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K P Roos

Publications and source records attributed to K P Roos.

At least 19 recordsLinked to original sources
PubMed1996

Mechanical measurements from isolated cardiac myocytes using a pipette attachment system.

Single rat left ventricular myocytes were attached at both ends using a newly described double-barreled micropipette technique. This attachment procedure enabled the measurement of the active and passive mechanical properties of chemically skinned cells that showed little structural deformation. The force and oscillatory stiffness (100 Hz) of the cells were measured with a high signal-to-noise ratio, and the sarcomere length throughout the entire cell was monitored using image analysis. The passive properties were investigated from the resting sarcomere length to > 3 microns. Analysis of the sarcomere behavior indicated a high level of homogeneity throughout the cell. The attachment method supported the full activation of the cells by increased free Ca2+ (pCa 4.5), which produced 22.3 mN/mm (mean sarcomere length 2.11 microns). A force/pCa relationship was determined, which, when fitted according to the Hill equation, gave parameters of nH = 2.62 and pCa50 = 5.58. The described techniques allow the accurate study of the mechanical properties of single myocytes with increased fidelity and reliability over the preexisting methods.

Animals↗
PubMed1993

Transformation of Helicobacter pylori by chromosomal metronidazole resistance and by a plasmid with a selectable chloramphenicol resistance marker.

Most strains of Helicobacter pylori are naturally competent for uptake of chromosomal DNA. Transformation frequencies for streptomycin resistance or rifampicin resistance markers ranged from 1 x 10(-4) to 1 x 10(-3) per viable cell using a plate transformation procedure. Transformation of a metronidazole resistance marker (MtrR) was demonstrated when either a laboratory-derived mutant or a MtrR clinical isolate were used as the source of donor DNA. MtrR was transformed at a frequency of 3 x 10(-5) per viable cell. All H. pylori strains tested produce large amounts of DNAase, which may reduce DNA available for transformation. Four H. pylori plasmids were isolated. DNA fragments from H. pylori plasmids were deleted or rearranged when cloned in pUC19 and propagated in Escherichia coli DH5 alpha. An H. pylori plasmid, pUOA26 which contained a chloramphenicol resistance determinant from Campylobacter coli, was constructed in H. pylori. This plasmid could be successfully introduced by natural transformation only into H. pylori recipients which contained a homologous resident plasmid. Transformation of pUOA26 into plasmid-free cells of H. pylori was achieved by electroporation. Transformation frequencies were 1 x 10(-4) transformants per viable cell when plasmid DNA was isolated from the same strain; however, introduction of pUOA26 DNA derived from H. pylori 8091 into a different H. pylori strain, NCTC 11639, resulted in transformation at much lower frequencies (< or = 1 x 10(-7) per viable cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1990

Osmotic compression and stiffness changes in relaxed skinned cardiac myocytes in PVP-40 and dextran T-500.

Sarcomere lengths, cell widths, indices of stiffness, and striation pattern uniformity were determined from radially compressed isolated adult cardiac myocytes from the rat. Single cells were bathed in a series of relaxing solutions containing 0-15% concentrations of nonpenetrating long chain polymers PVP-40 and dextran T-500. There were no significant changes observed in average sarcomere lengths or in striation pattern uniformity at any concentration. But cell widths decreased and stiffness increased in both polymers in a concentration-osmotic pressure-dependent relationship. Changes in cell width and stiffness were repeatable in either polymer, but only after an initial compression with a 10 or 15% concentration solution. The observed reduction in cell width after initial compression correlates well with known myofilament lattice spacing compression in rat cardiac muscle and is qualitatively similar to compressions seen in skeletal muscle preparations. But the cardiac myofilament lattice may not be as compressible as the skeletal lattice. Like skeletal muscle, stiffness exhibits a two-phase relationship where most of the increase occurs at solution osmotic pressures greater than 20 Torr. Finally, the inherently greater passive stiffness-length relationship of cardiac muscle is maintained at higher osmotic pressures such that the passive elastic modulus is strongly length dependent.

Actin Cytoskeleton↗
PubMed1989

Stiffness and shortening changes in myofilament-extracted rat cardiac myocytes.

Sarcomere lengths, cell widths, volumes, stiffness, and regional striation uniformity were determined from isolated adult cardiac myocytes. Single cells were examined in the control saline solution followed by a sequence of relaxing, membrane skinning, and myofilament extraction solutions. Cell size and shape parameters were determined from freely dispersed myocytes, whereas stiffness was measured from myocytes attached to a perturbator and tension transducer with micropipettes. There were small changes in cell appearance, size, shape, and stiffness in the relaxing and skinning solutions. However, in 0.17-0.56 M KCl myosin extraction media, cell length declined significantly to 1.19 microns, and stiffness fell to 5-10% of control. The rate of cell shortening and stiffness decline was dependent on KCl concentration and pH. Subsequent exposure to higher ionic strength 0.60 M KI thin filament extraction media elicited additional decreases in stiffness (less than 5% of control) and cell length (0.98 micron). Cell shortening and stiffness decline have similar time courses under the same conditions, and they appear to coincide with A-band disassembly as indicated by electron micrographs. These data suggest that cardiac myocyte stiffness, size, and shape are determined in part by a stressed cytoskeleton that is associated with the myofilament apparatus.

Actin Cytoskeleton↗
PubMed1989

Slowly relaxing caffeine responses in rat ventricle: relationships of ryanodine and caffeine actions.

Contractile responses to 20 mM caffeine were compared in arterially perfused rat right ventricle as tension, in intact rat myocytes as shortening, and in Triton X-100-"skinned" rat myocytes as shortening and stiffness. Responses in intact quiescent ventricle and membrane-disrupted myocytes at -log molar Ca concentration (pCa) of 7.0-6.8 were similar; force and stiffness rose quickly on application of caffeine and declined incompletely toward base line over 20 min. Subsequent caffeine responses were blunted. By contrast, in freshly isolated rat myocytes, rapid caffeine exposures induced large phasic contractures, which relaxed for the most part in 2-5 s. These responses were largely suppressed by 0.2 microM ryanodine pretreatment at 35 degrees C but not by high ryanodine concentrations (10 microM). Ryanodine was without effect on slowly relaxing contractile responses in both intact ventricle and Triton X-100-treated myocytes. The results are discussed in relation to the predicted effects of a calcium store leak in a simple model of cardiac excitation-contraction coupling. The results suggest that slowly relaxing caffeine contractures can be caused entirely by direct actions of caffeine on myofilaments.

Alkaloids↗
PubMed1987

Sarcomere length uniformity determined from three-dimensional reconstructions of resting isolated heart cell striation patterns.

A- and I-band striation positions have been obtained, three-dimensionally reconstructed, and statistically analyzed from the volumes of resting isolated heart cells. Striation patterns from optically discrete subvolumes are imaged along the length of these myocytes with a computer-interfaced optical microscope imaging system. Planar striation maps are reconstructed by the computer from sequentially obtained striation pattern images displaced across the width or depth of the cell in controlled steps. Multiple planar maps are combined to form full three-dimensional (3-D) reconstructions that illustrate the sarcomeric structure and ordering throughout the volume of the cell. These reconstructions demonstrate a high degree of striation registration throughout most regions of cardiac cells. The striation registration is often slightly (less than 10 degrees) skewed across the width or depth of nearly every cell and is occasionally disrupted between adjacent groups of sarcomeres. These disruptions in registration are always associated with the locations of the nuclei. Rigorous statistical analyses indicate small volumetric regions of the cell delineated by these disruptions can have significantly (0.014-0.113 micron) shorter or longer average sarcomere length periodicities. Unlike skeletal muscle "fibrillenstruktur," these data from cardiac cells exhibit no evidence of helical packing schemes for sarcomere order. These observations suggest that the relatively large nuclei displace and disrupt the normal registration of the sarcomeres, which is probably mediated by internal cytoskeletal structures.

Animals↗
PubMed1987

Theoretical Fraunhofer light diffraction patterns calculated from three-dimensional sarcomere arrays imaged from isolated cardiac cells at rest.

Sarcomere striation positions have been obtained throughout the volumes of calcium-tolerant resting heart cells by direct computer interfaced high-resolution optical imaging. Each sarcomere position is stored in a three-dimensional (3-D) matrix array from which Fraunhofer light diffraction patterns have been calculated using numerical methods based on Fourier transforms. Diffraction patterns have been calculated from heart cell data arrays oriented normal to a theoretical laser beam. Twelve characteristic features have been identified and described from these diffraction patterns that correlate to diffraction phenomena observed from both cardiac and skeletal muscle. This numerical approach provides the means to directly assess diffraction pattern formulation, the precision of layer line angular separation, layer-line intensity and angular asymmetries, line widths and fine structures in terms of the known diffracting source structures. These results confirm that theoretical calculations can predict real muscle diffraction patterns and their asymmetries.

Animals↗
PubMed1986

Length, width, and volume changes in osmotically stressed myocytes.

Sarcomere lengths, cell widths, and cell volumes were determined from photomicrographs and direct optical imaging of Ca2+-tolerant myocytes dispersed from collagenase-perfused whole rat myocardium. Individual unattached cells were examined at rest first in the normosmotic Ca2+-Tyrode saline solution and then in a sequence of hypotonic or hypertonic Tyrode solutions. Myocytes swell and shrink in hypotonic and hypertonic media but not as much as expected for an ideal uniform osmotic system. Furthermore, these changes were not proportional in the longitudinal and radial directions. These data suggest an 18% osmotically inactive volume and significant internal radial and longitudinal load-bearing structures within these cardiac myocytes.

Animals↗
PubMed1982

Individual sarcomere length determination from isolated cardiac cells using high-resolution optical microscopy and digital image processing.

Discrete sarcomere lengths have been determined from dynamically contracting isolated cardiac cells with a high-speed, high-resolution direct optical imaging system. Calcium-tolerant cardiac cells from the rat are isolated by perfusion with collagenase and hyaluronidase. Individual sarcomere lengths can be determined by directly imaging the cell's striation pattern onto a solid-state charge-coupled device (CCD) detector interfaced with a digital computer. The precision of detection in a real light microscopic optical system is discussed in relation to the type of image detector, optical contract enhancement techniques, and digital image processing. The optical performance of the direct striation pattern image apparatus has been determined empirically with test grids under standard bright-field and Nomarski-differential interference contrast (DIC) conditions for application to real muscle imaging. Discrete striation positions of isolated cells have been detected and followed with high precision during phasic contraction-relaxation cycles down to average sarcomere lengths as short as 1.43 +/- 0.053 microns. The maximum rates of contraction and relaxation are rapid and synchronous in time course along the length of the cell. These results indicate that direct optical imaging can provide an accurate means to monitor discrete striations and sarcomere lengths along the length of Ca2+-tolerant heart cells.

Animals↗
PubMed1982

Direct measurement of sarcomere length from isolated cardiac cells.

Isolated cardiac muscle cells enzymatically digested with collagenase and hyaluronidase from whole rat myocardium demonstrate the characteristics of an intact membrane in that they tolerate millimolar concentrations of free Ca2+ and exhibit phasic contractions with electrical excitation. These isolated cells maintain their characteristic A-I band striation patterns when at rest or during contraction. An apparatus has been developed to directly image these cells with phase-contrast micrography onto a 1,728-element charge-coupled device photodiode array for rapid data storage in a digital computer. The digitized striation pattern profile was analyzed for individual and average sarcomere spacing. In isotonic media the average resting sarcomere length ranged from 1.77 to 1.91 micrometers in 13 cells, with a mean length of 1.83 +/- 0.12 micrometers. Electrically stimulated phasic contractions in three cells demonstrated a synchronous 20% decrease in sarcomere spacing to a mean of 1.51 micrometers. Striation spacing decreased under hypertonic stress but elongated only up to 1.93 micrometers in hypotonic solutions, suggesting that some internal elastic constraint exists that limits elongation of the cell.

Animals↗
PubMed1980

Theory of light diffraction by single skeletal muscle fibers.

A theoretical discussion is presented describing the diffraction of laser light by a single fiber of striated muscle. The complete three-dimensional geometry of the fiber has been taken into consideration. The basic repeated unit is taken as the sarcomere of a single myofibril, including its cylindrical geometry. The single fiber is considered as the sum of myofibrils up to the fiber dimensions. When proper phasing is taken into account, three cases of interest are analyzed. (a) When the adjacent myofibrils are totally aligned with respect to their index of refraction regions (e.g., A and I bands), then the diffraction pattern reflects that of a larger striated cylinder with the dimensions of the fiber. (b) When a particular skew plane develops for the myofibril elements, additional Bragg reflection occurs at certain specific sarcomere lengths, and intensity asymmetry amongst the diffracted orders occurs. (c) When the myofibril phasing changes in a random fashion, while all sarcomeres remain at the same length, then intensity decrease is directly related to the phase deviation from a reference phase point. This condition may well describe a fiber undergoing active isometric contraction.

Chemical Phenomena↗
PubMed1979

Light diffraction study of single skeletal muscle fibres.

Light diffraction patterns from isolated frog semitendinosus muscle fibers were examined. When transilluminated by laser light, the muscle striations produce a diffraction pattern consisting of a series of lines that are projected as points onto an optical detector by a lens system. Diffraction data may be sequentially stored every 18 ms for later processing by digital computer systems. First- and second-order diffraction line intensities were examined from intact, chemically skinned, and glycerinated single fibers. The diffraction line intensities demonstrated a strong length dependence upon passive stretch from reference length to 3.6 micrometer. The first-order intensity linearly increased an average of 15-fold over the range examined. The magnitude of the second order intensity was less than the first order and showed an exponential rise with increasing length. Both first- and second-order intensities decreased upon muscle activation. Data from chemically skinned and glycerinated single fibers were not significantly different from intact fibers, indicating that the membrane structure has little effect upon the diffraction phenomenon in muscle. Theoretical model systems are examined in an attempt to find the basis of these results. Neither an analysis based on a diffraction grating with variable spacing nor the unit cell model of Fujime provides an explanation for the observed length dependency of intensity. Though the origin of the intensity decrease upon stimulation is not known, we have suggested that it could result from lateral misalignment of myofibrils and can occur upon activation.

Animals↗
PubMed1977

Light diffraction studies of sarcomere dynamics in single skeletal muscle fibers.

A position-sensitive optical diffractometer has been used to examine the diffraction spectra produced by single skeletal muscle fibers during twitch and tetanic contraction. First-order diffraction lines were computer-analyzed for mean sarcomere length, line intensity, and percent dispersion in sarcomere length. Line intensity was observed to decrease rapidly by about 60 percent during a twitch, with an exponential recovery to resting intensity persisting well beyond cessation of sarcomere shortening; recovery was particularly prolonged at zero myofilament overlap. A number of single fibers at initial lengths from 2.5 to 3.5 MICRON EXHIBITED a splitting of the first-order line into two or more components during relaxation, with components merging back into a single peak by 200 ms after stimulation. This splitting reflects the asynchronous nature of myofibrillar relaxation within a single fiber. During tetanus, the dispersion decreased by more than 10 percent from onset to plateau, implying a gradual stabilization of sarcomeres.

Animals↗
PubMed1976

Sarcomere length dispersion in single skeletal muscle fibers and fiber bundles.

Light diffraction patterns produced by single skeletal muscle fibers and small fiber bundles of Rana pipiens semitendinosus have been examined at rest and during tetanic contraction. The muscle diffraction patterns were recorded with a vidicon camera interfaced to a minicomputer. Digitized video output was analyzed on-line to determine mean sarcomere length, line intensity, and the distribution of sarcomere lengths. The occurrence of first-order line intensity and peak amplitude maxima at approximately 3.0 mum is interpreted in terms of simple scattering theory. Measurements made along the length of a singel fiber reveal small variations in calculated mean sarcomere length (SD about 1.2%) and its percent dispersion (2.1% +/- 0.8%). Dispersion in small multifiber preparations increases approximately linearly with fiber number (about 0.2% per fiber) to a maximum of 8-10% in large bundles. Dispersion measurements based upon diffraction line analysis are comparable to SDs calculated from length distribution histograms obtained by light micrography of the fiber. First-order line intensity decreases by about 40% during tetanus; larger multifibered bundles exhibit substantial increases in sarcomere dispersion during contraction, but single fibers show no appreciable dispersion change. These results suggest the occurrence of asynchronous static or dynamic axial disordering of thick filaments, with a persistence in long range order of sarcomere spacing during contraction in single fibers.

Animals↗
PubMed1975

Length-dependent optical diffraction pattern changes in frog sartorius muscle.

The length dependence of the laser light diffraction pattern produced by R. pipiens whole sartorius muscle has been examined at rest and during tetanic contraction. The muscle diffraction pattern was scanned by a vidicon camera; camera output was digitized and processed by an on-line digital computer to allow a real line display of a section through the diffraction pattern. Analysis of the first order diffraction line profiles yielded values for line amplitude, intensity, center of gravity, line width and percent dispersion. The calculated dispersion provided a measure of the muscle's sarcomere length distribution. First order line amplitudes and intensities were observed to increase, then decrease, with progressive stretch from 1.0 to 1.3 reference length. The percent dispersion among sarcomeres in resting muscle averaged 11% and was approximately proportional (coefficient = .0341) to length. The amplitude and intensity of zero and first order diffraction lines decreased during tetanus, while the line widths and dispersions increased. First order line intesity during tetanus was maximum at about the same length as during rest, i.e., at approximately 2.5 mu sarcomere length. Sarcomere dispersion increased by about 6% during tetanus.

Animals↗