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R Cooke

Publications and source records attributed to R Cooke.

At least 73 records · Page 4Linked to original sources

An Arabidopsis thaliana cDNA complementing a hamster apoptosis suppressor mutant.

Programmed cell death or apoptosis is a process in which unwanted cells are eliminated during growth and development. In mammals, several genes have been identified whose products are necessary to prevent entry into the apoptotic process. We have isolated a clone from an Arabidopsis thaliana cDNA library whose predicted translation product shows highly significant similarity to the mammalian defender against apoptotic death 1 (DAD1) protein. Transformation of the mutant hamster tsBN7 cells, which undergo apoptosis at restrictive temperature, demonstrates that the plant protein is as efficient as human DAD1 in rescuing these hamster cells from apoptosis. In contrast to mammals, Southern hybridisation and genomic data indicate that there are probably two genes in Arabidopsis thaliana. Northern blot analysis shows that AtDAD transcripts are present in all tissues examined, although the abundance of the transcripts is reduced in siliques during the maturation and desiccation phase of the seed. This is the first experimental proof that a homologue of an animal gene involved in apoptosis exists in plants and the first demonstration of complementation of a vertebrate mutant by a plant cDNA. Our results suggest that this process of suppression of apoptosis has been conserved in animals and plants.

Amino Acid Sequence↗

Myosin head interactions in Ca2+-activated skinned rabbit skeletal muscle fibers.

Interactions between the two myosin heads were studied in skinned rabbit slow-twitch muscle fibers activated in the presence of vanadate (Vi), a phosphate analog. The strong complex between Vi, MgADP, and myosin trapped the myosin in an inactivated myosin x MgADP x Vi state. Electron paramagnetic resonance spectroscopy was used to quantitate the fraction of myosin heads trapped in the presence of a spin labeled analog of ATP (SLATP). Force was found to depend directly on the fraction of untrapped heads. At high [Vi] (low force), most untrapped heads would have a trapped partner. The equivalence of force with the proportion of untrapped heads shows that the isometric force produced by a single untrapped myosin head on a molecule with a trapped partner is equivalent to that produced by either head of a myosin molecule with neither head trapped. The actin-activated MgATPase activities of one-headed and two-headed skeletal myosin species were inhibited similarly by Vi, suggesting that trapping one head did not preclude trapping its partner. These data indicate that the two skeletal muscle myosin heads can function without interacting during maximal Ca2+-activated force generation.

Adenosine Diphosphate↗

Relating physical activity to health status, social connections and community facilities.

An important public health goal is to increase the population's participation in regular, moderate physical activity. Descriptive epidemiological studies that focus only on associations between physical activity and demographic and psychological factors are not sufficient to inform exercise promotion strategies, and a broader view of health is required. This study investigates the additional factors of health status, social connections and satisfaction with local area facilities by analysing data from a 1987 community health survey of 1765 residents of Adelaide. Factors associated with low activity were age group, education, general health (women), reduced mobility, number of social connections (men) and degree of satisfaction with recreation facilities. Including social and structural factors is valuable for research into interventions, policy and theory relating to physical activity as it brings theoretical perspectives and links to other areas of public health and social research.

Adolescent↗

Response of compressed skinned skeletal muscle fibers to conditions that simulate fatigue.

During fatigue, muscles become weaker, slower, and more economical at producing tension. Studies of skinned muscle fibers can explain some but not all of these effects, and, in particular, they are less economical in conditions that simulate fatigue. We investigated three factors that may contribute to the different behavior of skinned fibers. 1) Skinned fibers have increased myofilament lattice spacing, which is reversible by osmotic compression. 2) A myosin subunit becomes phosphorylated during fatigue. 3) Inosine 5'-monophosphate (IMP) accumulates during fatigue. We tested the response of phosphorylated and unphosphorylated single skinned fibers (isometric tension, contraction velocity, and adenosinetriphosphatase activity) to changes in lattice spacing (0-5% dextran) and IMP (0-5 mM) in the presence of altered concentrations of P(i) (3-25 mM), H+ (pH 7-6.2), and ADP (0-5 mM). The response of maximally activated skinned fibers to the direct metabolites of ATP hydrolysis is not altered by osmotic compression, phosphorylating myosin subunits, or increasing IMP concentration. These factors, therefore, do not explain the discrepancy between intact and skinned fibers during fatigue.

Adenosine Diphosphate↗

Actomyosin interaction in striated muscle.

The mechanics of the actomyosin interaction have been extensively studied using the organized filament array of striated muscle. However, the extrapolation of these data to the events occurring at the level of a single actomyosin interaction has not been simple. Problems arise in part because an active fiber has an ensemble of myosin heads that are spread out through the various steps of the active cycle, and it is likely that only a small fraction of the heads are generating tension at any given time. More recently, two new approaches have greatly extended our knowledge of the actomyosin interaction. First, the three-dimensional crystal structures of both the actin monomer and the myosin head have been determined, and these structures have been fit to lower resolution images to give atomic models of the actin filament and of the actin filament decorated by myosin heads. Second, the technology to measure picoNewton forces and nanometer distances has provided direct determinations of the force and step length generated by a single myosin molecule interacting with a single actin filament. This review synthesizes the existing mechanical data obtained from the more-organized array of the muscle filament with the results obtained by these two technologies.

Actins↗

Orientation of paramagnetic probes attached to gizzard regulatory light chain bound to myosin heads in rabbit skeletal muscle.

The orientation of the myosin neck was monitored using electron paramagnetic resonance (EPR) spectroscopy. Gizzard regulatory light chain was labeled with a nitroxide spin probe and exchanged for the native subunit, located in the myosin neck, in rabbit psoas muscle fibers. The EPR spectra of rigor fibers indicated a substantial degree of probe immobilization and showed a strong dependence on the orientation of the fiber axis relative to the magnetic field, indicating that the neck was ordered in this state. Spectra of relaxed fibers at 24 degrees C showed that the neck was disordered, but the spectra of relaxed fibers at 4 degrees C indicated that the neck was partially ordered. Active fibers at the two temperatures produced spectra identical to relaxed fibers, indicating that no novel angles could be seen in the neck during the powerstroke. Proteolytic fragments of myosin, S1 and HMM, were exchanged with labeled light chains and bound to thin filaments in unlabeled fibers. The distribution of probe orientations for HMM was identical to that of labeled rigor fibers, while S1 showed a slightly different distribution, suggesting that the neck is distorted (by a few degrees) by the interactions of the two heads of myosin when bound to actin.

Animals↗

Crystal structure of the motor domain of the kinesin-related motor ncd.

Microtubule-based ATPases of the kinesin superfamily provide the motile force for many animated features of living cells. Kinesin motors differ in their direction of movement along microtubules. Kinesin and ncd, a kinesin-related motor involved in formation and maintenance of mitotic and meiotic spindles, move in opposite directions along microtubules, even though their motor domains are 40% identical in amino-acid sequence. Here we report the crystal structure of the MgADP complex of the Drosophila ncd motor domain determined to 2.5A by X-ray crystallography, and compare it to the kinesin structure. The ncd and kinesin motor domains are remarkably similar in structure, and the locations of conserved surface amino acids suggest these motors share a common microtubule-binding site. Moreover, structural and functional comparisons of ncd, kinesin, myosin and G proteins indicate that these NTPases may have a similar strategy of changing conformation between NTP and NDP states. We propose a general model for converting a common gamma-phosphate-sensing mechanism into opposite polarities of movement for kinesin and ncd.

Adenosine Diphosphate↗

Osmotic pressure probe of actin-myosin hydration changes during ATP hydrolysis.

Osmotic stress in the 0.5-5 x 10(6) dyne/cm2 range was used to perturb the hydration of actin-myosin-ATP intermediates during steady-state hydrolysis. Polyethylene glycol (PEG) (1000 to 4000 Da), in the 1 to 10 wt% range, which does not cause protein precipitation, did not significantly affect the apparent KM or the Vmax for MgATP hydrolysis by myosin subfragment 1 (S1) alone, nor did it affect the value for the phosphate burst. Consistent with the kinetic data, osmotic stress did not affect nucleotide-induced changes in the fluorescence intensities of S1 tryptophans or of fluorescein attached to Cys-707. The accessibility of the fluorescent ATP analog, epsilon ADP, to acrylamide quenching was also unchanged. These data suggest that none of the steps in the ATP hydrolysis cycle involve substantial hydration changes, which might occur for the opening or closing of the ATP site or of other crevices in the S1 structure. In contrast, KM for the interaction of S1.MgADP.Pi with actin decreased tenfold in this range of osmotic pressure, suggesting that formation of actin.S1.MgADP.Pi involves net dehydration of the proteins. The dehydration volume increases as the size of the PEG is increased, as expected for a surface-excluded osmolyte. The measured dehydration volume for the formation of actin.S1.MgADP.Pi was used to estimate the surface area of the binding interface. This estimate was consistent with the area determined from the atomic structures of actin and myosin, indicating that osmotic stress is a reliable probe of actin.myosin.ATP interactions. The approach developed here should be useful for determining osmotic stress and excluded volume effects in situ, which are much larger than those of typical in vitro conditions.

Actins↗

Massive thymic hemorrhage in a neonate: an entity revisited.

The authors describe a case of early neonatal death of a full-term infant who had respiratory distress and anemia after fetal distress during labor. Postmortem examination disclosed mediastinal compression by a large fresh hemorrhage into the left lobe of the thymus. Massive thymic hemorrhage is an extremely rare but sometimes lethal occurrence; it may represent a manifestation of early-onset hemorrhagic disease of the newborn.

Anemia, Neonatal↗

ADP release produces a rotation of the neck region of smooth myosin but not skeletal myosin.

Current theories of muscle cross-bridge function suggest that force is generated by a change in the orientation of the myosin neck region. We attached a paramagnetic probe to a subunit in the neck region and measured the orientation of the probe using electron paramagnetic resonance spectroscopy. The angle of the probes on smooth myosin S1 were changed by 20 degrees +/- 4 degrees on addition of ADP (50% effect at 5 +/- 2 microM), but ADP produced little effect on skeletal S1. The orientation of smooth myosin, +ADP, resembled that of skeletal myosin, +/- ADP, suggesting that the release of ADP generates an extra rotation of the neck region in smooth muscle at the end of its power stroke.

Adenosine Diphosphate↗

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs.

Nearly 7000 Arabidopsis thaliana-expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.

Amino Acid Sequence↗

Sequencing and mapping the Arabidopsis genome: a weed model for real crops.

Arabidopsis is a crucifer weed with a small genome of about 120 Mbp which has been chosen as a model species for plant molecular genetics. Four years ago, a consortium of nine French laboratories, including ours, initiated a project aimed at mapping the transcribed regions of the genome. The strategy employed was to systematically and randomly sequence cDNA clones isolated from libraries made from different tissues and organs of plants grown under various physiological conditions. The consortium released about 7,000 expressed sequenced tags (ESTs) in the dbEST database corresponding to approximately 3,500 unique genes. In the next phase of the programme, a YAC library with average inserts of 500 kbp has been prepared. We have now started to use the EST information to map the cDNA clones on these YACs. The most recent aspect of Arabidopsis sequencing is the ESSA (European Scientists Sequencing Arabidopsis) project, in which the aim is to describe 2.5 Mbp by the end of 1996. Genomic sequencing has revealed a very high gene density. Comparison of present genomic sequencing results with the EST data suggests that up to half of the genes might already be tagged with an EST. In collaboration with Carlos Quiros' group in Davis we have also analysed the conservation of a 30 kbp locus (Em 1, a late embryogenesis abundant protein gene) on chromosome 3 between Arabidopsis and several Brassica species. Progress on these various aspects will be reviewed. We shall also present some sequence comparisons between Arabidopsis and rice ESTs. These results suggest that it should be possible in the very near future to map a pool of common genes onto many different plant genomes. This should provide a common framework to integrate maps from different species and facilitate mapbased cloning of genes of agronomical importance.

Arabidopsis↗

Reduced effect of pH on skinned rabbit psoas muscle mechanics at high temperatures: implications for fatigue.

1. Inhibition of actomyosin function by decreased pH has been proposed to account for much of the depression of muscle function during fatigue. The clearest support for this hypothesis has been from studies of skinned skeletal muscle fibre mechanics at low temperatures (< or = 15 degrees C). 2. We re-examined the effect of decreased pH (7.0-6.2) on skinned mammalian skeletal fibre mechanics at low (10 degrees C) and high (30 degrees C) temperatures, using recently developed protocols that allow reproducible mechanical data to be obtained at higher temperatures. 3. At 10 degrees C we duplicated previous observations of a significant inhibition of maximum shortening velocity (Vmax) and isometric tension (Po) by acidosis. In contrast, at the higher temperature, we found only a very minimal effect of acidosis on Vmax and a threefold reduction in the decrease in Po. 4. Thus at temperatures only slightly below physiological for mammalian skeletal muscle systems, pH plays a much less important role in the process of muscle fatigue at the cross-bridge level than has been suggested by data obtained at physiologically unrealistic temperatures.

Acidosis↗

Photoaffinity ADP analogs as covalently attached reporter groups of the active site of myosin subfragment 1.

The enzymatic properties of rabbit skeletal myosin subfragment 1 (S1) have been determined after photoaffinity labeling the active site with two ADP analogs. These analogs, 2-[(4-azido-2-nitrophenyl)-amino]ethyl diphosphate (NANDP) and the fluorescent analog 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine diphosphate (Bz2 epsilon ADP), label the heavy chain residues Trp 130 and Ser-324, respectively. These residues in the crystal structure of chicken skeletal S1 are on either side of the entrance to the active site pocket (Rayment et al., 1993b). Here S1 was photolabeled with NANDP or Bz2 epsilon ADP after trapping with vanadate (Vi). Both of the photolabeled S1 preparations had normal MgATPase activities after removal of vanadate by actin treatment. These results show that the covalently tethered nucleotide analogs could move out of the active site and be replaced by MgATP. Experiments that monitored the fluorescence emission intensity, polarization, and quenching by acrylamide of S1 photolabeled with Bz2 epsilon ADP show that the covalently linked analog was displaced out of the active site cleft by MgATP (or MgATP and actin) but not by ATP in the absence of Mg2+ ions. The effective concentration of the tethered ethenoadenosine diphosphate at the active site, determined by competition with MgATP, was calculated to be 10 mM. In the absence of Mg2+ ions, ATP was unable to compete with the bound analog. Binding constants of the S1 photolabeled with Bz2 epsilon ADP to actin were 1.5 x 10(5) and 5.8 x 10(5) M-1 at 200 and 20 mM ionic strength, respectively, showing that actin binding affinities are similar to those obtained for S1.ADP. The binding of actin in the absence of MgATP did not produce any change in the emission intensity, polarization, or quenching by acrylamide of the tethered ethenoadenosine diphosphate, indicating that the conformation of the pocket around the adenine ring was unchanged. However, the binding of actin did destabilize Vi, which had been previously trapped in the form of photolabeled S1-Vi complexes. These results indicate that actin binding primarily affects the gamma-phosphate binding site but not the adenine ring binding site.

Acrylamides↗

A novel adenosine triphosphate analog with a heavy atom to target the nucleotide binding site of proteins.

We have synthesized 2'-deoxy-2'-iodoadenosine-5'-triphosphate (2'-IATP), a heavy-atom analog of adenosine-5'-triphosphate. This compound was made for X-ray structural studies to target the nucleotide site of ATP binding proteins. It was diffused successfully into crystals of the microtubule-based motor proteins ncd (non-claret disjunctional protein from Drosophila melanogaster) and kinesin. With ncd, the nucleotide binding site was 70% occupied and the crystals were able to diffract X-rays to 2.5 A. The iodo-analog provided a useful isomorphous derivative with overall phasing power 1.89 in the range of 25.0-2.5 A. With kinesin, 2'-IATP co-crystallized with the protein. The crystals diffracted to at least 2.8 A with a phasing power of 1.73 in the range of 20.0-5.0 A. The analog was also found to be a substrate for all of the enzymes tested, including creatine kinase, pyruvate kinase, hexokinase, and myosin, with values of Km and Vmax that were within a factor of 10 of those for ATP. The analog supported muscle contraction, relaxing fibers, and producing active tension with values not statistically different from those obtained with ATP. These results all suggest that this analog should be useful for providing a heavy-atom derivative for crystals of enzymes that bind ATP.

Adenosine Triphosphatases↗

The myosin catalytic domain does not rotate during the working power stroke.

Electron paramagnetic resonance spectroscopy of a spin probe attached to cys-707 on myosin cross-bridges was used to monitor the orientation of the myosin catalytic domain at the beginning and end of the working power stroke in active muscle. Elevated concentrations of orthophosphate and decreased pH were used to shift the population of cross-bridges from force-producing states into low force, pre-power-stroke states. The spectrum of probes in active fibers was not changed by conditions that reduced tension by 70%, indicating that the orientation of the catalytic domain was the same at the beginning and end of the power stroke. Thus the data show that the catalytic domain remains rigidly oriented on the actin filament during the power stroke.

Animals↗

Inhibition of muscle force by vanadate.

Vanadate (Vi), an analogue of inorganic phosphate (Pi), is known to bind tightly with a long half life to the myosin MgATPase site, producing a complex which inhibits force. Both of these ligands bind to an actin.myosin.ADP state that follows the release of Pi in the enzymatic cycle, and their effects on muscle fibers and proteins in solution provide information on the properties of this state. The inhibition of active force generation began to occur at a [Vi] of 5 microM and was 90% complete at a [Vi] of 1 mM. Hill plots of the inhibition of force by Vi approximated that expected for a simple binding isotherm. Similar plots were obtained at both 25 degrees C and 5 degrees C. A simple binding isotherm is not expected to occur in a muscle fiber where steric constraints imposed by the intact filaments should introduce more complexity into the energetics of ligand binding. The inhibition of MgATPase activity for acto-subfragment-1 to 50% of controls occurred at a [Vi] which was only 20-fold higher than that required to inhibit force generation in fibers to the same level. Some models of actomyosin interactions would predict that the range of [Vi] required for complete force inhibition in fibers and the difference in the [Vi] required for inhibition in fibers and of myosin in solution would both be much larger.

Actins↗