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Birefringence of oriented thin filaments in the I-bands of crab striated muscle and comparison with the flow birefringence of reconstituted thin filaments.

Birefringence of the I-band of glycerinated myofibrils of the walking legs of the crab, Plagusia dentipes was examined by rectified polarization optics. The coefficient of birefringence of the I-band was estimated to be 1.64 X 10(-3), which attained about 60% of the birefringence of the H-zone. The number ratio of thin filaments to thick filaments in the A-band was six according to electron microscopy and the spacing between neighbouring thick filaments was about 56 nm according to X-ray diffraction. From these values, the protein concentration in the I-band was estimated to be 91 mg/ml. Thus, the birefringence of the I-band was found to be of the same order as the birefringence of the reconstituted thin filaments oriented by flow at the same protein concentration. The ratio of protein concentration in the I-band to that in the H-zone was determined by interferometry. The ratio of birefringence of the I-band to that of the H-zone per unit protein concentration was found to be about 0.9, which was comparable to the ratio of birefringence of the thin filaments and thick filaments in solution. The imbibition experiment showed that 70% of the total birefringence of the I-band is form birefringence and remaining 30% comes from intrinsic birefringence.

Animals

Spindle birefringence of isolated mitotic apparatus: further evidence for two birefringent spindle components.

We studied sea-urchin zygote mitotic apparatus (MA) isolated in hexylene glycol, transferred immediately to a glycerol-dimethylsulphoxide medium, and stored for 2 weeks at room temperature. Treatment with 0-5 M KC1 caused loss of 45% of the birefringence, but microtubules remained intact (as seen electron microscopically in glutaraldehyde-fixed MA), and tubulin was not extracted (as determined by polyacrylamide gel electrophoresis). These results suggest that a non-tubulin component which is extracted by the KC1 contributes 45% of the MA birefringence. Further evidence for this conclusion came from indirect immunofluorescence experiments. Non-extracted (control) MA were fixed with formaldehyde and reacted with antibody against tubulin; there was intense staining of the spindle fibres and astral rays. Electron microscopically, however, microtubules were not present in formaldehyde-fixed MA. Since formaldehyde fixation caused breakdown of microtubules but the tubulin remained in the MA (as judged by reaction with antibodies) we suggest that after microtubule breakdown the tubulin remains in the MA because it is bound to a peri-microtubule spindle component (which we call 'substance gamma'). When KCl-extracted MA were fixed with formaldehyde and reacted with antibody against tubulin there was very little staining of spindle fibres and astral rays. Electron microscopically, formaldehyde caused microtubule breakdown, and since the tubulin is lost from formaldehydefixed, KC1-extracted MA (as judged by reaction with antibodies), we suggest that the tubulin-binding component, substance gamma, is extracted by the 0-5 M KC1. Pressure treatment caused the asters not to stain with antibody against tubulin, suggesting that the stability of substance gamma is different in different regions of the mitotic apparatus.

Animals

Transient electric birefringence of two small DNA restriction fragments of the same molecular weight.

The transient electric birefringence of two small DNA restriction fragments of the same molecular weight, one of which migrates anomalously slowly on polyacrylamide gels, has been investigated. Both fragments exhibit negative birefringence. The decay of the birefringence of the anomalously slowly migrating fragment is 8-9% faster than that of the normally migrating fragment. The faster birefringence decay of the anomalous fragment 12A persists under a variety of buffer conditions, suggesting that it is due primarily to static bending and/or curvature of fragment 12A. In reversing electric fields the absolute amplitude of the birefringence of fragments 12A and 12B decreased about 26% before returning to the steady state value. The minimum in the birefringence occurred faster than expected from the birefringence decay times and decreased with increasing electric field strength, suggesting that the minimum is due to a slow polarization of the ion atmosphere. For both fragments, the rise of the birefringence in the Kerr region is about 10% slower than the field-free decay. The buildup of the negative birefringence is preceded either by an interval when no birefringence is observed or by a small positively birefringent transient, suggesting that a small transverse ionic polarizability is also present. Both DNA fragments exhibit Kerr law behavior over most of the range of electric field strengths investigated. Analysis of the shapes of the saturation curves suggests that differences may exist in the polarization mechanisms of the two fragments.

Base Sequence

Birefringence of glycerinated crab muscle fiber under various conditions.

By using glycerinated single fibers of crab muscle (Sesarma haematocheir) which has long sarcomeres, the birefringence of the I band, H band and the overlapping region between thin and thick filaments was measured separately, under various environmental conditions. At the resting length, the birefringence of the fiber was decreased by the addition of Ca2+ in the absence of ATP, by about 0.35%. This birefringence decrease was found to take place in the overlapping region. The decrease corresponded to about 2% of the birefringence of thin filaments in this region. The birefringence of the fiber was increased by the addition of ATP in the absence of Ca2+, by about 6%. This birefringence increase also took place mostly in the overlapping region. The increase of birefringence by pyrophosphate was about half of that by ATP. The birefringence of the fiber was decreased by the increase of the ionic strength from 0.12 to 0.20. The origin of the observed changes of birefringence is discussed.

Adenosine Triphosphate

Quantitative studies on the polarization optical properties of striated muscle. I. Birefringence changes of rabbit psoas muscle in the transition from rigor to relaxed state.

The changes in birefringence in the rigor to relax transition of single triton-extracted rabbit psoas muscle fibers have been investigated with quantitative polarized light techniques. The total birefringence of rest lenght fibers in rigor was (1.46 +/- 0.08) x 10(-3) and increased to (1.67 +/- 0.05) x 10(-3) after Mg-ATP relaxation. Pyrophosphate relaxation increased the total birefringence only slightly, whereas subsequent Mg-ATP relaxation elicited the maximum increase in birefringence. Changes in lattice spacing did not account for the total increase in birefrigence during relaxation. Moreover, the increase in total birefringence was attributable to increases in intrinsic birefringence as well as form birefringence. No change in birefringence was exhibited upon exposure to a relaxation solution after myosin extraction. Synthetic myosin filaments were prepared and treated with relaxation and rigor solutions. The negatively stained filaments treated with a rigor solution had gross irregular projections at either end, while the filaments treated with a relaxing solution were more spindle shaped. The results are compatible with the view that the subfragment-2 moieties of myosin angle away from the myosin aggregates (light meromyosin) to permit the attachment of the subfragment-1 moieties to actin.

Adenosine Triphosphate

Myosin crossbridge orientation in demembranated muscle fibres studied by birefringence and X-ray diffraction measurements.

Muscle contraction is generally thought to involve changes in the orientation of myosin crossbridges during their ATP-driven cyclical interaction with actin. We have investigated crossbridge orientation in equilibrium states of the crossbridge cycle in demembranated fibres of frog and rabbit muscle, using a novel combination of techniques: birefringence and X-ray diffraction. Muscle birefringence is sensitive to both crossbridge orientation and the transverse spacing of the contractile filament lattice. The latter was determined from the equatorial X-ray diffraction pattern, allowing accurate characterization of the orientation component of birefringence changes. We found that this component decreased when relaxed muscle fibres were put into rigor at rest length, and when either the ionic strength or temperature of relaxed fibres was lowered. In each case the birefringence decrease was accompanied by an increase in the intensity of the (1,1) equatorial X-ray reflection relative to that of the (1,0) reflection. When fibres that had been stretched largely to eliminate overlap between actin- and myosin-containing filaments were put into rigor, there was no change in the orientation component of the birefringence. When isolated myosin subfragment-1 was bound to these rigor fibres, the orientation component of the birefringence increased. The birefringence changes at rest length are likely to be due to changes in the orientation of myosin crossbridges, and in particular of the globular head region of the myosin molecules. In relaxed fibres from rabbit muscle, at 100 mM ionic strength, 15 degrees C, the long axis of the heads appears to be relatively well aligned with the filament axis. When fibres are put into rigor, or the temperature or ionic strength is lowered, the degree of alignment decreases and there is a transfer of crossbridge mass towards the actin-containing filaments.

Adenosine Triphosphate

Form birefringence of muscle.

We investigate the sensitivity of measurements of muscle birefringence to cross-bridge dynamics in the resting, active, and rigor states. The theory of form birefringence is reviewed, and an optical model is constructed for the form birefringence of muscle. Values for the parameters in the model are selected or deduced from the literature. As an illustration of the use of the model, plausible distributions for the orientations of cross-bridges in the resting, active, and rigor states are constructed using a model for cross-bridge dynamics suggested by Huxley and Kress (1985). The general magnitude of the predictions of our model is comparable with that of published measurements of muscle birefringence. However, the precise values of the predicted birefringence for the resting, active, and rigor states are sensitive to the assumed orientations of cross-bridges. We also investigate the dependence of muscle birefringence on sarcomere length and on disorder in the orientation of the myofilament array. We conclude that measurements of muscle birefringence can play a useful role in distinguishing between proposed models of cross-bridge dynamics.

Actin Cytoskeleton

Electric birefringence of dilute agarose solutions.

The technique of transient electric birefringence was used to investigate the orientation of agarose solutions in pulsed electric fields. If the agarose was dissolved in deionized water, the sign of the birefringence was positive when the electric field was small, indicating that the agarose molecules were orienting parallel to the electric field lines. The decay of the birefringence was rapid, consistent with the orientation of individual agarose helices. The amplitude of the birefringence, but not the birefringence decay times, increased as the agarose solution aged, suggesting that the helices formed slowly from the sol state. Increasing the amplitude or duration of the pulsed electric field caused additional negative, and then positive, birefringence signals to appear, characterized by much slower rise and decay times, consistent with the formation of aggregates. The slowest decay times ranged from 7.5-9.0 s, suggesting that the aggregates were several microns in size. When agarose was dissolved in dilute Tris buffer instead of deionized water, the fast positive birefringence signal was not observed, suggesting that individual helices were not present in solutions containing dilute buffer.

Birefringence

Changes in birefringence as markers of thermal damage in tissues.

Light microscopy using polarized transmission illumination of routinely stained histologic sections shows changes of the native birefringence of certain tissue constituents when heated by laser irradiation or electrosurgical current. The naturally occurring birefringence of cardiac muscle disappears permanently when the muscle is frozen, thawed, and heated to temperatures in excess of 42 degrees C in vitro. This loss of birefringence is produced with temperatures at which other morphologic thermal changes are hard to detect; thus, it is a low-temperature tissue marker which can be used to observe the extent of thermal damage in tissues. Partial loss of the native birefringence of collagen occurs in canine urinary bladder coagulated by laser irradiation and pericardium heated with electrodes. In addition, thermally coagulated collagens have variable birefringence color shifts when compared to the adjacent unaffected collagens in stained histologic sections. The gradual birefringence color changes are seen at tissue temperatures higher than those at which the thermally induced hyalinization (coagulation) of collagen usually occurs (about 60-70 degrees C), but below those at which carbonization is seen (200+ degrees C). Birefringence changes can be measured to test mathematical models of thermal damage necessary for development of dosimetry models in medical applications of laser irradiation.

Animals

Monitoring rejection after heart transplantation: cytoimmunological monitoring on blood cells and quantitative birefringence measurements on endomyocardial biopsy specimens.

Cytoimmunological monitoring and quantitative birefringence measurements were used as potential aids in diagnosing acute rejection after heart transplantation instead of histopathological assessment of the endomyocardial biopsy specimen alone. Cytoimmunological monitoring was based on morphological inspection and quantitation of mononuclear cells, particularly activated lymphoid cells. Quantitative birefringence measurements comprise a variable for myocyte contractile function. Its read out is the ratio of the degree of birefringence before contraction to that after. Cytoimmunological monitoring indicated significantly higher concentrations of activated lymphocytes in moderate or severe acute rejection, and quantitative birefringence measurements indicated decreased myocyte function during severe and resolved or resolving rejection. Cytoimmunological monitoring and quantitative birefringence measurements were diagnostically most useful in terms of sensitivity, specificity, and predictive value, when only data gathered before the first episode of acute rejection were considered. For cytoimmunological monitoring, diagnostic relevance was optimal when the data were expressed as relative proportions of activated lymphocytes. The quantitative birefringence measurements correlated best with analysis of the endomyocardial biopsy specimen when a cut off value of 1.25 was used. When both methods for diagnosing acute rejection were analysed together, no improvement in sensitivity (value 0.44) was found, but the specificity increased to 0.98 and the predictive value to about 0.80. It is concluded that cytoimmunological monitoring is a useful, non-invasive additional method for diagnosing the first period of acute rejection after heart transplantation and that quantitative birefringence measurements give valuable information on the extent of myocyte damage.

Birefringence

A long-lasting birefringence change recorded from a tetanically stimulated squid giant axon.

A long-lasting birefringence change (the delayed response) was found to be produced in a tetanically stimulated squid giant axon. The change was independent of the concurrent membrane potential change, summated on repetitive stimulation, and always had a sign representing a decrease in resting birefringence. The axons was placed between a polarizer and an analyzer with their polarizing axes crossed, making an angle of 45 degrees with the longitudinal direction of the axon. The light beam that passed through the axon and the other optical elements was received by a photodiode. The change in light intensity evoked by repetitive stimulation was composed of brief initial responses, which took place in response to individual stimuli, and a delayed response, which developed gradually and lasted for several hundred msec. It was necessary to differentiate the effect of birefringence change from that of turbidity change. Formulas were derived on the assumption that the optical properties of the axon could be represented by a model of a uniaxial crystal that was not only birefringent but also dichroic, its extinction coefficients and the angle of retardation being changed independently on excitation. Calculations with them yielded the resting retardation, which agreed well with those obtained by the Senarmont's method, and the change in birefringence, which agreed well with the other calculated value derived from experiments using a quarter-wave plate. The results of the calculation confirmed the existence of the long-lasting birefringence change in the tetanically stimulated axon.

Action Potentials

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle.

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.

Animals

Spindle birefringence of isolated mitotic apparatus analysed by pressure treatment.

Sea-urchin zygote mitotic apparatus (MA) isolated in a glycerol/dimethylsulphoxide medium were treated with pressure. Pressure treatment had no effect on spindle birefringence when MA were in full-strength isolation medium. After placing MA in quarter-strength isolation medium, pressures of 4-0 X 10(3)-1-8 X 10(4) lbf in.-2 (2 X 76 X 10(4)-I X 24 X 10(5) k N m-2) for 15 min caused reduction of birefringence which occurred in 2 steps: firstly 20-30% of the birefringence was lost, and then, at higher pressures, the rest of the birefringence was lost. Electron microscopy suggested that pressure-induced changes were in non-microtubule material. Pressure treatment had no effect on MA isolated with hexylene glycol when the MA were pressurized in hexylene glycol; but pressure treatment did cause loss of birefringence when MA isolated in hexylene glycol were transferred immediately into glycerol/dimethylsulphoxide medium and were subsequently treated with pressure (after dilution into quarter-strength glycerol/dimethyl-sulphoxide). We discuss the differences in response between isolated MA and in vivo MA, and we discuss the possibility that 2 components contribute to MA birefringence.

Animals

Spindle birefringence of isolated mitotic apparatus analysed by treatments with cold, pressure, and diluted isolation medium.

Mitotic apparatus (MA) were isolated from sea-urchin zygotes using glycerol-dimethyl-sulphoxide. Cold treatment had no effect on MA birefringence when MA were in isolation medium, but caused a 10-15% reduction of MA birefringence when MA were in quarter-strength isolation medium. Pressure treatment also caused a reduction in MA birefringence, but the cold and pressure treatments were not additive, suggesting that both treatments affected the same MA component. MA were not stable in quarter-strength isolation medium, and birefringence gradually decayed, with a half-life of about 40 h. Electron microscopy after cold treatment, or after decay of 55% of the MA birefringence showed abundant, normal-looking microtubules, suggesting that alterations in non-microtubule components cause the reductions in birefringence. Addition of EGTA eliminates the effect of cold treatment, suggesting that Ca2+ has a role in maintenance of spindle structure. We discuss possible reasons why isolated MA do not respond to cold treatment like MA in vivo.

Animals

Birefringence of actin.

The total strain birefringence of F-actin isolated from chicken gizzards was measured as a function of elongation in thin transparent films. Each film held at a certain elongation in a jig was allowed to swell in a penetrating but nondissolving liquid. Seven liquids with different refractive indices were employed. The thickness of the film in each swelling liquid was obtained once equilibrium was established. At each elongation, from 0 to 16%, a Wiener curve was obtained. The minima of the Wiener curves yielded the intrinsic birefringence of F-actin as a function of elongation. The intrinsic birefringence increases with elongation up to 16%, above which the thin films break. The form birefringence at a set refractive index also increases with elongation. The implication of the strain birefringence of F-actin is discussed as it affects the optical properties, mainly light scattering, of tissues such as the fiber cells of lens of the eye.

Actins

Birefringence changes in vertebrate striated muscle.

The changes in birefringence in the rigor to relax transition of single Triton-extracted rabbit psoas muscle fibers have been investigated. The total birefringence of rigor muscle fibers was dependent on sarcomere length and ranged from (1.46 +/- 0.08) X 10(-3) to (1.60 +/- 0.06) X 10(-3) at sarcomere lengths from 2.70 mum to 3.40 mum. An increase in total birefringence was measured dependent on sarcomere length when 55 single fibers were relaxed from the rigor state with Mg-ATP. Pyrophosphate relaxation produced a smaller increase in retardation when compared to Mg-ATP. The expected change in intrinsic birefringence during the rigor to relax transition was calculated assuming a hinge function of the subfragment 2 moiety of myosin. The changes in birefringence during isometric contraction and relaxation have been discussed in relation to possible structural changes.

Adenosine Triphosphate

Positive and negative birefringence in chromosomes.

By using the optical properties of birefringence of DNA, the arrangement of these molecules has been studied in Dinoflagellate chromosomes and Dipteran polytene chromosomes. These latter are used, here, as a reference material. These observations have been made under a polarizing microscope on intact and stretched chromosomes.--Intact Dinoflagellate chromosomes show a positive birefringence, in contrast with polytene chromosomes bands which are negatively birefringent. From these observations one can deducd the preferential orientation of DNA filaments, in Dinoflagellates, normal to the chromosome axis, and in polytene chromosomes parallel to the same axis.--After stretching, these two kinds of chromosomes are negatively birefringent. In both cases, DNA molecules have been aligned along the stretch axis.--In Dinoflagellate chromosomes the passage from a positive to a negative birefringence is realized without any isotropic stage. The intermediary state presents a biaxial structure.

Animals