Non-invasive evaluation of pulmonary arterial and right ventricular pressures with contrast enhanced Doppler signals of tricuspid regurgitation flow using sonicated albumin solution.
(65/631)
To determine the feasibility of the non-invasive determination of systolic pressure of the pulmonary artery and the right ventricle in pediatric patients, the velocity of tricuspid regurgitation was measured in 30 patients using a contrast enhanced Doppler echocardiography. After sonicated albumin injection, trivial tricuspid regurgitation signals were enhanced in 27 patients (90%). Peak systolic velocity was not altered by before and after sonicated albumin injection in 2 patients. Right ventricular (RV) systolic pressure obtained by continuous wave Doppler during sonicated albumin enhancement corresponded very closely to that measured by catheter in 27 patients (r = 0.96). In 27 patients, difference of estimation of RV systolic pressure by non-enhanced Doppler and enhanced Doppler with sonicated albumin was statistically significant (32.3 +/- 27.6 mmHg versus 2.9 +/- 7.7 mmHg p < 0.001). Systolic pressure of pulmonary artery was estimated by RV systolic pressure measurement (by enhanced Doppler method) minus peak pressure gradient across the pulmonary valve (non-enhanced Doppler method). Pulmonary arterial systolic pressure measured by enhanced Doppler method and that by catheter method were highly significant (sonicated albumin method, r = 0.95). This technique may be a valuable non-invasive method for determining an accurate right ventricular and pulmonary arterial systolic pressures in this setting. (+info)
The topographical location and unique nature of a glucokinase associated with the Golgi apparatus of rat liver.
(66/631)
A particulate glucokinase was recovered in the Golgi-rich fraction of rat liver prepared by the method of Morre [Methods Enzymol. (1971) 22, 130-148], thus extending the demonstration by Berthillier et al. [Biochim. Biophys. Acta (1973), 293, 370-378] of particulate glucokinase activity in a microsomal subfraction that showed enrichment in Golgi characteristics. The purity of this fraction was examined and it was then subjected to several treatments, the action of Triton X-100, freezing and thawing, and sonication to establish the topographical location of the glucokinase activity thus solubilized. The evidence suggests that the glucokinase activity is either soluble in the lumen of the Golgi apparatus or loosely associated with the inside of the Golgi membranes. (+info)
Rapid shotgun cloning utilizing the two base recognition endonuclease CviJI.
(67/631)
A new approach has been developed for the rapid fragmentation and fractionation of DNA into a size suitable for shotgun cloning and sequencing. The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions which alter the specificity of this enzyme (CviJI**) yield a quasi-random distribution of DNA fragments from the small molecule pUC19 (2686 base pairs). To quantitatively evaluate the randomness of this fragmentation strategy, a CviJI** digest of pUC19 was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts PyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation. Advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 micrograms instead of 2-5 micrograms), fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed), and higher cloning efficiencies are obtained (CviJI** digested and column fractionated DNA transforms 3-16 times more efficiently than sonicated, end-repaired, and agarose fractionated DNA). (+info)
Adjuvant and mitogenic properties of a supernatant fraction of sonically treated Myobacterium bovis (BCG).
(68/631)
A water-soluble, oil-free supernatant fraction of sonically treated BCG (BCG-SS) was shown to be an immunological adjuvant and a mitogen. When BCG-SS and sheep erythrocytes (SRBC) were injected intravenously into mice, the plaque-forming cell (PFC) response was 10 times greater than that induced by injection of SRBC alone. Circulating antibody responses to SRBC and to bovine serum albumin were also enhanced by BCG-SS. The in vitro enhancement of PFC and circulating antibody responses did not require mineral oil or exogenous lipids. In vitro PFC responses by normal mouse spleen cells were also greatly increased in the presence of BCG-SS. Anti-theta serum-treated spleen cells from mice that had been lethally irradiated and reconstituted with normal bone marrow cells also gave a higher PFC response to SRBC in the presence of BCG-SS. This suggests that BCG-SS can stimulate B lymphocytes to develop an immune response when T lymphocytes are severely depleted or absent. BCG-SS also stimulated the uptake of 125IUdR by normal spleen cell cultures, indicating that it is a mitogen. (+info)
Protein kinase activity at the inner membrane of mammalian mitochondria.
(69/631)
This paper reports on the discovery of a protein kinase activity associated with the inner membrane of mammalian mitochondria. The enzyme does not respond to addition of cyclic AMP or cyclic GMP and has a preference for whole histone as phosphate acceptor. Some standard assay systems for the cyclic nucleotide-dependent cytosol protein kinases would be unable to pick up this activity of the orthophosphate concentration is higher than 25 mM and the pH or the assay lower than pH 6.5. The enzyme described here has an apparent pH optimum of 8.5. Activity in liver mitochondria is not evident unless the mitochondria are disrupted by either sonication or freezing and thawing. Distribution of kinase activity in centrifugal fractions of both liver and heart mitochondrial sonicates was parallel to that of the two inner membrane marker enzymes succinic dehydrogenase and cytochrome oxidase and quite different from that of the matrix enzyme malic dehydrogenase. Experiments with preparations enriched in outer or inner membranes confirmed the contention that this enzyme is located on the inner membrane. Since disruption of the inner membrane by a freeze-thaw treatment (after the outer membrane had been disrupted by swelling in phosphate) was necessary for full expression of activity by this enzyme, the tentative conclusion was reached that substrate is accepted only from the matrix side of the inner membrane. (+info)
Myocardial washout of sonicated iopamidol reflects coronary blood flow in the absence of autoregulation.
(70/631)
OBJECTIVES: The aim of the study was to evaluate the relation between measurements derived from myocardial contrast echocardiography and coronary blood flow. BACKGROUND: Contrast echocardiography has the potential for measuring blood flow. METHODS: In six open chest anesthetized dogs, the left circumflex coronary artery was cannulated and perfused with blood drawn from the left femoral artery. While adenosine was infused into the circuit, circumflex flow was generated by a calibrated roller pump to the point of abolishing coronary autoregulation. At each of 25 levels of coronary blood flow, paired bolus injections of sonicated iopamidol were performed proximal to a mixing chamber. The perfused area of the left circumflex coronary artery was labeled by radioactive microspheres injected into the perfusion line. Two-dimensional echocardiographic images of the left ventricular short axis were digitized off-line, and myocardial videodensity was measured in the area perfused by the left circumflex coronary artery to generate time-intensity curves. RESULTS: The washout slope of curves showed a good correlation with coronary blood flow, ranging from 0.5 to 12.5 ml/min per g of tissue. This correlation was good both in individual dogs (correlation coefficient [r] ranging from 0.78 to 0.96) and in the group of animals as a whole (r = 0.85). Washout slope also showed a good correlation with coronary diastolic pressure (r = 0.80), which ranged from 23 to 114 mm Hg, suggesting a possible primary effect of pressure on contrast washout. However, coronary blood flow appeared to be a stronger predictor of washout slope (partial F = 26.5, p < 0.001) than did perfusion pressure (partial F = 5.9, p < 0.05 by multiple regression). The injection to injection variability in myocardial washout slope appeared to be high (24%). The gamma variate fitting of curves did not improve the correlation with coronary flow (r = 0.78). CONCLUSIONS: Myocardial washout of sonicated iopamidol reflects coronary blood flow in a model in which coronary autoregulation is abolished. (+info)
Processing deep-sea particle-rich water samples for fluorescence in situ hybridization: consideration of storage effects, preservation, and sonication.
(71/631)
Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol for processing deep-sea particle-rich water samples is lacking. Some sample processing considerations are discussed in the present study, and different combinations of existing procedures for preservation, size fractionation sequential filtration, and sonication were tested in conjunction with FISH. Results from this study show that water samples should be filtered and processed within no more than 10 to 12 h after collection, or else preservation is necessary. The commonly used prefiltration formaldehyde fixation was shown to be inadequate for the rRNA targeted by FISH. However, prefiltration formaldehyde fixation followed by immediate freezing and postfiltration paraformaldehyde fixation yielded highly consistent cell abundance estimates even after 96 days or potentially longer storage. Size fractionation sequential filtration and sonication together enhanced cell abundance estimates by severalfold. Size fractionation sequential filtration effectively separated particle-associated microbial communities from their free-living counterparts, while sonication detached cells from particles or aggregates for more-accurate cell counting using epifluorescence microscopy. Optimization in sonication time is recommended for different specific types of samples. These tested and optimized procedures can be incorporated into a FISH protocol for sampling in deep-sea particle-rich waters. (+info)
Protein-only transmission of three yeast prion strains.
(72/631)
Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein. (+info)