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R Pasqualini

Publications and source records attributed to R Pasqualini.

80 records · Page 5Linked to original sources
PubMed1989

Determination of the putative binding site for fibronectin on platelet glycoprotein IIb-IIIa complex through a hydropathic complementarity approach.

We have applied the principle of complementary hydropathy to the prediction of the binding site for fibronectin (FN) and for the alpha-chain of fibrinogen in the platelet receptor complex glycoprotein (GP) IIb-IIIa. Since both ligands bind to it through their respective RGDS (Arg-Gly-Asp-Ser) domains and since both have been cloned, we were able to deduce the amino acid sequence of the binding site from the nucleotide sequence coding for RGDS in both proteins. The deduced peptides were very similar. Antibodies raised against a synthetic peptide WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) deduced from the cloned rat FN RGDS domain block ADP-mediated platelet aggregation; this block can be overcome by additional fibrinogen. In Western blots of whole cell platelet extracts run under reducing conditions, this antibody binds to a 108-kDa band. It also binds to affinity-purified GP IIIa. Furthermore, it reacts strongly with GP IIIa immunoprecipitated by a commercially available anti-GP IIb-IIIa monoclonal antibody. Binding of affinity-purified GP IIb-IIIa complex to fibronectin is inhibited by the 110-kDa FN fragment. Similar inhibitions can be effected by WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) and GAVSTA (Gly-Ala-Val-Ser-Thr-Ala) predicted from the rat and human fibronectin nucleotide sequences, respectively. GAGSTA (Gly-Ala-Gly-Ser-Thr-Ala) and GARSTA (Gly-Ala-Arg-Ser-Thr-Ala) related to the human peptide but with discrepant hydropathies are noninhibitory.

Adult↗
PubMed1988

Characterization of the cellular receptor for fibronectin through a hydropathic complementarity approach.

It has been shown that a significant correlation is seen when the hydropathy scores of amino acids encoded by the coding strand of double-helical DNA are plotted against those of the noncoding strand. Thus, peptides encoded by complementary DNA strands might form amphiphilic structures and bind one another. We have used this approach to study the interaction between fibronectin (FN) and its cell receptor. Taking into consideration the nucleotide sequence from published rat cDNA clones that corresponds to the cell binding site (Arg-Gly-Asp-Ser) in the FN molecule, the deduced amino acid sequence found for the putative receptor binding site was Trp-Thr-Val-Pro-Thr-Ala. This peptide was chemically synthesized and coupled to an AH-Sepharose column. FN bound appreciably to this column and was eluted much more efficiently by a solution of Arg-Gly-Asp-Ser-containing peptide than by a solution of related but inactive Arg-Gly-Glu-Ser-containing peptide. Binding of labeled FN to receptor-rich MG63 human osteosarcoma cells was inhibited by the hexapeptide. The hexapeptide Gly-Ala-Val-Ser-Thr-Ala predicted similarly from the nucleotide sequence of human FN was equally efficient in such inhibition. Antibodies produced against Trp-Thr-Val-Pro-Thr-Ala recognized with equal efficiency Gly-Ala-Val-Ser-Thr-Ala in an ELISA assay. Furthermore, they were able to recognize a single 140-kDa band in whole-cell extracts from Chinese hamster ovary cells, attesting to their specificity. Identification of the recognized protein was provided by showing that this antibody was also able to bind to affinity-purified FN receptor from human osteosarcoma MG63 cells.

Amino Acid Sequence↗
PubMedDate not supplied

Optimal timing for initial and redistribution technetium 99m-N-NOET image acquisition.

BACKGROUND: Bis (N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium-99m (V) (Tc-99m-N-NOET) is a new Tc-99m-labeled myocardial perfusion imaging agent that redistributes. We sought to determine the optimal timing for acquiring initial and delayed images to maximize sensitivity for the detection of coronary stenoses. METHODS: Twelve anesthetized dogs with critical stenoses of the left anterior descending coronary artery were infused with adenosine (250 microg/kg/min) or MRE-0470, an adenosine A2a agonist (0.6 microg/kg/min x 10 minutes), and Tc-99m-N-NOET (8 mCi; 296 MBq) was injected intravenously at peak flow. Myocardial and lung Tc-99m-N-NOET activities were determined by serial quantitative imaging and arterial blood sampling was performed over 2 hours. RESULTS: Left anterior descending/left circumflex artery defect count ratios showed rapid redistribution during the first 10 minutes after Tc-99m-N-NOET injection (0.66 +/- 0.02 at 2 minutes to 0.73 +/- 0.01 at 10 minutes; P < .01). Redistribution was nearly complete by 120 minutes (defect ratio = 0.87 +/- 0.03; P < or = .01 vs 2 minutes). Lung activity fell significantly during the first 10 minutes from a heart/lung activity ratio of 1.07 +/- 0.05 (2 minutes) to 1.44 +/- 0.09 (10 minutes; P < or = .01). CONCLUSION: Initial stress Tc-99m-N-NOET images should be acquired within 10 minutes after injection, whereas delayed images can be obtained as early as 2 hours later. Lung activity clears rapidly, permitting acquisition of good-quality poststress cardiac images. These Tc-99m-N-NOET uptake and redistribution kinetics after vasodilator stress provide important information for designing clinical imaging protocols for optimal identification of inducible ischemia.

Animals↗
PubMedDate not supplied

Clearance of technetium 99m N-NOET in normal, ischemic-reperfused, and membrane-disrupted myocardium.

BACKGROUND: Technetium 99m-labeled bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium(v) (99mTcN-NOET) is a new neutral cardiac perfusion imaging agent that has been shown to have very high uptake and retention in vitro. The purpose of this study was to determine the clearance kinetics of 99mTcN-NOET in control, ischemic-reperfused, and membrane-disrupted myocardium. METHODS AND RESULTS: After a 100 microCi (3.7 x 10(6) Bq) bolus of 99mTcN-NOET was injected, myocardial clearance was monitored for 1 hour by the use of a sodium iodide detector in 30 isolated, Krebs-Henseleit (KH) perfused rat hearts. Seven hearts were used as controls (group 1). In seven ischemic-reperfused hearts, tracer administration and uptake was followed by 30 minutes of no flow and 1 hour of reflow (group 2). In six additional ischemic-reperfused hearts, tracer administration was followed by deprivation of flow for 1 hour followed by 1 hour of reflow (group 3). Six hearts were perfused with a 0.5% Triton X-100 KH perfusate for 1 hour (group 4). Four hearts were perfused with KH for 10 minutes, followed by cyanide for 10 minutes (group 5). This cycle was repeated three times. Activities remaining in each heart at the end of each experiment were quantitated, and activity at peak uptake was calculated. The 99mTcN-NOET myocardial clearance was near linear in the control (0.6 +/- 0.4) and both ischemic-reperfused groups with virtually no fractional clearance (1.2% +/- 0.6% and 2.1% +/- 0.6%, respectively; p = NS). In the Triton X-100 membrane-disrupted hearts, clearance was substantial (94.2% +/- 4.0%; p < 0.0001 compared with the control and ischemic-reperfused groups). Cyanide treatment produced rapid clearance, which was arrested by a return to the standard KH perfusate. Peak uptake as a percentage of injected dose was 74.9% +/- 1.4% for all groups combined. CONCLUSION: Thus 99mTcN-NOET has extremely high myocardial retention after 1 hour in normal myocardium and is not significantly affected by ongoing myocardial ischemia or reperfusion injury in this model. Clearance is increased markedly in extreme conditions of membrane disruption. These data are consistent with the concept that 99mTc-NOET is localized predominantly in or on cell membranes. 99mTcN-NOET is a promising, new myocardial perfusion imaging agent that exhibits a stable myocardial distribution in the setting of acute developing injury.

Animals↗
PubMedDate not supplied

Planar imaging of 99mTc-labeled (bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium[V]) can detect resting ischemia.

BACKGROUND: (99m)Tc-labeled (bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium(V)) ([99m]TcN-NOET) is a new lipophilic, neutral-charge cardiac perfusion imaging agent that demonstrates apparent redistribution in animal models and humans. The purpose of this study was to determine whether the kinetics of (99m)TcN-NOET are suitable for the detection of resting ischemia. METHODS AND RESULTS: Microspheres were injected at baseline and simultaneously with (99m)TcN-NOET after a 90% reduction in resting flow in the left circumflex coronary artery in six open-chest canine experiments. The relationship of flow and activity early after injection was determined in one experiment by termination at 10 minutes. The flow ratio (left circumflex/left anterior descending coronary artery) after stenosis fell significantly (0.87 +/- 0.04 vs 0.46 +/- 0.04; p < 0.05). The end-tissue (99m)Tc ratio (0.78 +/- 0.05) was significantly higher than the flow ratio at injection (0.46 +/- 0.04; p < 0.05), indicating substantial redistribution. In vivo imaging was conducted during 2 hours in five experiments, followed by ex vivo imaging. Myocardial clearance from 10 minutes onward was biphasic in left anterior descending and monophasic in left circumflex coronary arteries. Myocardial clearance from 10 to 60 minutes was delayed in left circumflex (35.5% +/- 8.1%) versus left anterior descending coronary arteries (49.2% +/- 8.6%; p < 0.05). No significant difference was observed from 60- to 120-minute clearance. Five of five experiments demonstrated initial defects and complete fill-in at 90 to 120 minutes by qualitative assessment. Quantitation of ex vivo images confirmed significant redistribution. CONCLUSIONS: Resting ischemia caused by moderate to severe stenosis can be detected on scans with (99m)TcN-NOET. Redistribution was near complete in this model by 90 to 120 minutes. (99m)TcN-NOET is a promising new agent for the detection of coronary artery disease in viable myocardium and warrants further investigation.

Animals↗
PubMedDate not supplied

Influence of calcium channel inhibitors on the myocardial uptake and retention of technetium 99m N-NOET, a new myocardial perfusion imaging agent: a study on isolated perfused rat hearts.

BACKGROUND: Technetium 99m N-NOET is a new myocardial perfusion imaging agent currently in phase III clinical trials in Europe. In vitro studies on newborn rat cardiomyocytes have shown that calcium inhibitors, such as verapamil or diltiazem, inhibit its cellular uptake by 40%. To determine whether such a specificity exists ex vivo, we studied the effect of verapamil, diltiazem, and nifedipine on the myocardial uptake and retention of Tc-99m N-NOET in isolated perfused rat hearts. METHODS AND RESULTS: After a 15-minute baseline period, rat hearts were perfused with 0.5 micromol/L verapamil (n = 6), 0.75 micromol/L diltiazem (n = 6), or 0.1 micromol/L nifedipine (n = 6) for 10 minutes before the injection of a bolus (40 microCi/250 microL) of the tracer. Control hearts were perfused with either 1.5 mmol/L calcium (same concentration as in the treated groups; n = 7) or 0.75 mmol/L calcium (same contractility as in the treated groups; n = 6). Myocardial activity of Tc-99m N-NOET was monitored for 30 minutes. The functional parameters of the hearts were recorded throughout the experiments. Calcium inhibitors induced a 40% to 55% decrease in maximal first derivative of left ventricular pressure (dP/dt) (0.0001<P< .001 versus control, calcium 1.5 mmol/L). After 30 minutes, tracer retention was 51.6%+/-6.1% injected dose (ID)/g and 54.2%+/-10.2% ID/g for control hearts perfused with 1.5 mmol/L and 0.75 mmol/L calcium, respectively (P = not significant). Myocardial activity of Tc-99m N-NOET was not modified in hearts perfused with verapamil (50.0%+/-4.1% ID/g), diltiazem (47.2%+/-8.7% ID/g), or nifedipine (51.7%+/-3.4% ID/g; P = not significant versus control hearts perfused with 1.5 mmol/L or 0.75 mmol/L calcium). CONCLUSIONS: Verapamil, diltiazem, and nifedipine were not responsible for any variations in Tc-99m N-NOET uptake and retention in the isolated rat-heart model. This might be explained by the binding of the tracer to endothelial cells, which do not present voltage-dependent calcium channels.

Animals↗