A unitized enzyme-labeled immunometric digoxin assay suitable for rapid testing.

An enzyme-labeled immunometric assay has been developed for measuring digoxin concentrations in serum or plasma. Unitized, compartmentalized reagents are used with an automated sample-processing instrument. The enzyme activity of the processed sample, which is directly proportional to the digoxin concentration, is measured by using a reagent strip and the Ames Seralyzer reflectance photometer. The test takes less than 15 min, and digoxin concentrations are calculated from a two-point calibration line stored in the instrument. Within-run CVs for controls at four concentrations ranged from 2.3% to 3.8%; between-run CVs were from 1.5% to 2.6%. Results obtained with clinical serum samples correlated well (r greater than 0.96) with those obtained by fluorescent polarization immunoassay (Abbott TDx) and RIA (Clinical Assays and NML). This rapid and convenient method for monitoring digoxin concentrations in serum or plasma is particularly well suited for decentralized sites such as emergency rooms, urgent-care centers, and physicians' offices.

The effect of metacaine on the slow variation of peak sodium currents in potential clamped Ranvier nodes following changes in holding potential.

The effect of changes in the holding potential on peak sodium currents in isolated myelinated nerve fibres (peak INa) was investigated with the conventional sodium inactivation being kept at h infinity = 1. In Ringer solution no stationary values of peak INa could be obtained over the potential range tested. Near the normal resting potential, ER, peak INa changed with time clearly even after 10 min. Therefore, the individual values of peak INa as normalized by peak INa at ER and corrected for the unevitable run-down of peak INa could not serve as measure for stationary values of any membrane parameter. Under metacaine (1 mmol/l) peak INa changed comparably faster and proved to be less potential dependent as compared to peak INa of the untreated fibre. The effects observed are not necessarily governed by a specific process located inside the nodal membrane.

Effects of benzocaine and its isomers on sodium permeability and steady state sodium inactivation in the myelinated nerve, obtained by an improved dissection technique.

The blocking effects of benzocaine and its isomers (1 mmol/l) on sodium currents in myelinated nerve fibres were tested. As far as the so-called fast sodium inactivation is concerned, benzocaine shifted the h alpha-curve in negative direction to a stronger extent than did its isomers, while the potency of the isomers did not differ significantly from each other. The drug-induced reductions of maximum sodium permeability PNa were tested at constant test pulses at h alpha = 1. In this kind of experiments all the three isomers had the same potencies. The findings could not be correlated to the lipid solubilities of the drugs as measured by the corresponding octanol/water partition coefficients. In addition, efforts were undertaken to minimize any noxious pull during the isolation of the axon. Some consequences of the improvements introduced are discussed in terms of the reliability of ionic current measurements in Ranvier nodes.

[Effects of metacaine and its decomposition products on the excitation mechanism of isolated myelinated nerve fibers]. / Wirkungen von Metacain und seiner Zersetzungsprodukte auf den Erregungsmechanismus isolierter, myelinisierter Nervenfasern.

The blocking effects of metacaine-methanesulfonate (MS-222; abbreviated MMS) and of the corresponding hydrochloride (MHC) on the ionic currents in potential clamped myelinated nerve fibres were investigated. The dose-response relationships of MMS for blocking the Na-currents and K-currents could be satisfactorily fitted to the Langmuir adsorption isotherm with KNa = 0.7 mmol/l and KK = 1.8 mmol/l. The observed loss of efficacy in blocking ionic currents of 3-day-old solutions containing MMS was assumed to be due to a partial decomposition of the drug. We separated the water-soluble decomposition products by extraction of the metacaine base. The remaining aqueous solution showed an anti-blocking effect on metacaine blocked Na-currents. From corresponding experiments on the blocking effect of MHC we conclude that the anti-blocking effect is due to yet unknown decomposition products, formed in MMS but not in MHC solutions.

Changes in myelinated nerve fibres caused by insulating layers.

Single myelinated nerve fibres of the frog were investigated electron microscopically and electrophysiologically. The following results were obtained. Dissected internodes exhibited small deteriorations only compared to undissected fibres. In the vaseline seal the cross section area of the axoplasm was reduced by a factor of about 3. In the air gap the corresponding figure was about 5. With electrical measurements of up to 2 h the vaseline seal did not affect the axoplasmic resistance of the belonging internode. In corresponding experiments with an air gap, the axoplasmic resistance of the internode doubled after 49 +/- 14 min (mean +/- S.D.; n = 8). Thereafter, the resistance increased rapidly up to about 3,000 times normal, depending on the atmospheric humidity. The consequences of these findings for potential clamp experiments are discussed.

Interferon inducing activity of (A)n.(U)n complexes of varying chain length.

Evidence is presented that the interferon-inducing activity of (A)n.(U)n in primary rabbit kidney cells with respect to the chain length of the constituting (A)n and (U)n strands is governed by the following criteria: (1) the activity increases with the length of the uninterrupted double-stranded segment in the complex whereby both chains are equally important and the number of such segments for complex molecule is without effect, (2) at a constant total concentration of constituting nucleotides, the activity increases with the number of double-stranded molecular entities available to the cell, and (3) complexes with the (U)n strand considerably overlapping the (A)n strand are inactive due to the formation of triple stranded structures.

Effects of ionenes on interferon induction by poly(inosinic acid) . poly(cytidylic acid).

Depending on the spacing of their positive charges, ionenes, a class of quaternary ammonium polymers, increased the interferon-inducing activity of poly(inosinic acid).poly(cytidylic acid) in mouse L-929 cells, whereas they did not enhance poly(inosinic acid).poly(cytidylic acid) induced interferon production in primary rabbit kidney and human skin fibrolast cells.

Biological, biochemical and physicochemical evidence for the existence of the polyadenylate - polyuridylate - poly 2'-fluoro-2'-deoxyuridylate triple-stranded complex.

The interferon-inducing activity of the double-stranded complex poly(A) - poly(U) in primary rabbit kidney cell cultures is reduced when the cells are treated with poly(dUfl) either 1 h before, simultaneously with, or 1 h after the exposure to the double-stranded complex. It has been demonstrated in experiments involving sensitivity to hydrolysis by RNAase, UV absorbance-mixing curves, and UV absorbance-temperature profiles that this phenomenon is due to the formation of the triple-stranded complex poly(A) - poly(U) - poly(dUfl). The latter complex seems to be the principal product of interactions in the following systems: poly(A) - poly(U) + poly(dUfl); poly(A) - poly(dUfl) + poly(U); and poly(A) + poly(U) + poly (dUfl).

Effect of polyamines on the uptake of poly (2'-fluoro-2'-deoxyuridylic acid) by a mammalian cell line.

VERO cells can take up poly(dUfl)1 from the medium. The uptake involves surface adsorption and, most probably, intracellular penetration. Part of the poly(dUfl) is hydrolyzed during incubation with the cells but the hydrolysis products are not incorporated into de novo synthesized nucleic acids. The uptake is reduced by serum and stimulated by polycationic ionenes. The magnitude of stimulation depends on the structure of the ionene and the treatment regimen.

Polarography of polynucleotides. 3. Polyadenylic acid: the electrode process and interaction with polyamines.

Polyadenylic acid (poly A) was studied under various conditions using both DC polarography and phase sensitive AC polarography and by measuring the time-course of the current during the lifetime of a single drop of the dropping mercury electrode. Under certain conditions the current at potentials of the limiting portion of the DC polarographic wave does not reach its limiting value and in extreme situations peak-shaped curves are observed. This phenomenon is explained in terms of desorption and repulsion from the electrode of neutral poly A due to its polyanionic character. Consequently, the suppression of the current can be enhanced by increasing negative potential of the electrode and by exposing the negative charges of phosphate groups, e.g., by increasing pH and temperature and by decreasing ionic strength and buffer capacity; vice versa, the current suppression can be at least partially eliminated by reversing these conditions. Polyamines which seem to shield the phosphate groups through specific interactions are very effective in eliminating the current suppression. The effectiveness of a polyamine is determined by its chain length and by the density of its amino groups and the geometry of their distribution.