Gamma-Actinin, a new regulatory protein from rabbit skeletal muscle. I. Purification and characterization.
(1/338)
A new regulatory protein which we have designated as gamma-actinin has been isolated from native thin filaments of rabbit skeletal muscle. Depolymerized native thin filaments were fractionated by salting out with ammonium sulfate, and the precipitates obtained at 40--60% ammonium sulfate saturation were further subjected to DEAE-Sephadex and Sephadex G-200 column chromatography. The purified gamma-actinin was shown to have a chain weight of 35,000 daltons and had a strong inhibitory action on the polymerization of G-actin. The results of amino acid analysis indicated a unique amino acid composition of gamma-actinin as compared with other structural proteins of muscle. Non-polar and neutral amino acid residues were abundant. One cysteine residue was contained per one molecule of gamma-actinin and played a critical role in the maintenance of the inhibitory activity. Pelleting of gamma-actinin with F-actin showed that gamma-actinin binds to F-action. (+info)
Single-polymer dynamics in steady shear flow.
(2/338)
The conformational dynamics of individual, flexible polymers in steady shear flow were directly observed by the use of video fluorescence microscopy. The probability distribution for the molecular extension was determined as a function of shear rate, gamma;, for two different polymer relaxation times, tau. In contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition. Large, aperiodic temporal fluctuations were observed, consistent with end-over-end tumbling of the molecule. The rate of these fluctuations (relative to the relaxation rate) increased as the Weissenberg number, gamma;tau, was increased. (+info)
Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin.
(3/338)
Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer. (+info)
Designing conditions for in vitro formation of amyloid protofilaments and fibrils.
(4/338)
We have been able to convert a small alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 A in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions. (+info)
Electric field-induced transient birefringence and light scattering of synthetic liposomes.
(5/338)
The dynamics of electric field-induced transient birefringence Deltan(t) and light scattering (detected as turbidity) of 190 nm diameter unilamellar vesicles of dioleoylphosphatidylcholine are investigated as a function of applied field strength E, length of the square pulse Deltat, lipid concentration, mean hydrodynamic diameter , ionic strength, and temperature. Generally, induced birefringence exclusively is observed at low lipid concentration and below certain threshold values of E and Deltat, whereas concomitant induced turbidity appears at high lipid concentration and above thresholds values of E and Deltat. Turbidity is monitored through the change in transmitted intensity DeltaS parallel(t) and DeltaS perpendicular(t) of light polarized parallel and perpendicular to the applied field E. The field-induced structural changes are reflected in double-exponential forward relaxation and triple-exponential reverse relaxation of the positive birefringence, and in non-exponential relaxations of DeltaS parallel (t) and DeltaS perpendicular(t). Under the field, the associated physical events are interpreted as elongation of the spherical bilayer shells in the direction of E, linear chain formation (pearling) of the induced dipolar liposomes parallel to E, and partial fusion of adjoining vesicles within the chains. Under conditions where electroporation can be detected, pore opening succeeds the elongation of the vesicles. After termination of the field, the vesicles return to their original time average spherical shape, the oriented chains randomize and disintegrate, and the fused structures are converted either to unilamellar or multilamellar vesicles. (+info)
Biomechanical, histological and immunohistological studies of patellar cartilage in an ovine model of osteoarthritis induced by lateral meniscectomy.
(6/338)
OBJECTIVE: To evaluate the biomechanical, histological and immunohistochemical changes induced in patellar articular cartilage (AC) in ovine stifle joints 3 months after bilateral lateral meniscectomy, a procedure known to induce experimental osteoarthritis (OA) in the femoro-tibial joint (FTJ). METHODOLOGY: Fifteen mature adult Merino female sheep were used in this study. Ten were subjected to bilateral-lateral meniscectomy, while the remaining five were used as 'non-operated controls' (NOC). All animals were killed 3 months post-surgery. Topographical biomechanical indentation tests were performed on each patellae using a UMIS-2000 micro-indentation system. Initial load, relaxed and unload shear moduli were determined using an elastic analytical model, while the permeability was assessed by comparing the indentation response to a simulated indentation test conducted using a poroelastic finite element model. Immunohistochemical, normal and polarized histological studies were performed on each specimen after biomechanical testing. RESULTS: Patellar AC from meniscectomized joints exhibited an overall decrease in initial (-34%), relaxed (-32%) and unload shear modulus (-22%), and an increase in the permeability (+72%) relative to NOC cartilage (P< 0.01). The most significant differences in mechanical properties occurred on the lateral and central aspects of the patellae. There were no significant histological difference in staining between sections from NOC and meniscectomized joint AC using Toluidine Blue, a dye which binds to proteoglycans. However immunohistochemical staining with monoclonal antibody MAb 3B3(-), a putative marker of early OA change in PGs, demonstrated increased binding in the lateral and central regions of patellar sections from meniscectomized joints relative to the same regions of NOC AC. Moreover polarized light microscopy of Picro Sirius red stained sections revealed a significant decrease in birefringence intensity in the superficial-middle zones of the lateral and central regions of the patellar cartilage derived from the meniscectomized joints. CONCLUSION: This study has demonstrated that lateral meniscectomy is a procedure which was known to induce classical OA like changes in AC and subchondral bone of the FTJ also produced an early pathological response in the patellar AC. (+info)
Calculation of protein form birefringence using the finite element method.
(7/338)
An approach based on the finite element method (FEM) is employed to calculate the optical properties of macromolecules, specifically form birefringence. Macromolecules are treated as arbitrarily shaped particles suspended in a solvent of refraction index n1. The form birefringence of the solution is calculated as the difference in its refractive index when all the particles of refractive index n2 are either parallel to or normal to the direction of the polarization of light. Since the particles of interest are small compared to the wavelength of light, a quasi-static approximation for the refractive index is used, i.e., that it is equal to the square root of the dielectric constant of the suspension. The average dielectric constant of the mixture is calculated using the finite element method. This approach has been tested for ellipsoidal particles and a good agreement with theoretical results has been obtained. Also, numerical results for the motor domains of ncd and kinesin, small arbitrarily shaped proteins with known x-ray structures, show reasonable agreement with the experimental data obtained from transient electric birefringence experiments. (+info)
The effects of increasing the reverse curve of Spee in a lower archwire examined using a dynamic photo-elastic gelatine model.
(8/338)
This paper describes the development and testing of a dynamic in vitro photo-elastic model for evaluating the effects of orthodontic mechanics on an entire arch of teeth. A model of a mandibular arch was made and the teeth were embedded in a gelatine material with a high level of mechanical creep which permitted tooth movement in response to orthodontic forces. The excellent photo-elastic properties of this material also facilitated the analysis of the stress distribution around the roots of the teeth. The model of a mandibular arch was used to investigate the tooth movements and stress distributions produced by increasing the reverse curve of Spee in a 0.018 x 0.025-inch stainless steel archwire. The results revealed that a 1-mm reverse curve of Spee increased the arch length by 1.6 mm, but increasing the reverse curve of Spee to 5 mm did not increase arch length further. Photo-elastic analysis showed an increased stress distribution around the roots of the incisors and molars as the reverse curve of Spee was increased in the archwire. (+info)