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R Padrón

Publications and source records attributed to R Padrón.

At least 19 recordsLinked to original sources
PubMed2004

Helical order in tarantula thick filaments requires the "closed" conformation of the myosin head.

Myosin heads are helically ordered on the thick filament surface in relaxed muscle. In mammalian and avian filaments this helical arrangement is dependent on temperature and it has been suggested that helical order is related to ATP hydrolysis by the heads. To test this hypothesis, we have used electron microscopy and image analysis to study the ability and temperature dependence of analogs of ATP and ADP.Pi to induce helical order in tarantula thick filaments. ATP or analogs were added to rigor myofibrils or purified thick filaments at 22 degrees C and 4 degrees C and the samples negatively stained. The ADP.Pi analogs ADP.AlF4 and ADP.Vi, and the ATP analogs ADP.BeFx, AMPPNP and ATPgammaNH2, all induced helical order in tarantula thick filaments, independent of temperature. In the absence of nucleotide, or in the presence of ADP or the ATP analog, ATPgammaS, there was no helical ordering. According to crystallographic and tryptophan fluorescence studies, all of these analogs, except ATPgammaS and ADP, induce the "closed" conformation of the myosin head (in which the gamma phosphate pocket is closed). We suggest that helical order requires the closed conformation of the myosin head but is not dependent on the hydrolysis of ATP.

Adenosine Diphosphate↗
PubMed2001

Purification of native myosin filaments from muscle.

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.

Actin Cytoskeleton↗
PubMed2001

Mechanism of phosphorylation of the regulatory light chain of myosin from tarantula striated muscle.

Contraction is modulated in many striated muscles by Ca2+-calmodulin dependent phosphorylation of the myosin regulatory light chain (RLC) by myosin light chain kinase. We have investigated the biochemical mechanism of RLC phosphorylation in tarantula muscle to better understand the basis of myosin-linked regulation. In an earlier study it was concluded that the RLC occurred as two species, both of which could be phosphorylated, potentiating contraction. Here we present evidence that only a single species exists, and that this can be phosphorylated at one or two sites. In relaxed muscle we find evidence for a substantial level of basal phosphorylation at the first site. This is augmented on activation, followed by partial phosphorylation of the second site. We find in addition that Ca2+ has a dual effect on light chain phosphorylation, depending on its concentration. At low concentration (relaxing conditions) only basal phosphorylation is observed, while at higher concentrations (activating conditions) RLC phosphorylation is stimulated. At still higher Ca2+ concentrations we find partial inhibition of RLC phosphorylation, suggesting an additional mechanism by which the muscle cell can fine tune contractile activity by controlling the level of free Ca2+.

Adenosine Triphosphate↗
PubMed2000

A new model for the surface arrangement of myosin molecules in tarantula thick filaments.

Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 A have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head. We have modelled the surface array of myosin heads on the filaments using as a building block a model of a two-headed regulated myosin molecule in which the regulatory light chains of the two heads together form a compact head-tail junction. Four parameters defining the radius, orientation and rotation of each myosin molecule were varied. In addition, the heads were allowed independently to bend in a plane perpendicular to the coiled-coil tail at three sites, and to tilt with respect to the tail and to twist at one of these sites. After low-pass filtering, models were aligned with the reconstruction, scored by cross-correlation and refined by simulated annealing. Comparison of the geometry of the reconstruction and the distance between domains in the myosin molecule narrowed the choice of models to two main classes. A good match to the reconstruction was obtained with a model in which each ridge is formed from the motor domain of a head pointing to the bare zone together with the head-tail junction of a neighbouring molecule. The heads pointing to the Z-disc intermittently occupy the J-position. Each motor domain interacts with the essential and regulatory light chains of the neighbouring heads. A near-radial spoke in the reconstruction connecting the backbone to one end of the ridge can be identified as the start of the coiled-coil tail.

Actin Cytoskeleton↗
PubMed1998

Towards an atomic model of the thick filaments of muscle.

The thick filaments of muscle and non-muscle cells are polymers of myosin molecules whose energy-transducing heads lie on the filament surface, where they interact with actin to generate force. A key structural question is how the myosin heads are arranged in the relaxed state, and how this arrangement changes on activation of contraction. We have fitted the atomic structure of the myosin head to the three-dimensional structure of myosin filaments of tarantula muscle determined by electron microscopy to produce a near-atomic model of the head arrangement. A good fit is obtained only when the two heads from a myosin molecule run along the helical tracks antiparallel to each other. Oppositely oriented heads from axially adjacent molecules in a helix interact with each other, with their nucleotide-binding pockets opposed. This arrangement, supported also by crosslinking evidence, suggests a simple mechanism for the stabilization of myosin head helices in relaxed muscle via the formation of intermolecular "dimers" of heads from axially adjacent myosin molecules.

Actin Cytoskeleton↗
PubMed1998

Long-term therapy with policosanol improves treadmill exercise-ECG testing performance of coronary heart disease patients.

This study examined the effects of long-term lipid-lowering therapy with policosanol on the clinical evolution, and exercise-ECG testing responses of 45 coronary heart disease (CHD) patients with myocardial ischemia, documented by exercise 201T1-myocardial perfusion scintigraphy, in an overall randomized, double-blind, placebo-controlled trial, made for different test endpoints. Fifteen patients were treated with 5 mg of policosanol twice daily; another 15 patients were administered the same drug dose plus 125 mg aspirin; and the other 15 patients received placebo plus equal aspirin dose. They were followed for 20 months, previous baseline observations, with treadmill exercise-ECG, besides serum lipid test. Beneficial changes on proportions among the 2 policosanol groups and the placebo group, showed an increment on functional capacity class, a decrement on rest and exercise angina, and a significant decrease in cardiac events, and in ischemic ST segment response, especially in the policosanol plus aspirin group (p = 0.05, X2(2df) = 5.8; p = 0.04, p = 0.02; Fisher). After treatment, sets of mean changes revealed an increase on maximum oxygen uptake, and a decline on double product simultaneously in both policosanol groups (p < or = 0.02, p < or = 0.002; Pillais, Hotellings' T2), while the placebo group was impaired. Aerobic functional capacity percent showed an increment in policosanol groups (p < or = 0.05, paired T). Lipid levels improved as other endpoints already reported. A supposed ergogenic effect of octacosanol, policosanol's main active compound, was not detected with this design. These results show that policosanol-treated CHD patients improved clinical evolution, and exercise-ECG responses, owing to the amelioration of myocardial ischemia, even more when administered with aspirin.

Anticholesteremic Agents↗
PubMed1997

The action of local anesthetics on myelin structure and nerve conduction in toad sciatic nerve.

X-ray scattering and electrophysiological experiments were performed on toad sciatic nerves in the presence of local anesthetics. In vitro experiments were performed on dissected nerves superfused with Ringer's solutions containing procaine, lidocaine, tetracaine, or dibucaine. In vivo experiments were performed on nerves dissected from animals anesthesized by targeted injections of tetracaine-containing solutions. In all cases the anesthetics were found to have the same effects on the x-ray scattering spectra: the intensity ratio of the even-order to the odd-order reflections increases and the lattice parameter increases. These changes are reversible upon removal of the anesthetic. The magnitude of the structural changes varies with the duration of the superfusion and with the nature and concentration of the anesthetic molecule. A striking quantitative correlation was observed between the structural effects and the potency of the anesthetic. Electron density profiles, which hardly showed any structural alteration of the unit membrane, clearly indicated that the anesthetics have the effect of moving the pairs of membranes apart by increasing the thickness of the cytoplasmic space. Electrophysiological measurements performed on the very samples used in the x-ray scattering experiments showed that the amplitude of the compound action potential is affected earlier than the structure of myelin (as revealed by the x-ray scattering experiments), whereas conduction velocity closely follows the structural alterations.

Action Potentials↗
PubMed1992

Structure of the myosin filaments of relaxed and rigor vertebrate striated muscle studied by rapid freezing electron microscopy.

Rapid freezing followed by freeze-substitution has been used to study the ultrastructure of the myosin filaments of live and demembranated frog sartorius muscle in the states of relaxation and rigor. Electron microscopy of longitudinal sections of relaxed specimens showed greatly improved preservation of thick filament ultrastructure compared with conventional fixation. This was revealed by the appearance of a clear helical arrangement of myosin crossbridges along the filament surface and by a series of layer line reflections in computed Fourier transforms of sections, corresponding to the layer lines indexing on a 43 nm repeat in X-ray diffraction patterns of whole, living muscles. Filtered images of single myosin filaments were similar to those of negatively stained, isolated vertebrate filaments and consistent with a three-start helix. M-line and other non-myosin proteins were also very well preserved. Rigor specimens showed, in the region of overlapping myosin and actin filaments, periodicities corresponding to the 36, 24, 14.4 and 5.9 nm repeats detected in X-ray patterns of whole muscle in rigor; in the H-zone they showed a disordered array of crossbridges. Transverse sections, whose Fourier transforms extend to the (3, 0) reflection, supported the view, based on X-ray diffraction and conventional electron microscopy, that in the overlap zone of relaxed muscle most of the crossbridges are detached from the thin filaments while in rigor they are attached. We conclude that the rapid freezing technique preserves the molecular structure of the myofilaments closer to the in vivo state (as monitored by X-ray diffraction) than does normal fixation.

Animals↗
PubMed1992

[Value of combined electro- and vectorcardiography in the estimation of left ventricular mass in the elderly].

Electro- and vectorcardiographic methods describe left ventricle increases only when it has attained a significantly high magnitude, but even in such circumstances the exactitude of such methods is substantially lower than that of echocardiogram (ECHO). On the other hand, in comparison to other age groups, there have been relatively few reports relating electrocardiogram (ECG), vectorcardiogram (VCG) and ECHO with left ventricular mass (LVM) in healthy elderly subjects where increases of the left ventricle mass, if present, would be small or moderate. In this paper LVM as well as LVM index (iLVM) from a group of healthy subjects belonging to a physical training program for elderly, was studied by means of ECHO and computerized ECG and VCG. From ECG, voltage indexes and other LVM associated parameters were extracted; from VCG, planar maximum vectors, areas within VCG loops and maximal spatial magnitude of QRS (SM), were measured. Results of LVM (221 +/- 37.9, g) were higher than figures reported for others groups. Voltage indexes showed normal values, but QRS duration was somewhat prolonged. The best simple linear regression, combining variables from VCG and ECG was maximum horizontal vector (Vmax-Hor) vs Sokolow-Lyon index (SOK) and combining ECHO with ECG or VCG, LVM vs Area inside horizontal loop (AreaHor). A model for estimation of LVM from electrical variables was obtained by multiple linear regression; combining five variables from ECG and VCG. The best model included Sokolow-Lyon index and variables from horizontal and sagittal planes of VCG and spatial magnitude of QRS: LVM = 4.8 SOK-186 VmaxHOR-80 VmaxSAG + 126SM + 340 AreaH + 175.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1991

Direct determination of myosin filament symmetry in scallop striated adductor muscle by rapid freezing and freeze substitution.

Chemically skinned, relaxed bundles of fibers from the striated adductor muscle of the scallop Placopecten magellanicus were rapidly frozen and freeze-substituted. In the electron microscope, ultrathin transverse sections of embedded specimens showed, in many cases, clear regularly organized projections (crossbridges) protruding from the backbones of the myosin filaments. In the majority of cases the number of projections was directly observed to be seven: this was confirmed by alignment and averaging of the images using correlation methods. The rotational power spectrum of the average image showed a strong peak at N = 7. Tilting of sections in the electron microscope showed that the long-pitch crossbridge helices were right-handed. These and other observations confirm directly the essential features of the low-resolution three-dimensional helical reconstruction of negatively stained scallop filaments calculated previously.

Animals↗
PubMed1991

X-ray diffraction study of the structural changes accompanying phosphorylation of tarantula muscle.

Electron microscopy of negatively stained isolated thick filaments of tarantula muscle has revealed that phosphorylation of myosin regulatory light chains is accompanied by a loss of the helical order of myosin heads. From equatorial X-ray diffraction patterns of tarantula muscles in the phosphorylated state we have detected a mass movement in the myosin filaments that supports this finding.

Actin Cytoskeleton↗
PubMed1991

[Method for direct determination of the rotational symmetry of thick muscle filaments by digital image processing].

We present a method based on digital image processing techniques that permits to determine directly the rotational symmetry of muscle thick filaments. Electron micrographs of transverse sections of relaxed muscle rapidly frozen against a copper block cooled with liquid helium were digitized, aligned to give maximal cross correlation and averaged. The final averaged image permits to determine directly the number of projections on the backbone surface.

Animals↗
PubMed1989

Disorder induced in nonoverlap myosin cross-bridges by loss of adenosine triphosphate.

Adenosine triphosphate-dependent changes in myosin filament structure have been directly observed in whole muscle by electron microscopy of thin sections of rapidly frozen, demembranated frog sartorius specimens. In the presence of ATP the thick filaments show an ordered, helical array of cross-bridges except in the bare zone. In the absence of ATP they show two distinct appearances: in the region of overlap with actin, there is an ordered, rigorlike array of cross-bridges between the thick and thin filaments, whereas in the nonoverlap region (H-zone) the myosin heads move away from the thick filament backbone and lose their helical order. This result suggests that the presence of ATP is necessary for maintenance of the helical array of cross-bridges characteristic of the relaxed state. The primary effect of ATP removal on the myosin heads appears to be weaken their binding to the thick filament backbone; released heads that are close to an actin filament subsequently form a new actin-based, ordered array.

Actins↗
PubMed1988

A method for quick-freezing live muscles at known instants during contraction with simultaneous recording of mechanical tension.

We have developed a quick-freezing method, using a copper block cooled with liquid helium or nitrogen, which permits us to freeze muscles without any cryoprotectant at predetermined, precisely measured points in the recorded tension time-course of a single twitch or tetanus. Our aim is to arrest structural intermediates of the cross-bridge cycle for observation in the electron microscope. Chemically stimulated, demembranated muscles as well as electrically stimulated, live muscles can be frozen on the same apparatus. Good freezing of relaxed and contracting muscles has been obtained to a depth of 10-20 microns, with excellent structural preservation after freeze-substitution.

Animals↗
PubMed1987

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments.

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.

Actin Cytoskeleton↗