest increase in the number of infants who slept through the night by 12 weeks of age. The results are discussed in relation to other findings, which bear on the programme's adoption for routine health-care policy and practice.

Economic evaluation of strategies for managing crying and sleeping problems.

AIMS: To estimate the financial cost to the NHS of infant crying and sleeping problems in the first 12 weeks of age and to assess the cost effectiveness of behavioural and educational interventions aimed at reducing infant crying and sleeping problems relative to usual services. METHODS: A cost burden analysis and cost effectiveness analysis were conducted using data from the Crying Or Sleeping Infants (COSI) Study, a three armed prospective randomised controlled trial that randomly allocated 610 mothers to a behavioural intervention (n = 205), an educational intervention (n = 202), or existing services (control, n = 203). Main outcome measures were annual total cost to the NHS of infant crying and sleeping problems in the first 12 weeks, and incremental cost per interruption free night gained for behavioural and educational interventions relative to control. RESULTS: The annual total cost to the NHS of infant crying and sleeping problems in the first 12 weeks was 65 pound sterling million (US$104 million). Incremental costs per interruption free night gained for the behavioural intervention relative to control were 0.56 pound sterling (US$0.92). For the educational intervention relative to control they were 4.13 pound sterling (US$6.80). CONCLUSIONS: The annual total cost to the NHS of infant crying and sleeping problems is substantial. In the cost effectiveness analysis, the behavioural intervention incurred a small additional cost and produced a small significant benefit at 11 and 12 weeks of age. The educational intervention incurred a small additional cost without producing a significant benefit.

Elasticity of the red cell membrane and its relation to hemolytic disorders: an optical tweezers study.

We have used optical tweezers to study the elasticity of red cell membranes; force was applied to a bead attached to a permeabilized spherical ghost and the force-extension relation was obtained from the response of a second bead bound at a diametrically opposite position. Interruption of the skeletal network by dissociation of spectrin tetramers or extraction of the actin junctions engendered a fourfold reduction in stiffness at low applied force, but only a twofold change at larger extensions. Proteolytic scission of the ankyrin, which links the membrane skeleton to the integral membrane protein, band 3, induced a similar effect. The modified, unlike the native membranes, showed plastic relaxation under a prolonged stretch. Flaccid giant liposomes showed no measurable elasticity. Our observations indicate that the elastic character is at least as much a consequence of the attachment of spectrin as of a continuous membrane-bound network, and they offer a rationale for formation of elliptocytes in genetic conditions associated with membrane-skeletal perturbations. The theory of Parker and Winlove for elastic deformation of axisymmetric shells (accompanying paper) allows us to determine the function BH(2) for the spherical saponin-permeabilized ghost membranes (where B is the bending modulus and H the shear modulus); taking the literature value of 2 x 10(-19) Nm for B, H then emerges as 2 x 10(-6) Nm(-1). This is an order of magnitude higher than the value reported for intact cells from micropipette aspiration. Reasons for the difference are discussed.

In vitro motility speed of slow myosin extracted from single soleus fibres from young and old rats.

1. Isolated soleus muscle fibres from aged rats contract more slowly than those from young rats. To determine whether this effect is due to a difference between the myosin molecules, we measured the rate at which actin filaments are driven over a myosin coated surface in the presence of ATP by using a novel in vitro motility assay where myosin is extracted from single muscle fibre segments. 2. Motility was dependent on the myosin density on the coverslip. In regions of high myosin density, actin motility was orientated parallel and anti-parallel to the direction of flow during myosin adhesion to the coverslip. In contrast, in regions of lower myosin density, actin motility was more random. The speed was about 20 % higher in the high density regions (P < 0.001). Further, the speed of filaments in the high density region, moving away or towards the fibre was less variable (P < 0.05) than that of more randomly moving filaments in the low density region. 3. The speed with myosin from slow soleus fibres of young adult rats (3-6 months old; v = 1.43 +/- 0.23 microm s-1; mean +/- s.d.) was faster (P < 0.001) than with myosin from aged rats (20-24 months old; v = 1.27 +/- 0.23 microm s-1). 4. No difference in myosin isoforms between young adult and aged fibres could be detected using electrophoretic and immunocytochemical techniques. Fibres of both ages expressed the beta/slow myosin heavy chain (MyHC) isoform and slow isoforms of essential and regulatory myosin light chains (MyLCs). 5. It is concluded that an age-related alteration in myosin contributes to the slowing of the maximum shortening velocity (V0) observed in soleus muscle fibres expressing the beta/slow MyHC isoform.

The effect of age on in vitro motility speed of slow myosin extracted from single rat soleus fibres.

The effect of age on the motor protein myosin was examined in a novel in vitro motility assay. Myosin was extracted from soleus fibres of young (3-6 month) and old (20-24 month) rats. All fibres expressed the type I myosin heavy chain (MyHC) and the slow isoforms of the myosin light chains (MyLCs). In vitro motility speed was significantly (P < 0.001) faster in the young adult (1.43 +/- 0.23 microm s-1) than in the aged group (1.27 +/- 0.23 microm s-1). The result indicates that the age-related decrease in contractile speed observed in slow fibres may be the effect of a change in the properties of myosin with age.

The EMBL nucleotide sequence database.

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl. html ) constitutes Europe's primary nucleotide sequence resource. DNA and RNA sequences are directly submitted from researchers and genome sequencing groups and collected from the scientific literature and patent applications (Fig. 1). In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute. Database releases are produced quarterly and are distributed on CD-ROM. EBI's network services allow access to the most up-to-date data collection via Internet and World Wide Web interface, providing database searching and sequence similarity facilities plus access to a large number of additional databases.

Elasticity and unfolding of single molecules of the giant muscle protein titin.

The giant muscle protein titin, also called connectin, is responsible for the elasticity of relaxed striated muscle, as well as acting as the molecular scaffold for thick-filament formation. The titin molecule consists largely of tandem domains of the immunoglobulin and fibronectin-III types, together with specialized binding regions and a putative elastic region, the PEVK domain. We have done mechanical experiments on single molecules of titin to determine their visco-elastic properties, using an optical-tweezers technique. On a fast (0.1s) timescale titin is elastic and force-extension data can be fitted with standard random-coil polymer models, showing that there are two main sources of elasticity: one deriving from the entropy of straightening the molecule; the other consistent with extension of the polypeptide chain in the PEVK region. On a slower timescale and above a certain force threshold, the molecule displays stress-relaxation, which occurs in rapid steps of a few piconewtons, corresponding to yielding of internal structures by about 20 nm. This stress-relaxation probably derives from unfolding of immunoglobulin and fibronectin domains.

Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.

The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged for labeled cgRLC in a low [Mg2+] rigor solution at 30 degrees C. Troponin and troponin C removed in this procedure were replaced. RLC exchange had little effect on active force production. X-ray diffraction showed normal structure in rigor after RLC exchange, but loss of axial and helical order in relaxation. In isolated myofibrils labeled cgRLC was confined to the regions of the sarcomere containing myosin heads. The ATR dipoles showed a preference for orientations perpendicular to the fiber axis, combined with limited nanosecond rotational motion, in all conditions studied. The perpendicular orientation preference was more marked in rigor than in either relaxation or active contraction. Stretching relaxed fibers to sarcomere length 4 microns to eliminate overlap between actin- and myosin-containing filaments had little effect on the orientation preference. There was no change in orientation preference when fibers were put into rigor at sarcomere length 4.0 microns. Qualitatively similar results were obtained with ATR-labeled rabbit skeletal RLC.

Establishing priorities for research in education within one college of nursing and midwifery.

The educational provision for nurses and midwives is currently undergoing immense change. These innovations encompass the organizational structure, the process of delivery and the projected outcomes for professional education within the UK. There is, however, a dearth of published research evidence designed to evaluate these educational changes. Nevertheless, anecdotal evidence suggests that within individual institutions small-scale, educationally-focused research is currently being conducted. These are usually 'one off' studies which address questions of personal interest to individual researchers rather than issues of importance to the organization or to the professions as a whole. the net result is a diverse research portfolio which, by its very nature, is wasteful in terms of lost opportunity to commission larger-scale research which might influence educational practice and in failing to direct effectively the energies and skills of researchers. In an attempt to address these issues and to set an agenda for research priorities within one college of nursing and midwifery, a four-round Delphi survey was conducted. This is the first of its kind to be reported in the UK literature. Fifty six of the teaching staff (77% of the total number of teaching staff) initially identified 213 issues which were grouped into 14 categories. The interrater reliability of the categorization process was checked on three separate occasions. In successive rounds, high response rates were maintained. To test for the manipulative effect of providing respondents with controlled feedback of their score in round 2, the third questionnaire was completed "blind' to previous scores. In the final round, participants were provided with feedback and invited to re-score and rank the items. The final round yielded 28 prioritized items. It was notable that the top ten issues primarily focused upon the pre-registration (Project 2000) provision encompassing both the preparation of students for professional practice and the changing role of the nurse/midwife teacher. This paper explores the implications of these findings for setting a research agenda within the organization.

Inhibition of unloaded shortening velocity in permeabilized muscle fibres by caged ATP compounds.

The effects of both the P3-1-(2-nitrophenyl)ethyl ester of adenosine 5'-triphosphate (NPE-caged ATP) and its separate diastereoisomers, and the P3-3',5'-dimethoxybenzoin ester of ATP (DMB-caged ATP) were studied on the unloaded shortening velocity of glycerinated rabbit psoas muscle fibres. The unloaded shortening velocities of the active fibres were measured as a function of ATP concentration (0.1-5 mM) using the 'slack-test' with and without 2 mM caged ATP. Shortening velocity followed a Michaelis-Menten relationship with ATP concentration, the Km for ATP being 170 microM. The caged ATP compounds inhibited shortening velocity, in a manner consistent with competitive inhibition, with a Ki of 1-2 mM. The R- and S-diastereoisomers of NPE-caged ATP showed the same degree of competitive inhibition of the shortening velocity, as did DMB-caged ATP. These observations suggest that caged ATP compounds bind to the ATPase site of the actomyosin where they compete with the substrate, Mg2+ ATP.

Concentrated Tris solutions for the preparation, depolymerization, and assay of actin: application to erythroid actin.

High concentrations of Tris are effective in dissociating actin-containing complexes, such as the red cell membrane cytoskeleton. A preparative procedure for red cell actin is based on the dissociation of the membrane skeletal complex in a buffer containing 1 M Tris hydrochloride, followed by gel filtration chromatography in the same medium. The actin is recovered as the monomer and is fully native, as judged by its critical concentration of polymerization, inhibition of DNase I, stimulation of myosin ATPase, and the appearance in the electron microscope of filaments, both bare and decorated with heavy meromyosin, and of magnesium ion-induced paracrystals. The Tris solution causes rapid depolymerization of F-actin with no denaturation, and the solution of monomeric actin in this medium is stable for many weeks in the cold; concentrated Tris is more reliable than guanidinium chloride for the depolymerization of F-actin in the estimation of total actin concentration by the DNase I inhibition assay.

Sensing an improvement: an experimental study to evaluate the use of aromatherapy, massage and periods of rest in an intensive care unit.

There is widespread belief that the use of aromatherapy and massage in an intensive care environment offers a means of increasing the quality of sensory input that patients receive, as well as reducing levels of stress and anxiety. Despite a wealth of anecdotal evidence in support of these claims, there have been few objective studies to evaluate the effects of these therapies. In this experimental study 122 patients admitted to a general intensive care unit were randomly allocated to receive either massage, aromatherapy using essential oil of lavender, or a period of rest. Both pre- and post-therapy assessments included physiological stress indicators and patients' evaluation of their anxiety levels, mood and ability to cope with their intensive care experience. Ninety-three patients (77%) were able to complete subjective assessments. There were no statistically significant differences in the physiological stress indicators or observed or reported behaviour of patients' ability to cope following any of the three interventions. However, those patients who received aromatherapy reported significantly greater improvement in their mood and perceived levels of anxiety. They also felt less anxious and more positive immediately following the therapy, although this effect was not sustained or cumulative.

Inhibition of ATP binding to myofibrils and acto-myosin subfragment 1 by caged ATP.

The inhibitory effect of P3-[1-(2-nitrophenyl)ethyl]adenosine 5'-triphosphate (caged ATP) on the binding of Mg2+-ATP to myofibrils was investigated. The most sensitive method was found to be the monitoring of single turnovers of [gamma-32P] ATP hydrolysis using the quench flow technique. The method was tested using ADP, which was found to have an inhibition constant of 145 microM, in agreement with previous reports. Caged ATP behaved as a simple competitive inhibitor of ATP binding with an inhibition constant of 1.6 mM. The inhibitory effect of these ligands on the binding of ATP to acto-myosin subfragment 1 was investigated using the same method. The inhibition constants of caged ATP and ADP were found to be 0.35 mM and 50 microM, respectively. This inhibitory effect of caged ATP on ATP binding accounts for the lower rate of ATP binding to fibers, deduced from caged ATP [(0.5-1) x 10(6) M-1 s-1], than that reported for acto-S1 (3.5 x 10(6) M-1 s-1) [Goldman, Y. E., Hibberd, M. G., & Trentham, D. R. (1984) J. Physiol. (London) 354, 577].

A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking.

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)