Pathophysiologic concentrations of lysophosphoglycerides quantified by electron microscopic autoradiography

Lysophosphoglycerides accumulate in ischemic myocardium and appear to contribute to malignant dysrhythmia. Exposure of normoxic isolated Purkinje fibers or ventricular muscle strips to exogenous lysophosphatidyl choline (LPC) results in rapid, yet reversible, electrophysiologic derangements analogous to changes associated with ischemia in vivo. However, subcellular, local concentrations required for induction of electrophysiologic effects have not yet been elucidated unambiguously. The present study was designed to delineate the subcellular distribution and the sarcolemmal concentration of LPC by quantitative electron microscopic autoradiography after exposure of canine ventricular muscle strips to 3H-methyl LPC (200 microM) for 10 minutes. Tissue was processed for electron microscopy with a new procedure developed specifically to spatially fix choline phosphatides and render them insoluble in lipid solvents. Grain distributions were analyzed in the same group of cells that had been monitored electrophysiologically while superfused with LPC. LPC significantly decreased the resting membrane potential, action potential amplitude, duration, Vmax of phase 0, and conduction time. Grains were concentrated in the membranous organelles of cardiac myocytes. Myocyte sarcolemma exhibited the highest grain density (129 grains/100 micron2 versus a background of 0.27 grains/100 micron2). Calculations based on grain densities, emulsion sensitivity, exposure time, and specific activity indicated that the sarcolemma incorporated 5.4 X 10(6) LPC molecules/micron3 of membrane volume corresponding to approximately 1% of total sarcolemmal phospholipid. Since incorporation of only this small amount of lysophosphoglyceride into the sarcolemma was sufficient to elicit electrophysiologic disturbances, the observed accumulation of lysophosphoglycerides induced by ischemia appears to be sufficient to contribute to malignant dysrhythmia in vivo.     

Stress preconditioning attenuates oxidative injury to the alveolar epithelium of the lung following haemorrhage in rats

Inhibition of cAMP-dependent stimulation of vectorial fluid transport across the alveolar epithelium following haemorrhagic shock is mediated by reactive nitrogen species released within the airspaces of the lung. We tested here the hypothesis that the prior activation of the cellular heat shock or stress response, via exposure to either heat or geldanamycin, would attenuate the release of airspace nitric oxide (NO) responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Rats were haemorrhaged to a mean arterial pressure of 30–35 mmHg for 60 min, and then resuscitated with a 4 % albumin solution. Alveolar fluid clearance was measured by change in concentration of a protein solution instilled into the airspaces 5 h after the onset of haemorrhage. Stress preconditioning restored the cAMP-mediated upregulation of alveolar liquid clearance after haemorrhage. The protective effect of stress preconditioning was mediated in part by a decrease in the expression of iNOS in the lung. Specifically, stress preconditioning decreased the production of nitrite by endotoxin-stimulated alveolar macrophages removed from haemorrhaged rats or by A549 and rat alveolar epithelial type II cell monolayers stimulated with cytomix (a mixture of TNF-α, IL-1β and IFN-γ) for 24 h. In summary, these results provide the first in vivo evidence that stress preconditioning restores a normal fluid transport capacity of the alveolar epithelium in the early phase following haemorrhagic shock by attenuating NO-mediated oxidative stress to the lung epithelium.     

Test-bolus injection for optimization of arterial phase imaging during contrast-enhanced hepatic MR imaging

Contrast enhancement during the dynamic MR imaging is important for the detection and characterization of focal liver lesions. The purpose of this study was to determine whether or not a timing examination with a injection of a 1.0-mL bolus of gadopentetate dimeglumine into the antecubital vein followed by rapid dynamic scanning and measurement of signal intensity of the aorta could help to obtain proper arterial-dominant phase images for the characterization of focal hepatic lesions during subsequent multiphase dynamic MR imaging. The imaging delay to acquisition of the first gadolinium-enhanced image for multiphase dynamic MR imaging was set to equal the time to peak aortic enhancement during the test examination. The first contrast-enhanced images of 80 patients with 160 focal liver lesions (hepatocellular carcinoma, n = 79; cavernous hemangioma, n = 51; metastatic tumor, n = 30) were then retrospectively reviewed. Peak aortic enhancement occurred between 10 and 28 seconds (mean, 16.5 seconds +/- 3.1) after starting the infusion of contrast material in 80 patients during the test-examination. Depending on the findings of intrahepatic vascular enhancement on the full-scale dynamic images, hepatic arterial phase (n = 11, 14%) or sinusoid phase (n = 65, 81%) imaging was obtained during the first gadolinium-enhanced acquisition in 76 (95%) of 80 patients. Three different lesions were well characterized and easily distinguished from each other (p < .0001) on the first-phase images depending on their enhancement pattern. In the majority of patients, timing examination with test-bolus injection was helpful in obtaining qualified images for the characterization of various focal lesions.     

Monoclonal antibodies generated against recombinant ATM support kinase activity

We report on the rapid generation of two monoclonal antibodies, ATM A16.35 and ATM D16.11, that bind to the kinase domain of mutated ataxia telangiectasia (ATM). These antibodies were generated against E. coli-expressed recombinant protein using the RIMMS strategy. We show that ATM A16.35 binds ATM by Western blot analysis, and ATM D16.11 forms immune complexes with native ATM in immunoprecipitations without neutralizing kinase activity.     

Changes in dielectric properties at 460 kHz of kidney and fat during heating: importance for radio-frequency thermal therapy

We have developed a system to measure the changes due to heating to high temperatures in the dielectric properties of tissues in the radio-frequency range. A two-electrode arrangement was connected to a low-frequency impedance analyser and used to measure the dielectric properties of ex vivo porcine kidney and fat at 460 kHz. This frequency was selected as it is the most commonly used for radio-frequency thermal therapy of renal tumours. Tissue samples were heated to target temperatures between 48 and 78 degrees C in a hot water bath and changes in dielectric properties were measured during 30 min of heating and 15 min of cooling. Results suggest a time-temperature dependence of dielectric properties, with two separate components: one a reversible, temperature-dependent effect and the other a permanent effect due to structural events (e.g. protein coagulation, fat melting) that occur in tissues during heating. We calculated temperature coefficients of 1.3 +/- 0.1% degrees C(-1) for kidney permittivity and 1.6% degrees C(-1) for kidney conductivity, 0.9 +/- 0.1% degrees C(-1) for fat permittivity and 1.7 +/- 0.1% degrees C(-1) for fat conductivity. An Arrhenius model was employed to determine the first-order kinetic rates for the irreversible changes in dielectric properties. The following Arrhenius parameters were determined: an activation energy of 57 +/- 5 kcal mol(-1) and a frequency factor of (6 +/- 1) x 10(34) s(-1) for conductivity of kidney, an activation energy of 48 +/- 2 kcal mol(-1) and a frequency factor of 6 x 10(28) s(-1) for permittivity of kidney. A similar analysis led to an activation energy of 31 +/- 4 kcal mol(-1) and a frequency factor of (4.43 +/- 1) x 10(16) s(-1) for conductivity of fat, and an activation energy of 40 +/- 4 kcal mol(-1) and a frequency factor of 4 x 10(22) s(-1) for permittivity of fat. Structural events occurring during heating at different target temperatures as determined by histological analyses were correlated with the changes in the measured dielectric properties.     

Additive effect of TRAIL and p53 gene transfer on apoptosis of human lung cancer cell lines

TRAIL is a cytokine that can induce tumor-specific apoptosis through its specific death receptors (DR4 and DR5) and p53 has been proven to increase the expression of death receptors. To examine their interaction in tumor suppression, p53 and TRAIL genes were inserted in recombinant adenovirus vectors and transferred simultaneously into non-small cell lung cancer cell lines (NCI-H157, NCI-H358, NCI-H460 and A549). Western blot assay demonstrated production of TRAIL protein in NCI-H157 and A549 cell lines. Increased expressions of DR4 and DR5 of NCI-H157 and DR4 of A549 after p53 overexpression were confirmed by flow cytometry. p53 or TRAIL gene transfer increased sub-G1 fraction in cell cycle analysis and inhibited the tumor growth dose-dependently and the degree was potentiated by co-transfer. But isobologram analysis indicated an additive effect. Together, these data indicate that p53 and TRAIL interact additively on tumor apoptosis despite theoretical synergism.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.