The structure of a molecular motor.
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Journal Article↗
Biomedical subjects
R Cooke
Publications and source records attributed to R Cooke.
The use of differing nucleotides to investigate cross-bridge kinetics.
We have investigated the ability of the nucleotides GTP, CTP, and 1-N6-etheno-2-aza-ATP (aza-ATP) to support contraction of chemically skinned rabbit psoas fibers. Working at 10 degrees C, millimolar concentrations of all nucleotides relaxed fibers in the absence of calcium. In active fibers, GTP served as a very poor substrate with isometric tension, isometric GTPase rate, and maximum shortening velocity (Vmax) all less than 10% of those obtained with ATP. Aza-ATP was only a slightly better substrate. CTP, on the other hand, was an effective substrate with mechanical parameters which were 65-100% those obtained with ATP, and with a hydrolysis rate that exceeded that of ATP. For all three ligands, Vmax followed Michaelis-Menten saturation behavior with values for Km which were from 2.5 to 12 times greater than that for ATP, showing that the analogs bound slowly to myosin in the fibers. Increasing concentrations of orthophosphate inhibited tension with CTP, to a lesser extent with aza-ATP, but not all with GTP. A combination of the mechanical data obtained in fibers with the kinetic data obtained in solution (White, H.D., Belknap, B., and Jiang, W. (1993) J. Biol. Chem. 268, 10039-10045) is used to better define the actomyosin interaction in fibers.
Adenosine Triphosphate↗
Determination of the myosin step size from mechanical and kinetic data.
During muscle contraction, work is generated when a myosin cross-bridge attaches to an actin filament and exerts a force on it through some power-stroke distance, h. At the end of this power stroke, attached myosin heads are carried into regions where they exert a negative force on the actin filament (the drag stroke) and where they are released rapidly from actin by ATP binding. Although the length of the power stroke remains controversial, average distance traversed in the drag-stroke region can be determined when one knows both rate of cross-bridge dissociation and filament-sliding velocity. At maximum contraction velocity, the average force exerted in the drag stroke must balance that exerted in the power stroke. We discuss here a simple model of cross-bridge interaction that allows one to calculate the force exerted in the drag stroke and to relate this to the power-stroke distance h traversed by cross-bridges in the positive-force region. Both the rate at which myosin can be dissociated from actin and the velocity at which an actin filament can be translated have been measured for a series of myosin isozymes and for different substrates, producing a wide range of values for each. Nonetheless, we show here that the rate of myosin dissociation from actin correlates well with the velocity of filament sliding, providing support for the simple model presented and suggesting that the power stroke is approximately 10 nm in length.
Actins↗
Steroids for babies.
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Adrenal Cortex Hormones↗
Orientation and rotational dynamics of spin-labeled phalloidin bound to actin in muscle fibers.
We have used electron paramagnetic resonance spectroscopy (EPR) to investigate the orientational distribution of actin in thin filaments of glycerinated muscle fibers in rigor, relaxation, and contraction. A spin-labeled derivative of a mushroom toxin, phalloidin (PHSL), was bound to actin in the muscle fibers (PHSL-fibers). The EPR spectrum of unoriented PHSL-labeled myofibrils consisted of three sharp lines with a splitting between the outer extrema (2T parallel') of 42.8 +/- 0.1 G, indicating that the spin labels undergo restricted nanosecond rotational motion within an estimated half-cone angle of 76 degrees. When the PHSL-fiber bundle was oriented parallel to the magnetic field, the splitting between the zero-crossing points (2T') was 42.7 +/- 0.1 G. When the fiber bundle was perpendicular to the magnetic field, 2T' decreased to 34.5 +/- 0.2 G. This anisotropy shows that the motion of the probe is restricted in orientation by its binding site on actin, so that the EPR spectrum of PHSL-fiber bundles would be sensitive to small changes in the mean axial orientation of the PHSL-actin interface. No differences in the EPR spectra were observed in fibers during rigor, relaxation, or contraction, indicating that the mean axial orientation of the PHSL binding site changes by less than 5 degrees, and that the amplitude of nanosecond probe rotational motion, which should be quite sensitive to the local environment of the phalloidin, changes by no more than 1 degree.(ABSTRACT TRUNCATED AT 250 WORDS)
Actins↗
Fine structure and evolution of the rDNA intergenic spacer in rice and other cereals.
The intergenic spacer of a rice ribosomal RNA gene repeating unit has been completely sequenced. The spacer contains three imperfect, direct repeated regions of 264-253 bp, followed by a related but more highly divergent region. Detailed analysis of the sequence allows the presentation of an evolutionary scenario in which the 264-253-bp repeats are derived from an ancestral 150-bp sequence by deletion and amplification. Comparison of the rice sequence with those of maize, wheat, and rye shows that, despite considerable divergence from the ancestral sequence, several regions have been highly conserved, suggesting that they may play an important role in the structure and/or expression of the ribosomal genes.
Base Sequence↗
Synthesis of non-nucleotide ATP analogues and characterization of their chemomechanical interaction with muscle fibres.
To probe the substrate requirements for the actomyosin chemomechanical interaction, the effects of a series of eight new non-nucleotide ATP analogues on actomyosin-catalysed hydrolysis rates and on fibre mechanics have been investigated. These analogues have substitutions of new functional groups at the 2- and 4-positions of the ATP analogues, 2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate (NANTP), and 3-[(4-nitrophenyl)amino]propyl triphosphate (PrNANTP). Previous work has shown NANTP but not PrNANTP will support active tension and shortening in skinned muscle fibres in a manner almost identical to ATP. Here all 2- and 4-phenyl substituted analogues had myosin subfragment 1 (S1) NTPase hydrolysis rates higher than ATP and the rates were stimulated by addition of actin. In general, the replacement of the 4-azido group of NANTP with -H, -NO2 or -NH2 had small effects on fibre mechanics while replacement of 2-NO2 group with -H or -NH2 dramatically lowered the ability of the new analogues to support active tension and shortening. All PrNANTP-based analogues were ineffective in supporting active tension or shortening. We found no correlation between S1 or actoS1 NTPase rates and any mechanical parameters. However, for all analogues there was a strong correlation between the maximal velocity of shortening (Vmax) and isometric tension (P0). A three-state, chemomechanical model is proposed in which the analogues effect the transition rate into a strongly-bound, force-producing crossbridge state to account for this correlation. These studies identify 2-[(2-nitrophenyl)amino]ethyl triphosphate as the chemically simplest ATP analogue which closely mimics the effect of ATP in skinned muscle fibres.
Actins↗
Nucleotide sequence of a cDNA clone encoding ubiquitous beta-amylase in rye (Secale cereale L.).
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Amino Acid Sequence↗
cDNA encoding a putative 10-kilodalton chaperonin from Arabidopsis thaliana.
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Amino Acid Sequence↗
Outcome at 8 years for children with birth weights of 1250 g or less.
The educational, motor, and behavioural performance of a hospital based cohort of 51 children aged 8 years with birth weights of 1250 g or less is reported, as part of a longitudinal study. Compared with age, race and sex matched classmates, who were examined at school at the same visit, the very low birthweight (VLBW) group performed less well on the basic mathematics test A, the Schonell spelling test, and the test of motor impairment. Reading performance was also poorer in this group. Twenty three (45%) VLBW children were having difficulty with one or more school subjects compared with 11 (19%) controls, and 15 (26%) had difficulties in two or more areas, compared with three (5%) controls. Parents of VLBW children reported a similar frequency of behavioural problems to controls but teachers identified characteristics typical of emotional disorders and overactivity more frequently among the VLBW group. Motor testing at 6 years of age was the best predictor of school problems at 8 years, correctly identifying 15/16 children with multiple problems with a low (33%) positive predictive value but a high (98%) negative predictive value. Children with birth weights of 1250 g or less and no major impairment have a high frequency of learning difficulties that become more apparent with advancing age. Such problems may be predicted at an earlier age by detailed motor testing.
Child↗
A model of stress relaxation in cross-bridge systems: effect of a series elastic element.
Many experimental protocols employed in the study of muscle mechanics use tension transients as a probe of the magnitudes of the kinetic rates in the underlying cross-bridge dynamics. These transients could potentially be modified by the elastic elements that exist both within the fiber and at the points of attachment to the experimental apparatus. To better understand the magnitude of such modifications, we have used computer simulation to investigate the transients that would be expected for cross bridges acting on an actin filament attached to an elastic element. The original model of cross-bridge mechanics by A.F. Huxley was used (Prog. Biophys. 7: 255-318, 1957). After an isometric equilibrium is achieved, a tension transient is produced by changing the dissociation rate constant, g1, while holding the attachment rate constant, f1, fixed. This decreases the number of attached, force-producing cross bridges. We find that the tension transients are markedly slowed by the presence of even a few (> or = 2) nanometers of series elastic strain per half-sarcomere. Thus some rate constants inferred from mechanical transients (e.g., those induced by caged ligands) may underestimate the actual kinetic rates of the cross-bridge processes.
Animals↗
The symptom structure of panic attacks in depressed and anxious patients.
This study examined the panic symptom profiles of three diagnostic groups: those with panic disorder and no history of major depression; those with panic disorder with a history of major depressive episode but no current depression; and those current major depression with panic disorder. Patients were compared on the frequency of specific panic attack symptoms based on structured interview responses. The symptom profiles of all three groups were significantly correlated. The patients with past and current depressive episodes had the most similar symptom structure.
Adult↗
The changing incidence of chronic lung disease.
This paper reports an examination of the incidence of chronic lung disease in premature infants admitted to the neonatal intensive care unit at Liverpool Maternity Hospital. From January 1980 to December 1989 details were obtained for these infants such as gestation, weight, sex, survival to discharge and ventilatory support received. The findings show that a total of 242 infants had developed chronic lung disease; one-third of these were born in 1988 or 1989. Logistic regression suggests that chronic lung disease was significantly associated with being male, low birthweight, low gestation, surviving to discharge, and being born in 1988 or 1989. Among infants at 'high risk' of this condition, mortality had significantly decreased and the incidence had significantly increased. This increase is not fully explained by changes in the population admitted during the last decade. A rising workload should be anticipated in terms of the specialised follow-up and care required by these babies.
England↗
Insect crossbridges, relaxed by spin-labeled nucleotide, show well-ordered 90 degrees state by X-ray diffraction and electron microscopy, but spectra of electron paramagnetic resonance probes report disorder.
The structure of glycerinated Lethocerus insect flight muscle fibers, relaxed by spin-labeled ATP and vanadate (Vi), was examined using X-ray diffraction, electron microscopy and electron paramagnetic resonance (e.p.r.) spectra. We obtained excellent relaxation of MgATP quality as determined by mechanical criteria, using vanadate trapping of 2' spin-labeled 3' deoxyATP at 3 degree C. In rigor fibers, when the diphosphate analog is bound in the absence of Vi, the probes on myosin heads are well-ordered, in agreement with electron microscopic and X-ray patterns showing that myosin heads are ordered when attached strongly to actin. In relaxed muscle, however, e.p.r. spectra report orientational disorder of bound (Vi-trapped) spin-labeled nucleotide, while electron microscopic and X-ray patterns both show well-ordered bridges at a uniform 90 degrees angle to the filament axis. The spin-labeled nucleotide orientation is highly disordered, but not completely isotropic; the slight anisotropy observed in probe spectra is consistent with a shift of approximately 10% of probes from angles close to 0 degrees to angles close to 90 degrees. Measurements of probe mobility suggest that the interaction between probe and protein remains as tight in relaxed fibers as in rigor, and thus that the disorder in relaxed fibers arises from disorders of (or within) the protein and not from disorder of the probe relative to the protein. Fixation of the relaxed fibers with glutaraldehyde did not alter any aspect of the spectrum of the Vi-trapped analog, including the slight order observed, showing that the extensive inter- and intra-molecular cross-linking of the first step of sample preparation for electron microscopy had not altered relaxed crossbridge orientations. Two models that may reconcile the apparently disparate results obtained on relaxed fibers are presented: (1) a rigid myosin head could possess considerable disorder in the regular array about the thick filament; or (2) the nucleotide site could be on a disordered, probably distal, domain of myosin, while a more proximal region is well ordered on the thick filament backbone. Our findings suggest that when e.p.r. probes signal disorder of a local site or domain, this is complementary, not contradictory, to signals of general order. The e.p.r. spectra show that a portion of the myosin molecule can be disordered at the same time as the X-ray diffraction and electron microscopy show the bulk of myosin head mass to be uniformly oriented and regularly arrayed.
Animals↗
A study of L-thyroxine replacement.
We studied consecutive requests for in vitro thyroid function tests on patients receiving L-thyroxine (T4) for primary hypothyroidism for a three month period May to July 1990, and waited a further three months before ascertaining any changes made to the daily dose of T4. Serum sensitive TSH (sTSH) concentrations were normalised in only 43% of patients treated by a hospital general physician, 50% of patients treated by a specialist endocrinologist and 52% of patients treated by their primary care physician. The respective figures for elevated serum sTSH concentrations were 43%, 33% and 32% and for suppressed levels 15%, 17% and 15%. The dose of T4 required to normalise serum sTSH levels was approximately 110 micrograms/d. In 36% of those patients with suppressed serum sTSH levels the daily dose of thyroxine was reduced. This achieved a mean reduction in the daily dose of 20 micrograms. In 30% of those with raised serum sTSH levels the daily dose of thyroxine was increased. This achieved a mean increase in the daily dose of 15 micrograms. Patients receiving T4 may have raised serum free T4 (fT4) despite normal sTSH levels indicating that serum fT4 measurement is not the test of choice for screening for adequacy of T4 replacement.
Female↗
Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers.
We have measured the orientation of a region of the myosin head, close to the junction with the rod, during active force generation. Paramagnetic probes were attached specifically to a reactive cysteine (Cys 125) of purified myosin light chain 2 (LC2) and exchanged into myosin heads in glycerinated rabbit psoas muscle. Electron paramagnetic resonance spectroscopy was used to monitor the orientation of the probes. Previous work has shown that the LC2 bound spin probes are significantly ordered in rigor and muscle in the presence of adenosine diphosphate (ADP). In contrast, there is a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, all of the LC2 bound spin probes (98 +/- 1.6%) show an angular distribution similar to that of relaxed muscle. These findings contrast with results obtained from probes attached to Cys 707 on the cross-bridge, located close to the actin binding site, where, during active force generation, a proportion of the spin probes were ordered as in rigor, whereas the remaining probes were disordered as in relaxation. To test the hypothesis that this ordered component is due to modification of Cys 707, we measured the spectra obtained from probes attached to LC2 in fibers modified at Cys 707. The modification of Cys 707 did not produce an ordered component in these spectra. The absence of an ordered component at the LC2 site limits the populations of some states in active fibers. An actin/myosin/ADP state is thought to be the major force-producing state. Our present results show that the populations of states with ordered probes on LC2 are < 2% in active fibers; thus, the major force-producing state is different from the one obtained by addition of ADP to rigor fibers.
Adenosine Triphosphate↗
Contraction of glycerinated rabbit slow-twitch muscle fibers as a function of MgATP concentration.
We have measured the isometric tension and force-velocity relationships of glycerinated rabbit slow-twitch semimembranosus muscle as a function of MgATP concentration ([MgATP]) and have compared the results with those obtained previously from fast-twitch psoas muscle. We find that isometric tension decreases as [MgATP] increases. The magnitude of the decrease is not as great as observed in psoas. Maximum shortening velocity (Vmax) exhibits classical Michaelian saturation behavior with respect to [MgATP] with a Michaelis constant (Km) for half-maximal velocity of 18 microM and a value at saturating [MgATP] of 0.6 muscle lengths/s. Similar values were observed in fibers from soleus, another slow-twitch muscle. The corresponding values in rabbit psoas muscle are 150 microM and 1.6 lengths/s. Compared with psoas, in semimembranosus muscle Km decreases by a factor of approximately 10, whereas Vmax decreases by about a factor of 3. Thus, although in a nonphysiological regime, at low [MgATP], a "fast" muscle actually has a lower shortening velocity than a "slow" muscle.
Adenosine Triphosphate↗
Natural killer cell activity in very low birth weight infants.
The exact role of natural killer (NK) cells in host defense is unclear, but they may be important as an early response to certain infections. We evaluated NK cell phenotype and activity in premature very low birth weight infants (VLBWI) (n = 52) with an average gestational age of 29.3 wk (24-35 wk) and an average birth weight of 1124 g (537-1480 g). All patients initially were evaluated within 7 d of birth. Samples also were obtained at 2, 4, and 6 wk in some infants. The proportion of mononuclear cells expressing the phenotypic marker of NK cells (NKH-1; CD56) was significantly lower in VLBWI than in adults (2.5 +/- 1.4 versus, 12.5 +/- 7.8%, p less than 0.0001) or term infants (2.5 +/- 1.4 versus 9.5 +/- 7.1%, p less than 0.0001). VLBWI also had significantly diminished NK activity expressed as the percentage of specific lysis compared with adults (4.7 +/- 4.4 versus 32.3 +/- 14.5%, p less than 0.0001) or term infants (4.7 +/- 4.4 versus 15.5 +/- 10.8%, p less than 0.0001). Both the number of cells expressing the NK phenotype and the NK lytic activity in VLBWI increased in the 6 wk after birth. NK activity in VLBWI was enhanced by IL-2 and in most cases by interferon-gamma.
Adult↗