Biomedical subjects
Publications and source records attributed to R Cooke.
The complete nucleotide sequence of a very small plasmid whose presence and level in Pseudomonas cepacia have been linked to herbicide resistance is presented. The structural features of the plasmid are discussed.
The underlying stochastic nature of many models of the actomyosin interaction should result in fluctuations in both force and shortening velocity. In classical experimental approaches involving intact or glycerinated muscle preparations these fluctuations are too small to resolve owing to the large numbers of crossbridges involved. However, new experimental techniques allow mechanical measurements to be made in systems in which small numbers of myosin heads act on a single actin filament, or small numbers of kinesin molecules act on a single tubulin filament. In these systems, stochastic effects should be evident. To understand better the nature of the expected stochastic effects, we have used computer simulation to investigate the fluctuations predicted by the original model for muscle crossbridge mechanics proposed by A.F. Huxley. We consider three situations: (1) the translation of actin or tubulin filaments by myosin or kinesin motors immobilized on a fixed substrate, (2) the production of tension by ensembles of immobilized myosin which involve the displacement of an elastic load, and (3) the fluctuations in axial displacement of a single, bipolar myosin thick filament interacting with actin filaments as in a sarcomere. In all three cases, fluctuations are clearly evident in simulations involving small numbers of motors. For case (1), we show that translation velocities can vary with crossbridge density. Whether one motor translates a filament faster, slower or at the same speed as many motors depends on the relative magnitudes of the attachment and detachment rate functions. Analytical expressions are provided to quantitate this relationship. For case (2), we show that fluctuations predicted assuming perfectly isometric conditions differ form those observed when the 'isometric state' is achieved against an elastic load. 'Elastic damping' of the fluctuations in the system results from the presence of many attached motors. In case (3) we show that in spite of the presence of stochastic fluctuations which can destabilize the uniformity of filament overlap in a sarcomere, the magnitude of thick filament displacement is less than might be anticipated over time periods of in vivo contraction. Taken together, these simulations allow one to better interpret experimental data in terms of current models of motor function.
Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which labeled LC2 had been exchanged directly. However, the fraction of disordered probes was greater than seen in fibers. In summary, the above data indicate that the region of the myosin head proximal to the thick filament is ordered in rigor, and disordered in relaxation.
We have investigated the ability of the photoaffinity, nonnucleotide ATP analogues, 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP) and 2-[(4-azido-2-nitrophenyl) amino] propyl triphosphate (PrNANTP), to support active contraction in glycerinated rabbit psoas fibers. At millimolar concentrations, in the absence of calcium, both analogues relaxed fibers. In the presence of calcium, MgNANTP produced isometric tension and stiffness that were one-half to two-thirds the values obtained in MgATP. Maximum shortening velocity and the calcium-activated, myofibrillar catalyzed rate of hydrolysis were approximately the same for MgNANTP as for MgATP. With MgNANTP as the substrate, increasing concentrations of the diphosphate analogue, MgNANDP, inhibited shortening velocity but did not change isometric tension. The addition of increased concentrations of orthophosphate (P) decreased tension while shortening velocity increased. Thus, the effects of the hydrolysis products of NANTP were quite similar to those observed previously for ADP and P in the presence of MgATP. Taken together, these observations show that MgNANTP binds to, and functions in the active site of myosin in a manner quite analogous to MgATP. Thus, the aryl azido group should serve as a valid photoaffinity label for the purine portion of the active site. In contrast, MgPrNANTP, which differs from MgNANTP only in an extra CH2 spacer between the nitrophenyl ring and the triphosphate moiety did not support isometric tension or active shortening in the presence of calcium. Fiber stiffness increased in the presence of calcium and MgPrNANTP, with a calcium-activated, myofibrillar MgPrNANTPase which was about half that obtained with MgATP. Thus, in the presence of MgPrNANTP, cross-bridges appeared to be cycling through states that were attached to actin, but not producing force.
Trypsin inhibitors, alfa amylase inhibitors and hemagglutinins were determined in black beans (P. vulgaris) produced in Costa Rica. The effect of the traditional cooking on such antinutritional factors was also studied. The antinutritional factors were analyzed spectrophotometrically in the raw beans, as well as after several cooking periods of time. The results showed that alfa-amylase inhibitors were the most thermoresistant. After 30 min of cooking time there was a 33% of activity left from the initial activity of the raw beans. Approximately 80% of the antitryptic activity was destroyed at 9 min of cooking time. After 10 min of cooking time, only 1% of hemagglutinin activity was present.
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We have studied transcription from the cauliflower mosaic virus 19S and 35S promoters in a cell-free system derived from tobacco cells in suspension culture. While a whole-cell extract is incapable of detectable transcription from these promoters, successive purification by column chromatography allows the preparation of two fractions which contain all factors necessary for transcription from the 19S promoter. In contrast, transcription from the 35S promoter leads to the accumulation of short RNAs. This accumulation can only be partially alleviated by modifying the conditions of transcription.
STUDY OBJECTIVE: To examine (1) the effect of vitamin D intake (380 to 480 IU daily) on plasma 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)2D) concentrations and (2) the relationship of 1,25-(OH)2D to calcium and phosphorus absorption and retention in the very low birth weight infant receiving a preterm infant formula. SUBJECTS: Eleven "well" infants with a birth weight and gestational age (mean +/- SD) of 1078 +/- 128 gm and 29 +/- 1.9 weeks, respectively, were studied for a 3-week period. Weight and postnatal age (mean +/- SD) at the beginning of the study were 1132 +/- 56 gm and 16 +/- 6 days, respectively. All infants were fed a preterm infant formula and tolerated a full enteral intake (120 kcal/kg/day) for the duration of the study. INTERVENTIONS: Plasma 25-OHD and 1,25-(OH)2D concentrations were measured at the beginning of the study and at the beginning of each 48-hour balance period. Calcium and phosphorus balance studies (n = 33) were performed weekly. MAIN RESULTS: Plasma 25-OHD (30 +/- 10 ng/ml) and 1,25-(OH)2D (54 +/- 14 pg/ml) concentrations were normal at the beginning of the study. Plasma 25-OHD values did not change, but 1,25-(OH)2D values increased (p less than 0.001) throughout the study. Plasma 1,25-(OH)2D concentrations were not related to calcium or phosphorus absorption and retention, but were a linear function of postconceptional age. CONCLUSIONS: Normal vitamin D status and activity are maintained in the very low birth weight infant fed a high calcium formula (380 to 480 IU of vitamin D daily). Plasma 1,25-(OH)2D concentrations are not related to calcium absorption but are linearly related to maturity.
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Despite attempts to reduce their incidence, shunt infections remain a major complication of the treatment of hydrocephalus. Various forms of antimicrobial prophylaxis are in use, but no controlled, statistically valid trial has been conducted to assess their efficacy. Such a trial was therefore carried out and its design is described here. After a 1-year retrospective and prospective study by members of the United Kingdom Hydrocephalus Group to establish feasibility and infection rates, a statistical study showed that at least 712 patients would be required. Six centres were enrolled to fulfil these requirements, and ethical committee approval was obtained at each. The chosen prophylactic regimen was 10 mg vancomycin administered into the ventricular system during surgery. Adults and children undergoing insertion or revision of ventriculoperitoneal shunts were included unless they were receiving therapeutic antimicrobials. Randomisation was by computer-generated numbers. Controls received the antimicrobial regimen, if any, currently used in that centre, the only difference between the two groups being intraventricular vancomycin in the test group. Diagnosis of shunt infection included accepted clinical and microbiological criteria reinforced by measurement of serum C-reactive protein levels. Follow-up was for at least six months. After 2.5 years only 158 patients had been enrolled in the trial, 80 controls and 78 tests. There were 5 preventable infections in the control group and 2 in the test group. In view of the small total the planned statistical analysis was not possible. Therefore, while no problems were encountered with toxicity, the trial failed to enroll enough patients to answer the question of efficacy of antimicrobial prophylaxis in shunt surgery and the reasons for this are discussed.
Three hundred and twenty-four patients presenting to Wellington Women's Hospital with symptoms suspicious of an ectopic pregnancy were first studied by testing serum for HCG using a new simple and sensitive (25 mIU/mL) ICON pregnancy test. One hundred and thirty ICON tests were positive. Subsequent management: (a) Immediate laparoscopy noted 3 intrauterine and 15 ectopic pregnancies. (b) Twenty-five patients discharged on revised clinical grounds without pregnancies. (c) An immediate ultrasound examination of 87 women showed 58 intrauterine pregnancies (including 1 misdiagnosed ectopic pregnancy) and 29 ectopic pregnancies which were later confirmed at laparoscopy. One hundred and ninety-four ICON tests were negative including two false negative results in patients with ectopic pregnancies. To avoid both unnecessary laparoscopy or the discharge of patients with a possible ectopic pregnancy, patients with symptoms and signs suspicious of an ectopic pregnancy and who present to hospital should have a rapid sensitive HCG assay. Then, if the test is positive (sensitivity 96%), an ultrasound examination can assist in determining the likely site of the pregnancy.
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We have measured the effect of phosphate (Pi) on the tension and maximum shortening velocity of permeable rabbit psoas fibers. Work in a number of laboratories has established that addition of phosphate (0-25 mM) to active muscle fibers at physiological MgATP concentrations decreases isometric tension with little effect on the maximum shortening velocity. Here we extend these results to a wider range of Pi concentrations and to low MgATP concentrations. Low levels of Pi (approx. 150 microM-200 microM) were obtained by using sucrose phosphorylase and sucrose to reduce contaminating Pi in the solutions used to activate the fiber, and high levels (52-73 mM) were obtained by replacing acetate with Pi as the principal anion. In an activating solution containing either 50 microM or 4 mM MgATP, pH 6.2 or 7.0, isometric tension declines linearly with the logarithm of Pi concentration. Although the isometric tension decreases with increasing concentrations of H+ or MgATP, the slope of relative isometric tension as a function of log[Pi] is the same at the two values of pH and [MgATP]. At pH 7 and 4 mM MgATP, the velocity of contraction increased slightly as Pi increased from 0.2 to 52 mM. At 50 microM MgATP the velocity decreased slightly as Pi increased from 0.2 to 10 mM with a substantial decrease as Pi increased from 10 to 52 mM. These results are discussed in terms of models of cross-bridge energetics. The observation that force declines linearly with the logarithm of [Pi] is compatible with models in which a major force producing state occurs subsequent to Pi release.(ABSTRACT TRUNCATED AT 250 WORDS)
We have explored a model of crossbridge kinetics that explains many of the effects on steady-state muscle contraction of ligands that bind to the nucleotide site on myosin. The mathematical model follows the basic framework for crossbridge function first established by A. F. Huxley. In the model, detached crossbridges initially bind in a weakly attached, A.M.D.Pi state (A, actin; M, myosin; D, ADP; Pi, orthophosphate) at the beginning of the region of positive force production. Pi release then results in transition to a strongly-bound A.M.D state, as has been suggested by other investigators from both biochemical and mechanical data. Mg2+ ADP release and subsequent crossbridge detachment due to Mg2+ ATP binding to the A.M state occur at the end of the region of positive force production. Work in a number of laboratories has now defined the effects on steady-state contraction of variations in the concentrations of Mg2+ ATP, Mg2+ ADP and Pi. These data provide valuable constraints that can be used to further refine current models. The maximum velocity of shortening (V max) and ATPase activity of muscle fibres exhibit classical saturation behaviour with respect to Mg2+ ATP concentration, with Mg2+ ADP acting as a competitive inhibitor. The model can reproduce this behaviour. The model also explains the observations that increasing [Mg2+ ATP] decreases isometric tension and increasing [Mg2+ ADP] increases tension. As the concentration of Pi increases, model predictions suggest that tension should decrease approximately as log[Pi], that ATPase activity should decrease less than tension and that V max should be almost unchanged, as has been found experimentally. The model also demonstrates that the connection between the parameters of contraction and the free energy of hydrolysis of Mg2+ ATP can be complex.
Four pregnant and four castrated ewes were chronically instrumented for the measurement of external iliac blood flow to test the hypothesis that pregnancy alters alpha-adrenergic sensitivity in a major regional circulation. Complete dose-response curves were generated to methoxamine, phenylephrine, and norepinephrine. Pregnancy was associated with no change in methoxamine sensitivity, an increase in phenylephrine sensitivity, and a decrease in norepinephrine sensitivity. These differential changes in drug sensitivity suggest (1) the alpha 1-receptor population is functionally similar between the two groups of animals, (2) uptake 1 is inhibited, and (3) either catechol-O-methyltransferase activity is increased or the alpha 2- or beta-receptor population changes in this circulation during pregnancy. These data illustrate the complexity of the change in the adrenergic system during pregnancy.
To study long-lived mRNAs stored in radish (Raphanus sativus) seed, we have selected clones from a dry seed cDNA library by differential screening. One of these clones, p8B6, whose mRNAs are abundant in the dry seed, was characterized. This clone hybridizes to an RNA class of approximately 600 nucleotides whose accumulation begins during the desiccation phase, reaches its maximum level in the dry seed, and is no longer detectable in 12 hour old seedlings. mRNAs hybrid-selected by p8B6 encode four polypeptides, but only two are compatible with the size class of RNAs detected by Northern analysis. Three of them have previously been identified as major ;early germination' polypeptides, and their synthesis has been shown to be induced prematurely in immature embryos by a desiccation treatment. The protein deduced from the p8B6 nucleotide sequence is 9 kilodaltons in size, highly hydrophilic, rich in Gly and Glu, and contains no Cys, Trp, and lie. The amino acid sequence shares good homology with that of two recently described seed proteins: a cotton late embryogenesis abundant protein and the wheat early methionine-labeled protein. Southern blot analysis suggests that the p8B6 sequence belongs to a very small gene family. The exact function of the product encoded by p8B6 remains to be determined.
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The interaction between actin and myosin in the filament array of glycerinated muscle fibers has been monitored using paramagnetic probes and mechanical measurements. Both fiber stiffness and the spectra of probes bound to a reactive sulfydral on the myosin head were measured as the actomyosin bond was weakened by addition of magnesium pyrophosphate (MgPPi) and glycerol. In the absence of MgPPi, all myosin heads are attached to actin with oriented probes. When fibers were incubated in buffers containing MgPPi, a fraction of the probes became disordered, and this effect was greater in the presence of glycerol. To determine whether the heads with disordered probes were detached from actin, spin-labeled myosin subfragment-1 (MSL-S1) was diffused into unlabeled fibers, and the fractions bound to actin and free in the medium were correlated with the oriented and disordered spectral components. These experiments showed that the label was oriented when MSL-S1 was attached to actin in a ternary complex with the ligand and that all heads with disordered probes were detached from actin. Thus the fraction of oriented labels could be used to determine the fraction of heads attached to actin in a fiber in the presence of ligand. The fraction of myosin heads attached to actin decreased with increasing [MgPPi], and in the absence of glycerol approximately 50% of the myosin heads were dissociated at 3.3 mM ligand with little change in fiber stiffness. In the presence of 37% glycerol plus ligand, up to 80% of the heads could be detached with a 50% decrease in fiber stiffness. The data indicate that there are two populations of myosin heads in the fiber. All the data could be fit with a model in which one population of myosin heads (comprising approximately 50% of the total) sees an apparent actin concentration of 0.1 mM and can be released from actin with little change in fiber stiffness. A second population of myosin heads (approximately 50%) sees a higher actin concentration (5 mM) and is only released in the presence of both glycerol and ligand.