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Biomedical subjects

M Irving

Publications and source records attributed to M Irving.

At least 37 records · Page 2Linked to original sources
PubMed2000

Intestinal failure.

The recognition of intestinal failure (IF) as a distinct clinical entity over the past 20 years has primarily come about through the emergence of increasingly successful treatment based on use of intravenous nutrition delivered both in hospital and at home. Intestinal failure has many and varied causes, and may be complete or partial, acute and short lived, or chronic and permanent. If the latter, lifetime dependence on parenteral nutrition will usually be indicated and where possible patients are trained in the techniques of home parenteral nutrition (HPN). The success of HPN is itself primarily dependent on meticulous catheter care protocols. Sufficient numbers of such patients have now been entered into randomized controlled trials for there to be an evidence base for this aspect of IF treatment. The balanced delivery of nutrients, especially when an enteral component is included, can prevent many of the complications, such as liver and bone disease, recorded as being associated with prolonged total parenteral nutrition. However, nutritional balance is difficult to achieve in the presence of total gut resection, multiple intestinal fistulas or persisting sepsis. Surgical approaches involving bowel lengthening and reversed loops are indicated in selected patients. They have limited success and carry with them the risk of further loss of precious residual bowel. Greatest promise is held out by intestinal transplantation, but the problems associated with the powerful immunosuppression required reduce the value of this treatment. Specialized referral units for patients with severe and persistent IF can produce a high percentage of clinically effective and cost-effective outcomes.

Adaptation, Physiological↗
PubMed2000

Intestinal failure after surgery for complicated radiation enteritis.

Between 1983 and 1997, a total of 16 patients were referred to a tertiary Intestinal Failure Unit (IFU) following surgery elsewhere for complications of radiation enteritis. Eleven were female with a mean age of 43 years (range 21-71 years) and the most common primary site of malignancy was genitourinary (n = 13). Patients had undergone an average of two laparotomies (range 1-7 laparotomies) for complications of radiation enteritis prior to transfer to the IFU. On admission, the principal problem in eight patients was persisting intestinal fistulation, four patients had continuing intestinal obstruction and four had the short bowel syndrome after extensive intestinal resection. Only one patient had evidence of residual malignancy; this patient with short bowel syndrome was allowed home without invasive therapy. Of the remaining 15 patients, 12 required an abdominal surgical procedure, while three were discharged without further surgery after training for home parenteral nutrition (HPN). Following abdominal surgery, five patients died in hospital, but the remaining seven patients went home alive--including two further patients on HPN. Overall, of the 15 patients referred with intestinal failure after surgery for complications of radiation enteritis and actively treated, one-third died in hospital and a further third required institution of HPN before being able to be discharged home.

Adult↗
PubMed2000

Data acquisition, data storage, and data presentation in a modern genetics laboratory.

Modern genetic analysis can be divided into three main areas of investigation. The first is data acquisition, in the form of genomic sequence and the cataloguing of polymorphism data of the single nucleotide polymorphism variety (so called SNPs). Once identified, such genetic information can be adapted into high throughput tests to examine genetic information in large populations, making the analysis of sufficiently large numbers both cost and time effective so that relatively low-penetrant genetic effects can be accurately detected. The third step is correlating variation with phenotype (e.g. disease susceptibility or resistance) for a variety of disorders is paramount in our motivation and indeed is a common goal of modern human genetic analysis. While the technology to acquire vast amounts of genetic data is now well established and continues to expand, the ability to deal with such data, from the process of acquisition, storage, and analysis depends fundamentally on a solid informatics infrastructure as an essential component. Indeed, most of the major gains in productivity in this field are to be realized on the informatics front, and involve automating data acquisition, defining and sorting data in databases for quality control and analysis and facilitating access to data for the large variety of data analyses. Informatics-related issues including those relating to data acquisition, database structure, and analysis tools are summarized here in an effort to define some of the issues relevant to establishing informatics infrastructure in a small genetics laboratory focused on resequencing human immune response genes. From inherited diseases to drug efficacy to the specific genetic changes occurring during tumor development, this new field of medical genetics promises a profound impact on the state of human health. Ultimately, any and all advances in this field will continue to depend on major investments in informatics.

Computational Biology↗
PubMed1999

Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction.

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

Animals↗
PubMed1999

Changes in conformation of myosin heads during the development of isometric contraction and rapid shortening in single frog muscle fibres.

1. Two-dimensional X-ray diffraction patterns were recorded at the European Synchrotron Radiation Facility from central segments of intact single muscle fibres of Rana temporaria with 5 ms time resolution during the development of isometric contraction. Shortening at ca 0.8 times the maximum velocity was also imposed at the isometric tetanus plateau. 2. The first myosin-based layer line (ML1) and the second myosin-based meridional reflection (M2), which are both strong in resting muscle, were completely abolished at the plateau of the isometric tetanus. The third myosin-based meridional reflection (M3), arising from the axial repeat of the myosin heads along the filaments, remained intense but its spacing changed from 14.34 to 14.56 nm. The intensity change of the M3 reflection, IM3, could be explained as the sum of two components, I14.34 and I14.56, arising from myosin head conformations characteristic of rest and isometric contraction, respectively. 3. The amplitudes (A) of the X-ray reflections, which are proportional to the fraction of myosin heads in each conformation, changed with half-times that were similar to that of isometric force development, which was 33.5 +/- 2. 0 ms (mean +/- s.d., 224 tetani from three fibres, 4 C), measured from the end of the latent period. We conclude that the myosin head conformation changes synchronously with force development, at least within the 5 ms time resolution of these measurements. 4. The changes in the X-ray reflections during rapid shortening have two temporal components. The rapid decrease in intensity of the 14.56 nm reflection at the start of shortening is likely to be due to tilting of myosin heads attached to actin. The slower changes in the other reflections were consistent with a return to the resting conformation of the myosin heads that was about 60 % complete after shortening of 70 nm per half-sarcomere.

Animals↗
PubMed1999

Model-independent analysis of the orientation of fluorescent probes with restricted mobility in muscle fibers.

The orientation of proteins in ordered biological samples can be investigated using steady-state polarized fluorescence from probes conjugated to the protein. A general limitation of this approach is that the probes typically exhibit rapid orientational motion ("wobble") with respect to the protein backbone. Here we present a method for characterizing the extent of this wobble and for removing its effects from the available information about the static orientational distribution of the probes. The analysis depends on four assumptions: 1) the probe wobble is fast compared with the nanosecond time scale of its excited-state decay; 2) the orientational distributions of the absorption and emission transition dipole moments are cylindrically symmetrical about a common axis c fixed in the protein; 3) protein motions are negligible during the excited-state decay; 4) the distribution of c is cylindrically symmetrical about the director of the experimental sample. In a muscle fiber, the director is the fiber axis, F. All of the information on the orientational order of the probe that is available from measurements of linearly polarized fluorescence is contained in five independent polarized fluorescence intensities measured with excitation and emission polarizers parallel or perpendicular to F and with the propagation axis of the detected fluorescence parallel or perpendicular to that of the excitation. The analysis then yields the average second-rank and fourth-rank order parameters ( and ) of the angular distribution of c relative to F, and and , the average second-rank order parameters of the angular distribution for wobble of the absorption and emission transition dipole moments relative to c. The method can also be applied to other cylindrically ordered systems such as oriented lipid bilayer membranes and to processes slower than fluorescence that may be observed using longer-lived optically excited states.

Animals↗
PubMed1998

Elastic bending and active tilting of myosin heads during muscle contraction.

Muscle contraction is driven by a change in shape of the myosin head region that links the actin and myosin filaments. Tilting of the light-chain domain of the head with respect to its actin-bound catalytic domain is thought to be coupled to the ATPase cycle. Here, using X-ray diffraction and mechanical data from isolated muscle fibres, we characterize an elastic bending of the heads that is independent of the presence of ATP. Together, the tilting and bending motions can explain force generation in isometric muscle, when filament sliding is prevented. The elastic strain in the head is 2.0-2.7 nm under these conditions, contributing 40-50% of the compliance of the muscle sarcomere. We present an atomic model for changes in head conformation that accurately reproduces the changes in the X-ray diffraction pattern seen when rapid length changes are applied to muscle fibres both in active contraction and in the absence of ATP. The model predictions are relatively independent of which parts of the head are assumed to bend or tilt, but depend critically on the measured values of filament sliding and elastic strain.

Actins↗
PubMed1998

Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres.

Changes in the orientation of the myosin regulatory light chain (RLC) in single muscle fibres were measured using polarised fluorescence from acetamidotetramethylrhodamine (ATR). Mutants of chicken skeletal RLC containing single cysteine residues at positions 2, 73, 94, 126 and 155 were labelled with either the 5 or 6-isomer of iodo-ATR, giving ten different probes. The labelled RLCs were exchanged into demembranated fibres from rabbit psoas muscle without significant effect on active force generation. Fluorescence polarisation measurements showed that nine out of the ten probe dipoles were more perpendicular to the fibre axis in the absence of ATP (in rigor) than in either relaxation or active contraction. The orientational distribution of the RLC region of the myosin head in active contraction is closer to the relaxed than to the rigor orientation, and is not equivalent to a linear combination of the relaxed and rigor orientations. Rapid length steps were applied to the fibres to synchronise the motions of myosin heads attached to actin. In active contraction the fluorescence polarisation changed both during the step, indicating elastic distortion of the RLC region of the myosin head, and during the subsequent rapid force recovery that is thought to signal the working stroke. The peak change in fluorescence polarisation produced by an active release of 5 nm per half sarcomere indicates an axial tilt of less than 5 degrees for all ten probes, if all the myosin heads in the fibre respond to the length step. This tilting was towards the rigor orientation for all ten probes, and could be explained by 14% of the heads moving to the rigor orientation. An active stretch tilted the heads away from the rigor conformation by a similar extent.

Animals↗
PubMed1998

On the working stroke elicited by steps in length and temperature.

Muscle contraction is generally thought to involve tilting of the light chain region of the myosin head. This could account for 5-10 nm of axial displacement as it moves from nearly perpendicular to the filament axis (the state at the beginning of the working stroke) to the rigor conformation (at the end of the working stroke). According to the kinetic model of Huxley and Simmons, the extent that a cross-bridge progresses through the working stroke depends on the mechanical conditions. A large tilting occurs only when the fibre is allowed to shorten. Evidence for such tilting was provided by the changes in intensity of the third myosin meridional reflection (M3) following a step release. However, there is little change in the M3 intensity when a force increase is elicited by a 10 degrees C temperature jump, and these results were interpreted to indicate that tilting is not the structural transition responsible for force generation. Here we present a simulation of the changes in the intensity of the M3 reflection elicited by step changes in either length or temperature, based on the atomic model of the actin-myosin head complex. The results show that the same set of assumptions for the motions associated with the working stroke can predict the response to both kinds of perturbation. The main difference is due to the larger extent of the working stroke elicited by the length step.

Animals↗
PubMed1998

Myosin head movements during isometric contraction studied by X-ray diffraction of single frog muscle fibres.

Time resolved X-ray diffraction experiments in single muscle fibres of the frog at 2.15 microns sarcomere length and 4 degrees C were performed at ID2 (SAXS), ESRF, Grenoble (France) to investigate the structural aspects of cross-bridge action during the development of the isometric tetanic tension (T0). Changes in the low angle myosin-based reflections were measured with 5 ms time resolution by signal averaging data collected with a 10 m camera length and a 2D gas-filled detector. Upon activation the intensity of the first order myosin layer line reflection, I(M1), and the intensity of the second order meridional reflection, I(M2), reduced practically to zero with a half-time which leads the tension rise by 15-20 ms. The complex changes of the intensity of the third order myosin meridional reflection, I(M3), and the increase of its axial spacing from 14.34 nm (at rest) to 14.57 nm (at T0) could be analysed by assuming that they were the result of the combination of the time dependent modulation in intensity of two closely spaced periodicities, one at 14.34 nm, characteristics of the myosin molecule at rest and the other at 14.57 nm, assumed by the myosin as a consequence of the activation and force production. I(14.34) drops monotonically in advance to isometric tension development with a half-time similar to that of I(M1) and I(M2), while I(14.57) rises from zero to a maximum in parallel with tension.

Animals↗
PubMed1998

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.

Actins↗
PubMed1998

Steady-state fluorescence polarization studies of the orientation of myosin regulatory light chains in single skeletal muscle fibers using pure isomers of iodoacetamidotetramethylrhodamine.

The regulatory light chain (RLC) from chicken gizzard myosin was covalently modified on cysteine 108 with either the 5- or 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLCs were purified by fast protein liquid chromatography and characterized by reverse-phase high-performance liquid chromatography (HPLC), tryptic digestion, and electrospray mass spectrometry. Labeled RLCs were exchanged into the native myosin heads of single skinned fibers from rabbit psoas muscle, and the ATR dipole orientations were determined by fluorescence polarization. The 5- and 6-ATR dipoles had distinct orientations, and model orientational distributions suggest that they are more than 20 degrees apart in rigor. In the rigor-to-relaxed transition (sarcomere length 2.4 microm, 10 degrees C), the 5-ATR dipole became more perpendicular to the fiber axis, but the 6-ATR dipole became more parallel. This orientation change was absent at sarcomere length 4.0 microm, where overlap between myosin and actin filaments is abolished. When the temperature of relaxed fibers was raised to 30 degrees C, the 6-ATR dipoles became more parallel to the fiber axis and less ordered; when ionic strength was lowered from 160 mM to 20 mM (5 degrees C), the 6-ATR dipoles became more perpendicular to the fiber axis and more ordered. In active contraction (10 degrees C), the orientational distribution of the probe dipoles was similar but not identical to that in relaxation, and was not a linear combination of the orientational distributions in relaxation and rigor.

Animals↗
PubMed1998

Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.

Animals↗
PubMed1996

Steady-state polarization from cylindrically symmetric fluorophores undergoing rapid restricted motion.

Steady-state fluorescence polarization measurements provide a relatively simple method for investigating the orientation of molecular components in ordered biological systems. However, the observed fluorescence polarization ratios also depend on any mobility of the fluorophores on the time scale of the fluorescence lifetime. Such mobility commonly arises from incomplete immobilization of a fluorescent probe on the macromolecule of interest. A theoretical formalism is presented for the interpretation of steady-state fluorescence polarization ratios in the presence of such rapid fluorophore motion. It is assumed that the fluorophores move freely within a cone between absorption and emission of a photon. Only one new parameter is introduced to describe fluorophore motion, the semi-angle of the cone, and this can be measured in separate experiments on an isotropic system. The fluorophore orientations are assumed to have cylindrical symmetry, but the symmetry axis need not be in the same plane as the center axes of the absorption and emission cones. This geometry applies to muscle and other biological fibers.

Animals↗