In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site. (1/82)

For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412-417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure-function studies and analysis.  (+info)

Severe thrombocytopenia caused by digitoxin intoxication in a patient with heart failure associated with Sjogren's syndrome. (2/82)

Congestive heart failure (CHF) related to Sjogren's syndrome is extremely rare. This report concerns a patient who presented with CHF and severe thrombocytopenia (5,000/microl). Serum concentrations of K, Mg and digitoxin were 3.2mmol/L, 1.4mg/L and 57.2ng/ml, respectively. Digitoxin intoxication was evident, seemingly evoked by hypokalemia, hypomagnesemia, hepatorenal dysfunction and hypothyroidism. The severe thrombocytopenia was considered to have been caused by this intoxication, as it disappeared soon after the digitoxin was discontinued and potassium was supplemented.  (+info)

Haemodynamic effects of the antiarrhythmic quaternary ammonium compound QX-572 in man. (3/82)

The haemodynamic effects of N, N-bis(phenyl-carbamoylmethyl) dimethylammonium chloride (QX-572) in man were studied. A controlled study was performed to rule out a possible influence of the catheterization procedure as such on the results. Ten patients with mild to moderate aortic regurgitation were studied: based on clinical data the patients were divided into 2 groups of 5. Randomly it was decided that one group should constitute a control group receiving saline while the second group received QX-572 , MG/KG BODY WEIGHT. In both groups the administration was performed as a slow intravenous infusion during 30 minutes. Heart rate, pressures in brachial artery and right atrium, cardiac output, stroke volume, and systemic vascular resistance were determined before, during, and up to 30 minutes after completion of placebo or QX-572. These variable remained stable in the control group while QX-572 produced an increase in heart rate most pronounced at the end of the infusion period. A transient decrease in systolic and mean brachial artery pressure during the infusion, and during the same period a decrease in right atrial pressure. Cardiac output and systemic vascular resistance were unchanged by QX-572 but they were not measured during the infusion when the changes in pressures were most pronounced. QX-572 was thought to act as a peripheral vasodilator during the infusion. Left ventricular contractility was studied by means of pressure curves obtained from a catheter tip manometer placed in the left ventricle. The first derivative of the isovolumic left ventricular pressure at the highest level (45mmHg) common to all patients was used (dp/dt-45). No significant difference could be observed when comparing mean changes of dp/dt-45 for the two groups. In the control group there was a slight but significant increase in dp/dt-45 during the time of observation. In the QX-572 group the results varied between individuals. Two of the patients differed from all other patients in the control and QX-572 groups showing a decrease in dp/dt-45 which, when most pronounced at the end of the infusion period, was -31 and -28 per cent of the preinfusion levels, respectively. This decrease probably reflects reduction of contractility. It was concluded that QX-572 in a dose of 8 mg/kg body weight did not have any major haemodynamic drawbacks.  (+info)

Chronic hypertension induced by ouabain but not digoxin in the rat: antihypertensive effect of digoxin and digitoxin. (4/82)

Elevated circulating levels of an endogenous ouabain (EO) have been associated with essential hypertension. To investigate structure-activity relationships relevant to blood pressure, we infused either ouabain, ouabagenin, digoxin or digitoxin at 30 microg/kg/day in normal Sprague Dawley rats. After five weeks, the ouabain and ouabagenin infused rats were hypertensive, whereas blood pressures declined below their vehicle controls in rats infused with digoxin or digitoxin. In a second study, mean blood pressures were 118.5+/-1.7 mmHg in rats infused with ouabain (15 microg/kg/day) on day 35 vs. 98.3+/-1.8 and 100.3+/-1.1 mmHg in the digoxin (30 microg/kg/day) and vehicle infused groups (both p<0.005 vs. ouabain), respectively. Plasma and kidney levels of ouabain immunoreactivity were increased 4-8 fold in ouabain infused rats while blood pressure and plasma levels of ouabain returned to normal one week following discontinuation of the steroid infusion. In rats with ouabain-dependent hypertension, secondary infusions of digoxin or digitoxin (30 microg/kg/day) normalized blood pressure even though circulating ouabain remained elevated. In digoxin infused rats, neither blood pressure nor kidney digoxin immunoreactivity was raised whereas plasma digoxin was increased. Collectively, the results show that the hemodynamic effects of these sodium pump inhibitors differ dramatically during prolonged administration and that tissue rather than circulating levels of these agents appear to better explain their effects on blood pressure. These studies suggest that sodium pump inhibition is not the exclusive mediator of the hemodynamic effects of these cardiac glycosides and demonstrate the presence of structure-specific mechanisms that regulate their tissue levels and effects on long-term blood pressure.  (+info)

Studies on the kinetics of (3H)-ouabain uptake and exchange in the isolated papillary muscle of the guinea-pig. (5/82)

1 The uptake, wash-out and exchange of [3H]-ouabain was studied in isolated, resting, and electrically stimulated papillary muscles of the guinea-pig. 2 At the equilibrium level of uptake, a different tissue/medium ratio was obtained for each of the concentrations used, i.e. 3.4, 1.8 and 0.82 for 1 times 10-minus 7M, 7.7 times 10-minus 7M, and 5 times 10-minus 6M ouabain, respectively. Equilibrium was reached more rapidly at high concentrations of ouabain. 3 The maximum number of binding sites for ouabain was estimated to be 1 times 10-15 binding sites/g wet weight. 4 No difference in [3H]-ouabain uptake could be detected between resting and electrically stimulated papillary muscles. 5 The kinetics of the ouabain uptake, wash-out and exchange are discussed. The results suggest that there is a saturable compartment in papillary muscle which can best be demonstrated if low concentrations of ouabain are used. Because of its small size, the saturable compartment submerges in the process of the filling of the extracellular space at high concentrations (5 times 10-minus 6M).  (+info)

Digoxin-like and digitoxin-like immunoreactive substances in elderly people. Impact on therapeutic drug monitoring of digoxin and digitoxin concentrations. (6/82)

We compared digoxin-like (DLIS) and digitoxin-like (DTLIS) immunoreactive substance concentrations for 30 people older than 65 years with those for 25 people younger than 50. None received digoxin or had liver disease, uremia, or volume expansion. We found no DTLIS in any specimen, and only 1 specimen from an elderly person demonstrated a low DLIS concentration. In addition, for 22 non-volume expanded patients (8 younger than 50 years and 14 older than 65) receiving digoxin, the fluorescence polarization (FPIA) and the microparticle enzyme (MEIA) immunoassays revealed comparable serum digoxin concentrations, indicating an insufficient DLIS concentration to interfere with digoxin immunoassay results. Therefore, elderly people who are not volume expanded do not have elevated DLIS or DTLIS concentrations. Furthermore, for patients with liver disease or uremia (18 older than 65 years and 20 younger than 50), the DLIS and DTLIS concentrations were elevated. Finally, for 5 patients with liver disease who received digoxin, serum digoxin concentrations were lower by MEIA and higher by FPIA, indicating the patients had elevated DLIS levels that interfered with the assays. Elevated DLIS and DTLIS concentrations are associated with volume expansion and not age.  (+info)

Direct inhibitory effect of digitalis on progesterone release from rat granulosa cells. (7/82)

Digoxin (10(-7) - 10(-5) M) or digitoxin (10(-7) - 10(-5) M) decreased the basal and human chorionic gonadotropin (hCG)-stimulated release of progesterone from rat granulosa cells. Digoxin (10(-5) M) or digitoxin (10(-5) M) attenuated the stimulatory effects of forskolin and 8-bromo-cyclic 3' : 5'-adenosine monophosphate (8-Br-cAMP) on progesterone release from rat granulosa cells. Digoxin (10(-5) M) or digitoxin (10(-5) M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450(scc)) activity (conversion of 25-hydroxyl cholesterol to pregnenolone) in rat granulosa cells but did not influence the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Neither progesterone production nor P450scc activity in rat granulosa cells was altered by the administration of ouabain. Digoxin (10(-5) M) or digitoxin (10(-5) M), but not ouabain, decreased the expression of P450scc and steroidogenic acute regulatory (StAR) protein in rat granulosa cells. The present results suggest that digoxin and digitoxin decrease the progesterone release by granulosa cells via a Na(+),K(+)-ATPase-independent mechanism involving the inhibition of post-cyclic AMP pathway, cytochrome P450scc and StAR protein functions.  (+info)

Standardization of incubation time and temperature for eight radioimmunoassays. (8/82)

We wanted to know if incubation time and temperature for many radioimmunoassay methods could be standardized, to decrease assay time and so improve efficiency. Percentage binding was determined for triiodothyronine, thyroxine, digoxin, digitoxin, testosterone, aldosterone, estradiol, and diphenylhydantoin when the reaction mixtures were incubated at 6-8, 22, or 37 degrees C for various periods of time. In all methods tested, binding of antigen to antibody was greatest when the reaction mixtures were incubated at 6-8 degrees C, less at 22 degrees C, and least at 36 degrees C. Maximum binding occurred within 30-60 min at each temperature for all methods tested. Hence, it is possible to standardize the incubation time and temperature for these eight radioimmunoassay methods to 60 min at 6-8 degrees C.  (+info)