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Biomedical subjects

T C Irving

Publications and source records attributed to T C Irving.

At least 19 recordsLinked to original sources
PubMed2004

The BioCAT undulator beamline 18ID: a facility for biological non-crystalline diffraction and X-ray absorption spectroscopy at the Advanced Photon Source.

The 18ID undulator beamline of the Biophysics Collaborative Access Team at the Advanced Photon Source, Argonne, IL, USA, is a high-performance instrument designed for, and dedicated to, the study of partially ordered and disordered biological materials using the techniques of small-angle X-ray scattering, fiber diffraction, and X-ray absorption spectroscopy. The beamline and associated instrumentation are described in detail and examples of the representative experimental results are presented.

Academies and Institutes↗
PubMed2003

X-ray diffraction of the molecular substructure of human articular cartilage.

The molecular substructure of human articular cartilage has been difficult to study because of its complex composition and high degree of hydration. Using newly available small-angle X-ray diffraction (SAX) instrumentation that allows very short exposure times (0.1 to 10 sec), we have obtained spatially resolved information concerning the disposition of collagen fibers in the matrix of cartilage from the normal and osteoarthritic ankle and knee joints of human cadavers. Surprisingly, in zones of cartilage damage, such as in preosteoarthritic lesions or in the severely degenerated cartilage of osteoarthritic joints, collagen fibers of the deeper layers tended to be reoriented from the vertical. The SAX technique represents a nondestructive method of analyzing the collagen network in cartilage. Taken together, the data suggest a rigid control mechanism for the fiber network and an extensive passive reorganization of the collagen fiber orientation in diseased joint cartilage.

Ankle Joint↗
PubMed2001

Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes.

We studied the effect of titin-based passive force on the length dependence of activation of cardiac myocytes to explore whether titin may play a role in the generation of systolic force. Force-pCa relations were measured at sarcomere lengths (SLs) of 2.0 and 2.3 microm. Passive tension at 2.3 microm SL was varied from approximately 1 to approximately 10 mN/mm(2) by adjusting the characteristics of the stretch imposed on the passive cell before activation. Relative to 2.0 microm SL, the force-pCa curve at 2.3 microm SL and low passive tension showed a leftward shift (pCa(50) [change in pCa at half-maximal activation]) of 0.09+/-0.02 pCa units while at 2.3 microm SL and high passive tension the shift was increased to 0.25+/-0.03 pCa units. Passive tension also increased pCa(50) at reduced interfilament lattice spacing achieved with dextran. We tested whether titin-based passive tension influences the interfilament lattice spacing by measuring the width of the myocyte and by using small-angle x-ray diffraction of mouse left ventricular wall muscle. Cell width and interfilament lattice spacing varied inversely with passive tension, in the presence and absence of dextran. The passive tension effect on length-dependent activation may therefore result from a radial titin-based force that modulates the interfilament lattice spacing.

Actin Cytoskeleton↗
PubMed2001

The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.

Collagen Type I↗
PubMed2000

In vivo x-ray diffraction of indirect flight muscle from Drosophila melanogaster.

Small-angle x-ray diffraction from isolated muscle preparations is commonly used to obtain time-resolved structural information during contraction. We extended this technique to the thoracic flight muscles of living fruit flies, at rest and during tethered flight. Precise measurements at 1-ms time resolution indicate that the myofilament lattice spacing does not change significantly during oscillatory contraction. This result is consistent with the notion that a net radial force maintains the thick filaments at an equilibrium interfilament spacing of approximately 56 nm throughout the contractile cycle. Transgenic flies with amino-acid substitutions in the conserved phosphorylation site of the myosin regulatory light chain (RLC) exhibit structural abnormalities that can explain their flight impairment. The I(20)/I(10) equatorial intensity ratio of the mutant fly is 35% less than that of wild type, supporting the hypothesis that myosin heads that lack phosphorylated RLC remain close to the thick filament backbone. This new experimental system facilitates investigation of the relation between molecular structure and muscle function in living organisms.

Amino Acid Substitution↗
PubMed2000

Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium.

The Frank-Starling relationship of the heart has, as its molecular basis, an increase in the activation of myofibrils by calcium as the sarcomere length increases. It has been suggested that this phenomenon may be due to myofilaments moving closer together at longer lengths, thereby enhancing the probability of favorable acto-myosin interaction, resulting in increased calcium sensitivity. Accordingly, we have developed an apparatus so as to obtain accurate measurements of myocardial interfilament spacing (by synchrotron X-ray diffraction) as a function of sarcomere length (by video microscopy) over the working range of the heart, using skinned as well as intact rat trabeculas as model systems. In both these systems, lattice spacing decreased significantly as sarcomere length was increased. Furthermore, lattice spacing in the intact muscle was significantly smaller than that in the skinned muscle at all sarcomere lengths studied. These observations are consistent with the hypothesis that lattice spacing underlies length-dependent activation in the myocardium.

Actin Cytoskeleton↗
PubMed1998

High-Resolution X-ray Diffraction of Muscle Using Undulator Radiation from the Tristan Main Ring at KEK.

High Energy Accelerator Research Organization (KEK), Tsukuba, Japan.High-resolution X-ray diffraction studies on striated muscle fibres were performed using a hard X-ray undulator installed in the Tristan main ring at KEK, Tsukuba, Japan. The performance of the undulator, along with an example experiment which exploited the unique characteristics of undulator radiation, are reported. The vertical divergence angle of the first harmonic of the undulator was approximately 10 micro rad under 8 GeV multi-bunch operating conditions and the peak photon flux density was estimated to be approximately 3 x 10(16) photons s(-1) mrad(-2) (0.1% bandwidth)(-1) (10 mA)(-1). The well collimated X-ray beam from the undulator made it possible to resolve clearly, with high angular resolution ( approximately 700 nm), the closely spaced diffraction peaks on the meridional axis in the X-ray patterns arising from the thick filaments of a striated muscle under static conditions. By fitting the meridional intensity pattern, a model for the molecular arrangement of the constituent proteins in the thick filaments is proposed. These studies of muscle demonstrate the promise of undulator radiation from third-generation sources for high-resolution diffraction studies.

Journal Article↗
PubMed1998

X-ray diffraction indicates that active cross-bridges bind to actin target zones in insect flight muscle.

We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigor's lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor.

Actins↗
PubMed1998

Z/I and A-band lattice spacings in frog skeletal muscle: effects of contraction and osmolarity.

A-band and Z-line/I-band lattice spacings were measured by small-angle X-ray diffraction from relaxed and isometrically-contracting whole frog sartorius muscles with lattice spacings reduced or swollen by changing the osmolarity of the bathing solution. A-band spacing increased by approximately 3% upon isometric contraction at reduced lattice spacings (245-356 mOsm) and decreased by approximately 1% at swollen spacings (172 mOsm), similarly to the behaviour of skinned muscles upon changing from the relaxed state to rigor. The Z/I lattice underwent a significant lattice expansion (3-8%) upon isometric contraction at all osmolarities, in qualitative agreement (but quantitative disagreement) with results from electron microscopy on mammalian skeletal muscle. Lattice areas calculated for the Z/I and A-band lattices indicate a barrel-shaped sarcomere in the resting state, which may provide a partial explanation for how longitudinal forces produced in the A-band can produce a radial expansive force in the Z-line during contraction. The radial component of cross-bridge stiffness was calculated from the A-band data for contracting muscle, using a lattice stability model incorporating structural, osmotic and electrostatic forces. The calculations gave a radial cross-bridge stiffness during contraction of about 9 x 10(5) N m-2, and outward radial force per thick filament in normal Ringer's solution of 6 x 10(-9) N, corresponding to a radial force per cross-bridge of 10(-11) N.

Animals↗
PubMed1997

Cerebrosides alter the lyotropic and thermotropic phase transitions of DOPE:DOPC and DOPE:DOPC:sterol mixtures.

Freezing injury in rye and oat is a consequence of the formation of the inverted hexagonal (H(II)) phase in regions where the plasma membrane is brought into close proximity with cytoplasmic membranes during freeze-induced dehydration. Susceptibility to plasma membrane destabilization and H(II) phase formation during freezing is associated with alterations in plasma membrane lipid composition. This paper examines the influence of lipid composition and hydration on the propensity of lipid mixtures of DOPE:DOPC and DOPE:DOPC:sterols with added cerebrosides (CER) to form the H(II) phase during dehydration. The addition of CER to DOPE:DOPC:beta-sitosterol mixtures decreased the water content of the dispersions in a manner suggesting that most or all of the water in the dehydrated mixtures was associated with the phospholipids. The addition of CER significantly decreased the osmotic pressure at which the L(alpha) --> H(II) phase transition occurred from an osmotic pressure of 76.1 MPa for DOPE:DOPC (50:50) to 20 MPa in DOPE:DOPC:beta-sitosterol:CER (22.5:22.5:50:5) and 8 MPa in DOPE:DOPC:beta-sitosterol:CER (15:15:50:20). Experiments examining the effects of CER on the thermally-induced formation of the H(II) phase in fully hydrated mixtures and examining the influence of CER on the formation of the H(II) phase in DOPE:DOPC mixtures lacking beta-sitosterol suggested that CER facilitated the L(alpha) --> H(II) phase transition by effecting a decrease in bilayer hydration and by increased lateral packing pressures within the acyl domain of the bilayer. Taken in sum, these data indicate that the differential propensity of the rye and oat plasma membranes to undergo freeze-induced formation of the L(alpha) --> H(II) phase cannot be attributed to one lipid species. Rather, the propensity towards freeze-induced membrane destabilization is a consequence of the summation of physical characteristics of the membrane lipid components that included bilayer hydration, packing pressures within the hydrophobic domain of the membrane, the propensity of the lipid components to demix, and the relative proportions of the various lipid components.

Avena↗
PubMed1996

Small-Angle X-ray Diffraction of Muscle Using Undulator Radiation from the Tristan Main Ring at KEK.

Time-resolved X-ray diffraction of muscle has demanded ever-increasing flux into small sample volumes with low beam divergence. Results are reported of static and time-resolved small-angle X-ray diffraction studies on muscle fibers using a hard X-ray undulator installed in the Tristan main ring at KEK, Tsukuba, Japan, as an innovative source of synchrotron radiation more intense and better collimated than that available with the Photon Factory bending-magnet beamline. Static studies used the low divergence of the source to obtain detailed high-quality diffraction patterns of stable muscle states. The diffraction patterns from live skeletal muscles showed the numerous (over 100) meridional reflections. The well collimated beam from the undulator made it possible to clearly resolve, with an angular resolution of ca 700 nm, the closely spaced diffraction peaks arising from the two halves of the thick filaments centred on the M lines in a sarcomere, in addition, the diffraction peaks from the thin filaments on opposite sides of the Z bands could be resolved with an angular resolution of ca 1000 nm. The detailed structure of the meridional pattern defines the nature of the molecular packing in the thick and thin filaments. Time-resolved experiments using a focusing mirror aimed to prove cross-bridge states in striated muscle fibers by collecting X-ray diffraction data at a 0.185 ms time resolution from sinusoidally oscillating chemically skinned rabbit muscle fibers during active contraction and in rigor. When sinusoidal length changes at 500 Hz with a peak-to-peak amplitude of 0.6% of the muscle length were applied to a small fiber bundle, the tension showed a simple elastic response during the length oscillation. In the active muscle the intensity of the 14.5 nm myosin-based meridional reflection changed out of phase with the tension change during the oscillating length change. In contrast, in the rigor muscle it occurred in phase with the tension change. The high time-resolved experiments provide an insight into the coupling between conformational changes and force generation of the actomyosin cross-bridges. These studies provide a preview of the expected gains for muscle studies from the more widespread use of undulator radiation at third-generation synchrotron sources.

Journal Article↗
PubMed1996

Small-angle x-ray scattering from lipid bilayers is well described by modified Caillé theory but not by paracrystalline theory.

X-ray scattering data at high instrumental resolution are reported for multilamellar vesicles of L alpha phase lipid bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine at 50 degrees C under varying osmotic pressure. The data are fitted to two theories that account for noncrystalline disorder, paracrystalline theory (PT) and modified Caillé theory (MCT). The MCT provides good fits to the data, much better than the PT fits. The particularly important characteristic of MCT is the long power law tails in the scattering. PT fits (as well as ordinary integration with no attempt to account for the noncrystalline disorder) increasingly underestimate this scattering intensity as the order h increases, thereby underestimating the form factors used to obtain electron density profiles.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Effects of plant sterols on the hydration and phase behavior of DOPE/DOPC mixtures.

Freeze-induced injury of protoplasts of non-acclimated rye and oat is associated with the formation of the inverted hexagonal (HII) phase in regions where the plasma membrane and various endomembranes are brought into close apposition as a result of freeze-induced dehydration. The influence of lipid composition and hydration on the propensity of mixtures of DOPE:DOPC containing either sterols or acylated steryl glucosides to form the HII phase was determined by DSC, freeze-fracture electron microscopy and X-ray diffraction. The addition of plant sterols to a mixture of DOPE/DOPC (either 1:1:1 or 1:1:2 mole ratio of DOPE/DOPC/sterols) reduced the total hydration of the mixture (expressed as wt% water) after desorption over a range of osmotic pressures of 2.8 to 286 MPa. However, most or all of the water remaining in the dehydrated lipid mixtures was associated predominantly with the phospholipids. Both sterols and acylated steryl glucosides significantly promoted both the dehydration-induced and thermally induced L alpha-->HII phase transitions in DOPE/DOPC mixtures however, acylated steryl glucosides were much more effective. In mixtures containing plant sterols, the HII phase occurred after dehydration at 20 MPa (20 degrees C), which resulted in a water content of 11.7 wt%. In contrast, mixtures containing acylated steryl glucosides were in the HII phase in excess water, i.e., they did not require dehydration to effect the L alpha-->HII phase transition. The results indicate that genotypic differences in the lipid composition of the plasma membrane of rye and oat leaves have a significant influence on the propensity for formation of the HII phase during freeze-induced dehydration.

Acylation↗
PubMed1995

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.

Animals↗
PubMed1993

Filamentous aggregates of native titin and binding of C-protein and AMP-deaminase.

In solutions of high ionic strength, native titin-2, a large extractable fragment of the sarcomere matrix protein titin, appears as extremely long, flexible, and slender beaded strings. We report here that in solutions of lower ionic strength near neutral pH, titin-2 assembles into higher-order aggregates with surface projections. Solid phase binding assays show that two myosin-binding proteins, C-protein and AMP-deaminase, are also titin-binding proteins. Both proteins decorate titin aggregates, producing filaments of more uniform appearance. Numerical Fourier transforms of these decorated aggregates show approximately 12-nm periodicities. The interaction of titin with myosin-associated proteins such as C-protein may take part in the anchoring mechanism that prevents the stretching and extension of titin filaments in the A band.

AMP Deaminase↗
PubMed1992

Z-line/I-band and A-band lattices of intact frog sartorius muscle at altered interfilament spacing.

Muscle contraction has long been known to be affected by the osmolarity of the bathing solution. Part of this effect is caused by changes in interfilament spacing in the A-band. We have investigated the variation in spacing of the square lattice of thin filaments within and near the Z-line (the Z-line/I-band or Z-I lattice) in intact frog sartorius muscle over a wide range of osmolarities and compared it with the corresponding changes in the A-band lattice. Both lattices have a lower limit for compression and an upper limit for swelling. The spacing of the Z-I lattice is nearly proportional to that of the A-band, but shows a 2-3% variation at extreme shrinkage or swelling. In normal intact muscle, the osmotically-inactive volume of both lattices is between 20 and 30%. These in vivo measurements of lattice spacing differ significantly from those observed in electron micrographs. With moderate variations in osmolarity, lattice spacing and muscle fibre width show similar behaviour, but at extreme osmolarities, the lattice spacing changes less than the fibre width. An equatorial reflection was observed in intact muscle, previously identified in skinned muscle, which does not index on the A-band and which changes with osmolarity in a manner different from that observed for the A-band and Z-I lattices. This reflection may arise from changes in the ordering of the Z-I lattice or may involve components additional to the thick and thin filaments.

Actin Cytoskeleton↗
PubMed1989

Changes in thick filament structure during compression of the filament lattice in relaxed frog sartorius muscle.

Equatorial X-ray diffraction patterns from relaxed, chemically-skinned frog sartorius muscles under a range of external osmotic pressures from 0 to 290 torr have been analysed and compared to the pattern from the relaxed intact muscle. Lattice spacings and electron density diagrams were determined as a function of external pressure. Reflection intensities, averaged over small ranges of pressures, were determined out to the 4,0 reflection; phases for the first five orders were established as ++--+ over the whole pressure range. As external pressure was increased, lattice spacing decreased, as did full width at half maximum density for both thick and thin filaments. Most of the lattice spacing and thick filament compression occurred at low pressure, whereas thin filaments were compressed proportionally to pressure over the whole pressure range. These conclusions were confirmed by fitting cylindrical models for filament density to the X-ray diffraction patterns. Axially-projected electron density across the A-band filament lattice showed that in relaxed muscle the thick filament projections (myosin heads) are concentrated in regions between adjacent thick filaments, as far as possible from the thin filaments, and they tend to become pushed against the thick filament backbone as the lattice is compressed. Both thick and thin filament axes can be displaced randomly from their lattice positions; on average this displacement is about twice as great for the thin filaments, accounting for their larger projected size as compared to isolated thin filaments and for their apparent decrease in diameter as the lattice is compressed.

Absorptiometry, Photon↗