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PubMed1975

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↗
PubMed1978

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↗
PubMed1988

Analysis of the birefringence of the smooth muscle anococcygeus of the rat, at rest and in contraction. I.

The birefringence of the rat anococcygeus muscle was measured at rest and in contraction. A large increase (+30%) of the optical retardation was observed in muscles fully stimulated by Noradrenaline, in isometric conditions. This was associated with a reduction of the muscle thickness (-12%), so that the birefringence increased by 48%. These changes were reversed upon relaxation. The relationship between the birefringence increase and the mechanical response was investigated as a function of time and of Noradrenaline concentration. Possible origins for the birefringence increase are considered: an increased density of birefringent material, mainly filaments, seems the most likely explanation of the results.

Anal Canal↗
PubMed1990

Chloramine-T alters the nerve membrane birefringence response.

The change in birefringence during depolarizing voltage-clamp pulses of internally perfused squid giant axons are biphasic. There is a rapid decrease in birefringence with a 220-microsec half time at 8 degrees C followed by a slow decrease over the next several milliseconds. After the pulse there is a rapid recovery which is smaller than the initial rapid decrease followed by a slow recovery phase. The rate of change of the slow phase during the pulse is more rapid for larger depolarizations. After the pulse the rate of change is more rapid for more negative potentials. 3.6 mM chloramine-T, applied externally until the sodium currents were prolonged and inactivation was removed, removed the slow phase of the birefringence response both during and after the pulse and made the fast 'off' response as large as the fast 'on' response. Two anesthetics reduced the birefringence response by about 20%. A rocking helix model is presented which relates the birefringence findings and earlier gating current experiments.

Animals↗
PubMed1987

Optical birefringence of phosphatidylcholine liposomes in gel phases.

A simple and rapid method for studying optical anisotropic properties of liposomes was proposed. Intensities of transmitted light through one spherical liposome of dipalmitoylphosphatidylcholine placed between two polarizers were measured at various wavelengths by a microscopic spectrophotometer. Large increases in the intensities were observed at the prephase-transition temperature, which were caused by an increase in the birefringence of the multilayer of the liposome. The birefringence values obtained from the intensity data were about 0.020 below the pretransition temperature and 0.028 above that temperature. These values are in good agreement with the results reported for the plane samples in which lipid bilayers are stacked. The obtained values of the birefringence were far lower than the values estimated from polarizabilities. This lower birefringence is attributed to disordering of the tilt direction in the multilayer. The degree of order of the liposome multilayers calculated from the birefringence increased by 38% at the pretransition. This simple method is applicable to the study of the multilayer structure of liposomes in water.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1985

Histone phosphorylation in native chromatin induces local structural changes as probed by electric birefringence.

In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.

Animals↗
PubMed2004

Analysis of the contraction of an organelle using its birefringency: the R-fibre of the Ceratium (Dinoflagellate) flagellum.

Some organelles responsible for contraction consist of bundles of 2-4 nm filaments called nanofilaments. Such organelles are present in the longitudinal flagellum of Ceratium (Dinoflagellate): the R-fibre is the motor system for contraction and parallels the axoneme, which is responsible for wave generation. We used a highly sensitive polarization microscope developed by one of the authors to measure the birefringence of these nanofilament bundles during contraction in vivo. Our results show that the R-fibre gives a highly birefringent signal, retarding the polarization to much the same extent irrespective of the direction of polarization. By rotating the axis of the microscope compensator we confirmed that the birefringence is positive, suggesting that the bundles run parallel to the longitudinal axis of the flagellum. Conversely, when the compensator was rotated contrary to the direction of retardation, the bundle appeared dark (except when the organelle was in a fully contracted state). Experiments performed on detergent-treated and ATP-reactivated flagella show that a portion of the flagella regained activity with the addition of ATP in the presence of low Ca(2+) concentrations. This demonstrates the ability to reactivate flagellar motility after permeabilization and that axonemal microtubules were not responsible for the strong flagellar birefringence. Combined with complementary data from DIC microscopy of demembranated flagella and electron microscopy, these findings have led to the development of a model of the R-fibre and a comparison with other types of birefringent nanofilament bundles, such as the myoneme of Acantharia.

Animals↗
PubMed2004

Simultaneous birefringence and scattered light measurements reveal anatomical features in isolated crustacean nerve.

Simultaneous fast birefringence and scattered light changes associated with crustacean nerve activation have different time courses and are produced by separate biophysical mechanisms. Technological advances in illumination, photodiodes and amplification circuitry achieved better signal-to-noise than earlier studies revealing optical signals in axonal nerve bundles as small as crayfish ventral cord and claw. The birefringence measurements yielded signals that could be observed in single trials, with temporally separated peaks associated with axonal populations of different diameters. A slit aperture placed perpendicular to the nerve reduced the spatial-temporal integration and enhanced the temporal structure of the separate peaks in the birefringence signal. Moving the slit aperture farther from the stimulation point delayed the signal in time, and also enhanced the separation between peaks. Different propagation velocities of the separate peaks provided evidence for at least three neuronal populations in the bundle. These studies underscore the advantages of birefringence over scattering measurements. Application of birefringence methods can optimize non-invasive imaging techniques being developed to detect fast optical responses associated with electrical neural activity in humans.

Animals↗
PubMed1963

Birefringence of protein solutions and biological systems. I.

The quantitative interpretation of birefringence of biological structures such as muscle requires a knowledge of intrinsic birefringence of the components. The intrinsic birefringence of fibrous structures as determined by variation of solvent index is positive while the intrinsic birefringence of proteins in solution is negative as calculated by the Peterlin-Stuart theory. As a first step in clarifying this discrepancy the basis of the Peterlin-Stuart theory has been re-examined. The theory has been recalculated from the standpoint of light scattering and extended to particles whose length is not small compared to the wavelength. The birefringence of a system of particles possessing a shell with index different from the bulk solvent has been obtained in order to interpret measurements in mixed solvents.

Biophysical Phenomena↗
PubMed1978

Birefringence measurements of structural inhomogeneities in Rana pipiens rod outer segments.

The birefringence (deltan) of Rana pipiens rod outer segments (ROS) reveals microstructure inhomogeneities not seen with other techniques. In the basal 20-30-micron length of the ROS there is a nearly linear axial gradient in deltan of approximately equal to -2 x 10(-5)/micron. No consistent deltan gradients are found in the distal 20-30 micron of the ROS. Using glycerol imbibition to separate the intrinsic and form birefringence components, we found that the basal deltan gradient was principally due to a gradient of the intrinsic birefringence component. The disk membrane volume fraction decreases uniformly from the basal end to the distal end, while the disk membrane refractive index increases. The contributions of these changes to the form birefringence approximately cancel, so that the form component is fairly uniform along the ROS axis. Because its axial distance from the inner segment is a measure of the time since a disk membrane was formed, these gradients may reflect a disk membrane aging process. Occasionally a highly birefringent, 2-micron-wide band is seen at the basal end ot the ROS, possibly where the envelope membrane folds to form new disk membranes.

Animals↗
PubMed1994

Simultaneous measurement of electric birefringence and dichroism. A study on photosystem 1 particles.

We have developed a straightforward method to separate linear-dichroism and birefringence contributions to electric-field induced signals in a conventional birefringence setup. The method requires the measurement of electric birefringence for three different angular positions of the analyzer. It is demonstrated that the presence of linear dichroism can significantly influence the measured signals and lead to completely erroneous calculations of the birefringence signal and field-free decay times if its contribution is not taken into account. The new method is used to determine electric birefringence and linear dichroism of trimeric Photosystem 1 complexes from the cyanobacterium Synechocystis PCC 6803 in the detergents n-dodecyl-beta-D-maltoside and n-octyl-beta-D-glucoside. It is concluded that the orientation of the particles in the field is predominantly caused by a permanent electric dipole moment that is directed parallel to the symmetry axis of the particles. Comparison of the decay times obtained with dodecylmaltoside and octylglucoside supports a model in which the thickness of the disc-like complexes remains similar (7-8 nm) upon replacing dodecylmaltoside by octylglucoside, whereas the diameter increases from 14.4 +/- 0.2 to 16.6 +/- 0.2 nm because of an increased thickness of the detergent layer. This change in diameter is in good agreement with electron-microscopy results on Photosystem 2 complexes in dodecylmaltoside and octylglucoside (Dekker, J. P., E. J. Boekema, H. T. Witt, and M. Rögner. 1988. Biochim. Biophys. Acta 936:307-318). The value of approximately 16.6 nm for the diameter of Photosystem 1 trimers in dodecylmaltoside is in good agreement with recent results obtained from electron microscopy in combination with extensive image analysis (Kruip, J., E. J. Boekema, D. Bald, A. F. Boonstra, and M. Rögner. 1993. J. Biol. Chem. 268:23353-23360).

Birefringence↗
PubMed1989

Temperature-dependent birefringence patterns in Xenopus rod outer segments.

Results reported here show that the birefringence of disk membranes in Xenopus rod photoreceptors depends upon the temperature at which the disks were assembled. Portions of the outer segments produced by the assembly of new disks while frogs are exposed to constant darkness and an ambient temperature of 25 degrees C have a higher birefringence than portions assembled at 18 degrees C. The intensity of birefringence reflects molecular-level structure and macromolecular-level organization of the disk membranes. It has been shown previously that increasing the ambient temperature enhances the rate of assembly of disk membranes in Xenopus rod outer segments. It also has been shown that disk membranes are assembled most frequently in the hours following light onset, and that disks assembled in the light have a lower birefringence than disks assembled in the dark. Therefore, although light and increased temperature both enhance the rate of disk membrane assembly, they have opposite effects upon the birefringence of Xenopus rod outer segments.

Animals↗
PubMed1979

Lymphocyte activation and the increase of nuclear birefringence.

Nuclear birefringence of neutral red or rivanol stained cell suspensions from rat spleen has been investigated. Polyclonal or monoclonal mitogens produced an increased birefringence of the nucleus following 30 min in vitro stimulation at 37 degrees C. The nuclear birefringence increased by 62.2% [p less than 0.001], when the cells were incubated in the supernatants of a previously phytohemaglutinin stimulated culture. Amantadine, a potent phytohemagglutinin inhibitor, was unable to prevent the effect of the supernatant, but heating for 1 h at 56 degrees C destroyed its activity. The results suggest that increase in nuclear birefringence is mediated by a soluble factor which is released in the course of lymphocyte activation. The nuclear birefringence of surviving cells from human spleens obtained within 6 to 24 h post mortem increases after in vitro stimulation.

Animals↗
PubMed1986

Structural aspects and orientation mechanism of mitochondrial F1 adenosinetriphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle.

The electric birefringence technique was used to investigate the steady-state birefringence, the orientational relaxation time, and the orientation mechanism of pig heart mitochondrial F1 adenosine-5'-triphosphatase (F1-ATPase). The electrooptical properties of this enzyme in solution were studied as functions of pH, protein concentration, and applied electric field. The F1-ATPase exhibits a surprising negative electric birefringence with a specific Kerr constant of -1.5 X 10(-3) esu cgs. The field-independent relaxation time was found to be 0.65 +/- 0.05 microseconds, corresponding to a rotational diffusion constant of 2.55 X 10(5) s-1. The overall size and shape of F1-ATPase have been calculated from both translational and rotational diffusion constants. The enzyme may be assumed to be an oblate ellipsoid of revolution with dimensions of about 170 X 170 X 70 A. The orientation mechanism of F1-ATPase was analyzed by fitting experimental birefringence rising curves with theoretical rising functions. The ratio of the permanent to induced dipole moment is found to be very high; therefore, the birefringence of F1-ATPase is due to a strong permanent dipole moment in a direction perpendicular to the long axes of the particle. These particular electric properties can be explained by the oligomeric structure of the protein and seem likely to play a role in its mechanism of functioning.

Animals↗
PubMed1999

Layered structure of saccular aneurysms assessed by collagen birefringence.

Cerebral aneurysms are composed principally of collagen, a birefringent protein which is responsible for withstanding the forces of blood pressure. The known correlation between collagen birefringence and its mechanics provides the basis for using polarizing microscopy to evaluate the strength of collagen, layer by layer across the aneurysmal wall. In order to obtain better quantitative measurements, several birefringent enhancement stains were investigated. We concluded that sirius red F3B, at a concentration of 0.05% in saturated picric acid, is an excellent stain to enable measurement of both birefringence and directional organization on the same tissue sections. Six aneurysms from autopsy, fixed at 120 mmHg, and one surgical specimen were cut at 4 microns to provide sets of tangential sections. The polarizing optics emphasizes the multi-layered structure of the aneurysmal wall with the mean fiber alignments distinguishing one layer from another. Birefringence measurements showed that the outer third of the wall had mainly higher strength collagen, although not as high as nearby artery adventitia. The inner layers of the aneurysms had intermediate values, similar to the artery media and subendothelium. Our results are consistent with a model of aneurysmal enlargement that requires the reorganization of higher strength outer fibers while new collagen is added to the inner layers.

Birefringence↗
PubMed1975

Microtubular origin of mitotic spindle form birefringence. Demonstration of the applicability of Wiener's equation.

Meiosis I metaphase spindles were isolated from oocytes of the sea-star Pisaster ochraceus by a method that produced no detectable net loss in spindle birefringence. Some of the spindles were fixed immediately and embedded and sectioned for electron microscopy. Others were laminated between gelatine pellicles in a perfusion chamber, then fixed and sequentially and reversibly imbibed with a series of media of increasing refractive indices. Electron microscopy showed little else besides microtubules in the isolates, and no other component present could account for the observed form birefringence. An Ambronn plot of the birefringent retardation measured during imbibition was a good least squares fit to a computer generated theoretical curve based on the Bragg-Pippard rederivation of the Wiener curve for form birefringence. The data were best fit by the curve for rodlet index (n1) = 1.512, rodlet volume fraction (f) = 0.0206, and coefficient of intrinsic birefringence = 4.7 X 10(-5). The value obtained for n1 is unequivocal and is virtually as good as the refractometer determinations of imbibing medium index on which it is based. The optically interactive volume of the microtubule subunit, calculated from our electron microscope determination of spindle microtubule distribution (106/mum2), 13 protofilaments per microtubules, an 8 nm repeat distance and our best value for f, is compatible with known subunit dimensions as determined by other means. We also report curves fitted to the results of Ambronn imbibition of Bouin's-fixed Lytechinus spindles and to the Noll and Weber muscle imbibition data.

Animals↗
PubMed1983

Birefringence signals in mammalian and frog myocardium. E-C coupling implications.

Birefringence signals from mammalian and frog hearts were studied. The period between excitation and the onset of contraction in which optical signals were free of movement artifact was determined by changes in scattered incandescent light and changes in laser diffraction patterns. The birefringence signal preceding contraction was found to behave as a change in retardation and was not contaminated measurably by linear dichroic or isotropic absorption changes. There were two components of the birefringence signal in mammalian heart muscles but only one component in the frog heart. The first component of the birefringence signals in both mammalian and frog hearts had a time course coincident with the action potential upstroke. The second component in mammalian preparations was sensitive to inotropic interventions, such as variation of extracellular Ca2+, stimulation frequency, temperature, and epinephrine, in a manner that correlated with the maximum rate of rise of tension. Caffeine (2-10 mM) not only failed to generate a second component in the frog heart, but also suppressed the second component in the mammalian heart while potentiating twitch tension. The results suggest that the second component of the birefringence signal in the mammalian myocardium is related to Ca2+ release from the sarcoplasmic reticulum.

Action Potentials↗
PubMed1993

Structural analysis of Trypanosoma brucei brucei chromatin by limited proteolysis. An electrical-birefringence study.

The sensitive electric-birefringence method was used to reveal structural differences between the soluble chromatin of procyclic Trypanosoma brucei brucei and the chromatin of the higher eukaryotes. The orientation of the nucleosomal chains and the presence of extended DNA were analysed from the sign and amplitude of the steady-state birefringence, and the conformational properties (overall dimensions and flexibility) were studied in relation to the orientational relaxation times. In contrast to the higher eukaryotes, the birefringence of T. brucei brucei is negative and of low amplitude, corresponding to that of H1-depleted rat liver nucleosomes. Furthermore, the relaxation times are very small, about 10 microseconds. If salt is added, the birefringence as well as the relaxation time decreases dramatically, indicating that condensation affects T. brucei brucei chromatin although it behaves like nucleosome filaments, with less stable DNA-protein interaction than for the higher eukaryotes. However, this condensation does not induce the formation of regular higher-order structure. This complies with the hypothesis that typical histone H1 is absent from T. brucei brucei chromatin and that a protein or protein domain fulfils the role of histone H1. The accessibility and structural role of histone-like proteins in T. brucei brucei chromatin were also investigated using limited proteolysis with enzymes covalently bound to nylon spheres. The analysis of protein products obtained after digestion with immobilized trypsin and subtilisin shows that proteins a and d, which are classified as H3 and H4 histones, respectively, are the first to be attacked. The changes in chromatin conformation indicate that chromatin undergoes a structural transition, leading to decondensation, as indicated by increases in negative birefringence and relaxation time, and to a change in its orientation mechanism, indicated by the appearance of a permanent moment. This result is very interesting since, in rat liver, H4 was very resistant and was the last histone to be attacked, suggesting internal location and its involvement in nucleosome stabilization rather than higher-order condensation. Therefore, in T. brucei brucei chromatin, the characteristic properties of proteins a and d (their composition and interaction with DNA), as well as their external location on the nucleosome surface, suggest that if these proteins play a role similar to that played by H3 and H4 in higher eukaryotes, probably through their N-terminal regions and interaction either with DNA or protein domains, the mechanisms involved in chromatin compaction are quite different.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗