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Biomedical subjects

R Cooke

Publications and source records attributed to R Cooke.

At least 163 records · Page 9Linked to original sources
PubMed1985

The inhibition of muscle contraction by adenosine 5' (beta, gamma-imido) triphosphate and by pyrophosphate.

We have studied the inhibition of the contraction of glycerinated rabbit psoas muscle caused by ligands that bind to the ATPase site of myosin. Two ligands, adenosine 5' (beta, gamma-imido) triphosphate (AMPPNP) and pyrophosphate (PPi), decreased the force and stiffness developed in isometric contractions and the velocity of shortening of isotonic contractions. The force exerted by isometric fibers was measured as a function of MgATP in the presence and absence of a constant concentration of the ligands. As the MgATP concentration decreased, the inhibition of tension caused by the ligand increased, reaching approximately 50% at 25 microM MgATP and either 2 mM MgPPi or 2 mM MgAMPPNP. The maximum velocity of shortening was also measured as a function of MgATP concentration in the presence of 1 and 2 mM MgPPi and 2.5 and 5 mM MgAMPPNP. Both ligands acted as pure competitive inhibitors with Ki = 3.0 mM for PPi and 5.1 mM for MgAMPPNP. These data show that both ligands are weak inhibitors of the contraction of fibers. The results provided information on the energetics of actin-myosin-ligand states that occur in the portion of the cross-bridge cycle where MgATP binds to myosin. A simple analysis of the inhibition of velocity suggests that MgAMPPNP binds to the actomyosin complex at this step of the cycle with an effective affinity constant of approximately 2 X 10(2) M-1.

Adenosine Triphosphate↗
PubMed1985

Urinary free cortisol levels and dexamethasone suppression testing in organic affective disorder associated with hyperthyroidism.

Eleven of 32 newly diagnosed untreated patients with hyperthyroidism met DSM-III criteria for organic affective syndrome. Thirty of these patients submitted 24-hour urine specimens for measurement of urinary free cortisol levels, and 31 were given a 1-mg dexamethasone suppression test (DST) before antihyperthyroidism therapy was started. There was no difference in the mean +/- SD urinary free cortisol excretion levels between depressed and nondepressed hyperthyroid patients. One nondepressed patient demonstrated nonsuppression (greater than 5 micrograms/dl) at 8:00 a.m. These results suggest that cortisol abnormalities as reflected by urinary free cortisol levels and DST findings are uncommon in patients with hyperthyroidism whether they are depressed or nondepressed.

Depressive Disorder↗
PubMed1984

Fluorescence energy transfer between probes on actin and probes on myosin.

The structural relationship between F-actin filaments and the biologically active fragments of myosin (either as myosin subfragment-1 or heavy meromyosin) has been investigated using the technique of fluorescence energy transfer. Donor and acceptor probes were used to obtain the following inter- and intramolecular distances. Energy transfer was measured: (1) from the SH1 groups of the myosin 'heads' to the nucleotide sites of F-actin (in the absence of free nucleotide); (2) from the SH1 groups of myosin to multiple probes on the surface of the actin filament; (3) from the nucleotide-binding sites of F-actin to the ATPase sites of myosin; (4) from the ATPase sites of myosin to the nucleotide-binding sites of F-actin; (5) from the SH1 sites of myosin to the nucleotide-binding sites of F-actin; and (6) from the Cys-373 residues of F-actin to the nucleotide binding sites of F-actin. We observed very little energy transfer between the probes on actin and the probes on myosin (10% or less) and we observed a large transfer between the actin Cys-373 and the actin nucleotide. These data strongly suggest that both the SH1 moiety and the ATPase site of myosin are located more than 6 nm from the actin sites. When these distances are combined with similar measurements by other authors and inserted into the most recent three-dimensional reconstruction of electron micrographs of the acto-subfragment-1 complex, it is apparent that the SH1 and the ATPase sites on myosin are not located adjacent to actin and are most probably located in the half of the myosin head that is distal from actin in the actomyosin complex.

Actins↗
PubMed1984

Muscle cross-bridges: do they rotate?

We have used electron paramagnetic resonance (EPR) spectroscopy to monitor the orientation of spin labels attached specifically to a reactive sulfhydryl on the myosin heads in glycerinated rabbit psoas skeletal muscle. Previous work has shown that the paramagnetic probes are highly ordered in rigor muscle and display a random angular distribution in relaxed muscle (Thomas and Cooke , 1980). Addition of ADP to rigor fibers caused no spectral changes, while addition of AMPPNP or PPi increased the fraction of disordered probes. We show here that the application of stress to fibers in the presence of ADP, AMPPNP or PPi causes no change in their spectra. During the generation of isometric tension approximately 80% of the probes display a random angular distribution as in relaxed muscle while the remaining 20% are highly oriented at the same angle as found in rigor muscle. In each of the above cases the spectrum consists of two components, one highly ordered as in rigor and one highly disordered. Saturation transfer EPR has shown that the ordered component is rigid while the disordered component is mobile on the microsecond time scale (Thomas, Ishiwata , Seidel and Gergely , 1980). These data lead to the conclusion that the disordered spectral component arises from myosin heads that are detached from actin while the ordered component comes from heads that are attached to actin. The observation that the ordered component displays an identical angular distribution under all conditions indicates that its orientation is not linked to force generation.

Adenosine Diphosphate↗
PubMed1984

The effect of myosin sulphydryl modification on the mechanics of fibre contraction.

Glycerinated rabbit psoas fibres have been modified with paramagnetic probes ( IASL and MSL) which react selectively with the reactive sulphydryl on the myosin head. The extent of SH-1 modification was monitored by extracting myosin and measuring its ATPase activity in the presence of EDTA and of Ca2+. The isometric tension, stiffness, maximum velocity of contraction (slack test), and the force-velocity relation was measured as a function of the degree of SH-1 modification. Reaction of up to 50% of SH-1, i.e. 50% reduction in the K+-EDTA ATPase activity of extracted myosin, produced little change (less than 10%) in any of the fibre parameters. Modification of 75% of the SH-1 sites produced small decreases (15-30%) in the magnitude of all parameters, while reaction of more than 90% of SH-1 required long reaction times and produced decreases of 40-75%. In all cases the velocities of contraction decreased in parallel with the decrease in tension, while the decrease in stiffness was less pronounced. We conclude that a large fraction of muscle fibre SH-1 groups can be modified without greatly affecting the mechanical performance of the fibre. At least a portion of the decrease in fibre parameters that is observed at high levels of SH-1 modification can be attributed to modification of other sulphydryls by the probes. The reaction of both SH-1 and nonspecific sulphydryls abolishes myosin ATPase activity, and can account for approximately one half of the decrease in fibre parameters that is observed at high degrees of sulphydryl modification. We conclude that the modification of SH-1 does not greatly affect the function of a myosin head in the filament array of a fibre. This is in contrast to results obtained in vitro where SH-1 modification alters several rates in the interaction of myosin with ATP and decreases the actin-activated ATPase activity of myosin subfragments.

Adenosine Triphosphatases↗
PubMed1984

Myosin phosphorylation decreases the ATPase activity of cardiac myofibrils.

Our previous work showed that myosin phosphorylation decreased the ATPase activity of skeletal muscle myofibrils that were lightly fixed with glutaraldehyde. The fixation process prevented sarcomere shortening and destruction of the ordered filament array upon the addition of ATP. We have now extended these results to myofibrils prepared from hearts of rabbits, dogs and rats. Myofibrils were phosphorylated by incubation with myosin light chain kinase, calmodulin and either ATP-gamma s or ATP, for 15 minutes at 25 degrees C. The extent of myosin light chain phosphorylation was 50% to 80%. The ATPase activity of unphosphorylated myofibrils was not altered by reaction with 0.01% glutaraldehyde for 5 minutes at 0 degrees C, and the ATPase activity of unfixed myofibrils was not changed by phosphorylation. However, phosphorylation decreased the ATPase activity of fixed myofibrils by 50%. The effect on myocardial myofibrillar ATPase activity of phosphorylation was similar in the three animal species. These results suggest that in both skeletal and cardiac muscle, myosin phosphorylation decreases the rate of cross-bridge cycling resulting in decreased energy expenditure. It also appears that the effect of myosin light chain phosphorylation on ATPase activity requires an ordered myofilament structure.

Adenosine Triphosphatases↗
PubMed1984

The effect of chemotherapy on hepatoblastoma.

A 15-month-old girl had a case of mixed hepatoblastoma. The unresectable tumor became surgically resectable after treatment with doxorubicin hydrochloride and cisplatin, which was also continued postoperatively. This regimen had a remarkable necrotizing effect on the fetal epithelial component of the hepatoblastoma. The patient's high alpha-fetoprotein level and extreme thrombocytosis, which resolved with treatment, were used as indicators of the disease's activity. An unaffected mesenchymal component and a few microscopic foci of undisturbed embryonal hepatoblastoma were found. The finding of a mature intestinal epithelial island in this case is unique.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1983

Actin-myosin interactions visualized by the quick-freeze, deep-etch replica technique.

A new method of preparing biological samples for electron microscopy has been used to re-examine the structure of actin filaments, actin filaments decorated by myosin subfragment-1 (S1), and insect flight muscles. Samples were quick-frozen by contact with a block of copper cooled to approximately 4 K; then were freeze-fractured, deep-etched, rotary-replicated with platinum, and viewed in a transmission electron microscope. By this approach, actin filaments display prominent transverse bands whose repeat (approximately 5.5 nm) and pitch (approximately 15 to 20 degrees) fit with the expected left-handed "genetic" helix. Freeze-etched actin filaments do not, however, display the usual two-start helix as prominently as is seen after negative staining, and they also appear substantially thicker than after negative staining (9 to 10 nm versus 8 nm). The latter two-start helix appears very clearly after S1 decoration. Nevertheless, freeze-etched acto-S1 does not display the "arrowheads" that are seen after negative staining. Instead it displays the outer envelope of the helically deployed S1, and as would be expected from current models derived from optical reconstruction of negatively stained samples, this surface view looks only slightly polarized. Finally, the quick-freeze, deep-etch approach provides particularly distinct images of the crossbridges in insect flight muscles. These are plentiful and regularly arranged in rigor muscles, but rare in muscles relaxed with ATP before freezing. In rigor muscles fixed with aldehydes, these crossbridges assume a broad distribution of inclination, ranging from 45 degrees to 90 degrees with a mean of approximately 80 degrees, which is less tilt than has been seen before in thin-sectioned muscles. However, when aldehyde fixation is followed by exposure to tannic acid with or without uranyl acetate block-staining, crossbridges assume a more acute angle with respect to the fiber axis, centering around 45 degrees. This is associated with a commensurate reduction in interfilament spacing within the muscle fibers, such that tilted crossbridges are not any longer than untilted ones (both measuring approximately 15 nm). At the opposite extreme, crossbridges often become stretched in unfixed muscles, owing to an unnatural increase in interfilament spacing that occurs during sample preparation; in such regions, crossbridges display narrow "stalks", which invariably emerge from the thick filaments at close to 90 degrees. We conclude that crossbridge shape and orientation is strongly affected by different methods of sample preparation, and this will make it difficult to visualize natural crossbridge movements by electron microscopy.

Actins↗
PubMed1983

Penile block for circumcision? A comparison with caudal blockade.

A prospective blind trial was conducted comparing the analgesia and adverse effects produced by caudal extradural blockade (caudal block) with blockade of the dorsal nerves of the penis (penile block) in 38 boys undergoing elective circumcision. Analgesia was assessed by senior nurses using a linear analogue scale and by the response to direct questioning over the first 7 hours after surgery. The degree and duration of postoperative analgesia was similar in the two groups. However, the caudal block group had a higher incidence of motor blockade (p = less than 0.01). One technical failure occurred in the penile block group. It is concluded that a penile block is a satisfactory alternative to caudal blockade with regard to postoperative analgesia and offers several specific advantages.

Anesthesia, Caudal↗
PubMed1982

Orientation of spin labels attached to cross-bridges in contracting muscle fibres.

Electron micrographs showing different cross-bridge orientations in different states of muscle fibres, and X-ray diffraction patterns indicating axial cross-bridge disorder in contracting muscle first suggested that force generation in the contracting muscle involved a change in orientation of the myosin heads that form cross-bridges between thick and thin filaments. This has been supported by subsequent work; the myosin molecule has the required flexibility for changes in orientation. The orientation of muscle tryptophans and of probes attached to the myosin heads of permeable muscle fibres depends on the state of the muscle. Recently, fluorescence polarization fluctuations and time-resolved X-ray diffraction patterns have suggested that cross-bridges of a contracting muscle can rotate. We have used electron paramagnetic resonance (EPR) spectroscopy to monitor the orientation of spin labels attached specifically to a reactive sulphydryl on the myosin heads in glycerinated rabbit psoas skeletal muscle. Previously, it has been shown that the paramagnetic probes are highly ordered in rigor muscle, with a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, approximately 80% of the probes display a random angular distribution as in relaxed muscle while the remaining 20% are highly oriented at the same angle as found in rigor muscle. These findings indicate that a domain of the myosin head does not change orientation during the power stroke of the contractile interaction.

Actins↗