A disulfide conjugate between anti-tetanus antibodies and HIV (37-72)Tat neutralizes tetanus toxin inside chromaffin cells.

Conjugates between anti-tetanus F(ab')2 fragments and the (37-72) fragment of the HIV Tat protein were taken up by chromaffin cells, NG108-15 neurohybridoma cells and Rev-2-T-6 lymphoma cells. The uptake could not be inhibited by competition with (37-72)Tat, but was reduced in the presence of metabolic inhibitors or at low temperature. The disulfide as well as the thioether conjugate were translocated to the cytoplasmic space, but only the disulfide conjugate moderately restored the stimulated transmitter release inhibited by tetanus toxin. Therefore, disulfide conjugates are more promising than thioethers for the neutralization of intracellular antigens. These conjugates provide new tools to study neuroprotection against bacterial neurotoxins.

Translocation of acylated pardaxin into cells.

Acylated pardaxin is translocated through the cytoplasmic membrane and is accumulated in the nucleoli of NG108-15 and chromaffin cells. The uptake is time- and dose-dependent and temperature-sensitive. However, the binding of acylated 125I-pardaxin cannot be reduced by competition with pardaxin acylated with Rudinger's reagent. In this respect, acylated pardaxin resembles the Tat protein 37-71 fragment. Metabolic inhibitors do not significantly reduce the uptake of acylated 125I-pardaxin. Acylated pardaxin might be useful as a vector to translocate other molecules.

Isolation, characterization and synthesis of a novel paradaxin isoform.

We report the isolation of a novel pardaxin isoform from the toxic secretion of the Red Sea Moses sole (Pardachirus marmoratus). Mass spectrometrical analysis of the newly purified peptide revealed a different primary structure compared to the previously known pardaxin isoforms. Sequence analysis disclosed an aspartic acid residue instead of glycine at position 31 of the new isoform. According to the novel sequence, a synthetic Asp-31-peptide was compared with the native compound as well as with synthetic Gly-31-pardaxin. The isolated Asp-31-pardaxin isoform and its synthetic analog exhibited identical elution properties during reverse-phase HPLC, as well as similar dose-dependent lytic effects on human erythrocytes at a concentration of 10(-6) to 10(-5)M. The hemolytic activity of Asp-31-pardaxins was lower than that of Gly-31-pardaxin and no synergistic effect between these peptides was found. The additional negative charge introduced by Asp-31 is likely to affect the selectivity of pardaxin pores towards a variety of ions.

Acidification of the cytosol inhibits the uptake of tetanus toxin in NG108-15 and NBr-10A neurohybridoma cells.

The influence of cytosol acidification on the uptake of two-chain tetanus toxin (TeTX)1 by neurohybridoma cells NG 108-15 and NBr-10A was investigated with two established techniques, the NH4Cl pulse method and the pH-clamp method. With the former, the extracellular pH is maintained at its physiological value, but is set to different values with the latter. Acidification of the cytoplasm with an NH4Cl pulse retarded the uptake of TeTX by both NG 108-15 and NBr-10A cells. This result provides further evidence for a vesicular endocytotic uptake of TeTX. In contrast, acidification of both the external medium and the cytoplasm (pH-clamp method) resulted in a net increase of toxin uptake. This result is explained as follows: Acidification of the extracellular environment has been shown to facilitate the uptake of tetanus toxin, and under pH clamp conditions, this effect is stronger than the simultaneous retardation of the toxin uptake by acidification also of the cytosol.

High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples.

A quantitative, highly sensitive HPLC-based method for the direct measurement of azathioprine is described, introducing a newly synthesized 9-methyl derivative of this immunosuppressant as internal standard in combination with isocratic HPLC and UV-absorbance measurement at 285 nm. Analysis was performed on a RP18 select B column with acetonitrile-0.01 M potassium phosphate buffer (12:88, v/v) at pH 2.3 as mobile phase. Results of precision analysis from serum samples spiked with 3.125, 12.5, and 25 ng azathioprine, respectively, were (mean +/- S.D.): 3.148 +/- 0.259 ng (8.22%), 12.594 +/- 0.571 ng (4.53%), and 25.016 +/- 0.658 ng (2.63%) with C.V. values in parentheses for n = 5. The accuracy of the assay ranged from -7.6 to 0.7% (expressed as % bias) tested on five consecutive days. The limit of quantification was at 2.5 +/- 0.256 ng (C.V. 10.25%), thus allowing drug monitoring in long-term patients. The method can also be used to evaluate individual pharmacokinetic parameters of a single patient, as well as for drug monitoring of a cohort of patients who suffer from azathioprine-induced symptoms of toxicity. An example of the pharmacokinetic behaviour in an individual is given in this paper.

Improved method for autoradiographic localization of beta-adrenoceptors using photoaffinity labelling.

Contact autoradiography of tissue sections, using emulsion coated coverslips or X-ray films, is widely used to provide information about the regional distribution of receptors. This easy to perform, standard technique has the disadvantage of an image spread due to the gap between the radioactive source and the film. The present study describes a new technique which combines photoaffinity labeling of beta-adrenoceptors with "dipping" autoradiography and a modified trichrome stain. Incubation of 16 microns cryosections of rat lung tissue with the iodinated, photoaffinity labeling, non-selective, beta-adrenergic agonist [125I]-cyanopindololazide II ([125I]-CYPA II) (100 pmol/l) in the absence or presence of 1 mumol (+/-)-propranolol revealed strong, specific beta-adrenoceptor binding to alveolar parenchyma and bronchial epithelium of large and small bronchioles, lesser binding to smooth muscle bundles of large airways and only sparse binding to the smooth muscle of small bronchioles or peripheral branches of pulmonary artery. With standard autoradiographic techniques, a similar distribution of the label was obtained, although resolution and sensitivity were inferior. Staining of tissue sections through the photoemulsion by means of a modified Mallory's trichrome dye facilitated the discrimination between alveolar and bronchial epithelium, muscular and collagenous tissues. In conclusion, the photoaffinity labeling of beta-adrenoceptors with [125I]-CYPA II allows the use of "dipping" autoradiography. This technique, in combination with trichrome staining through the photoemulsion, results in an improved autoradiographic image together with a better association of the label with distinct histological structures and the higher sensitivity of the method.

Uptake and concentration of bioactive macromolecules by K562 cells via the transferrin cycle utilizing an acid-labile transferrin conjugate.

The transferrin cycle was used to attempt the import of bioactive macromolecules into cells with the aid of an acid-labile cross-linking agent. Anti-tetanus F(ab')2 fragments were iodinated and then conjugated to transferrin with a newly developed acid-labile cleavable cross-linking reagent, bismaleimidoethoxy propane, following thiolation of both proteins. Noncleavable conjugates were also prepared. At saturating conjugate concentrations, the uptake rate for both conjugates averaged over the first 2 h is about 6.5 fmol/million cells/min. Incubation of loaded cells in fresh medium for 30 min and analysis of cell pellets and supernatants reveal that 1) of the previously cell-associated label, only intact conjugate (about 50% of the label) is returned to the medium; 2) most of the remaining cell-associated material for the cleavable conjugate is chromatographically coincident with free Fab with some contribution from free F(ab')2 fragments. In contrast, the cell pellets loaded with noncleavable conjugates contained intact transferrin-F(ab'), conjugates. These results are consistent with transferrin receptor-mediated uptake of acid-labile conjugate followed by hydrolysis in acidified endosomes and resulting in concentration of free F(ab')2 and Fab within a prelysosomal intracellular compartment. A protein shuttle such as transferrin may therefore be used with ketal based acid-labile cross-linkers to load foreign molecules into an intracellular compartment. In addition, these data provide independent confirmation of the low pH compartment within the transferrin cycle. This new methodology is applicable to other cases of receptor/ligand trafficking to report low pH compartments independent of morphological analysis. Since transferrin receptors are overexpressed in tumors, antineoplastic agents could be targeted to tumors as transferrin acid-labile conjugates. This import system might be particularly useful in combatting the tumor cell export of antitumor agents occurring in multidrug resistance.

Uptake of antitetanus F(ab')2 fragments into eukaryotic cells.

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.

Tetanus antitoxin binds to intracellular tetanus toxin in permeabilized chromaffin cells without restoring Ca2(+)-induced exocytosis.

Tetanus toxin blocks Ca2(+)-evoked catecholamine release from permeabilized bovine adrenal chromaffin cells preloaded with gangliosides. Tetanus toxin preincubated with its specific antibodies F(ab')2 is without any effect on exocytosis. Specific antitetanus F(ab')2 presented to chromaffin cells which are pretreated with tetanus toxin and permeabilized by digitonin cannot restore exocytosis. Under the same conditions, however, 125I-labeled F(ab')2 accumulates in chromaffin cells. The accumulation depends on the presence and concentration of tetanus toxin and can be prevented by an excess of unlabeled F(ab')2. Once tetanus toxin has initiated block of exocytosis, it cannot be neutralized by binding to its specific antibody.

Effect of acyclovir on herpes simplex virus infected neuronal, glial, and neurohybridoma cells in vitro.

Herpesvirus type 1 could be propagated most efficiently in cultured fetal neurons, to a lesser extent in NG108-15 neurohybridoma cells, and with the lowest titer in glial cells. Herpesvirus type 2 could not be cultured in neurohybridoma cells, and in fetal neurons a titer 100-fold lower than for herpesvirus type 1 was obtained. Cells infected with herpesvirus type 1 were used in an infectivity assay for acyclovir dose-response studies. The ED50 values were 7.4 nmol/l for fetal neurons, 180 for neurohybridoma cells, and 275 nmol/l for glial cells.

Fast determination of demeton-S-methylsulfoxide (Metasystox R) in blood plasma.

An HPLC method for the detection of demeton-S-methyl-sulfoxide (Metasystox R) in human plasma was developed. DSMSO was extracted by passing the acidified plasma sample through an Extrelut 3 column and eluted from the column with dichloromethane. An aliquot of the extract was injected onto the HPLC nitrile reversed-phase column. The mobile phase was a solution of 3% acetonitrile in 0.01M phosphoric acid. The eluent was monitored at 215 nm. With this method, plasma concentrations between 0.5 and 50 mg/L were determined. Within this range, the concentration-response relation was linear (r = 0.9991). The within-run and between-run coefficients of variation were concentration dependent, with maximal values less than 10%. The analysis took about 45 minutes.

Tetanus toxin binds with high affinity to neuroblastoma x glioma hybrid cells NG 108-15 and impairs their stimulated acetylcholine release.

Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under "physiological" conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.

The in situ perfused spinal cord of the rat. Applicability of drugs and chemicals, sodium-lithium exchange, and calcium reduction to functional intact central nervous system tissue.

A new forequarters preparation, the in situ perfused cervical spinal cord of the adult rat, is described with respect to its suitability for pharmacological and toxicological investigations. The dose-dependent actions of strychnine, p-chloromercuriphenylsulfonate, ouabain, dinitrophenol, bicuculline, and (-)-nipecotic acid ethyl ester on mass reflex discharges were studied. The study was extended to sodium-lithium exchange and calcium reduction in the perfusion medium. With the intent to discriminate between primary central actions of substances and ion changes and their secondary metabolic effects, the flow rate, the sodium and potassium concentrations, the pH, the pO2, and the pCO2 of the perfusion medium were measured. The new perfused forequarters preparation, including the intact cervical spinal cord, has a number of advantages over an experimental design using an intact animal dependent on its physiological circulation. These advantages are: the composition of fluids supplying the preparation is under control; the oxygen supply to the cord is no longer dependent on the cardiovascular function, which may be impaired by the substance under study; a complete dose-response curve may be obtained from each animal; washout experiments may be performed; the action of substances can be studied in the presence of extreme concentrations of drugs, ions, etc.

Predicative calculation of the efficiency of hemodialysis or hemoperfusion for the removal of drugs from the body.

Formula are derived for the calculation of the amounts of drug which, during a defined time interval, are eliminated from the body with and without the help of hemodialysis or hemoperfusion (HD/HP). A programmable pocket calculator like the TI-59 suffices to perform all calculations. The pharmacokinetic system constants of the drug, the effective plasma flow, and the HD/HP plasma extraction rate are entered into the program. Additional information can be obtained if the plasma concentration of the drug at the onset of HD/HP is known. Worked examples are presented.

Toxicokinetics of labeled amatoxins in the dog.

Radioactivities were measured in serum, urine, and bile of dogs at different times after intravenous injection of 14C-methyl-gamma-amanitin (14C-A) and 3H-O-methyl-dehydroxymethyl-alpha-amanitin (3H-A). For either substance, the relation between the specific plasma activity C and the time t could be best described with the function C = C1 X e- lambda 1 X t + C2 X e- lambda 2 X t. Therefore the linear open two-compartment system was selected as an adequate toxicokinetic model. Most important, the distribution volumes (in the steady state) were in the range of the extracellular space, and the total body clearances were in the range of the dog creatinine clearance. In accordance with former findings for 3H-A, 14C-A was not bound to plasma proteins. More than 80% of 14C-A was eliminated in the urine; less than 10% was found in the bile. From these data, two suggestions may be derived for the therapy of Amanita intoxication in man. First, detection in the urine of amatoxins 2 or 3 days after mushroom ingestion points to an ongoing amatoxin absorption or reabsorption from the intestine, and should lead to therapy with adsorbents and, in the absence of diarrhea, with laxatives. Second, hemoperfusion will remove significant amounts of amatoxins during the time of ongoing absorption or reabsorption and a few hours thereafter.

Liposome-entrapped [125I]anti-tetanus immunoglobulin G: evidence for entry into spinal cord neurons of the rat.

Intrathecally administered free anti-tetanus immunoglobulin G (IgG) diffuses through the spinal cord but does not enter nerve cells. In order to facilitate entry into neurons, 125I-labeled anti-tetanus IgG was entrapped in liposomes. After injection into the cerebrospinal fluid of rats, however, only a very low specific radioactivity of the spinal cord could be calculated from gross counts and no neuronal labeling was seen in autoradiographs. Therefore, it was assumed that the liposomes were unable to cross the basement membrane of the spinal cord surface. To circumvent this barrier the liposome-entrapped [125I]IgGs were injected directly into the grey matter. Histoautoradiographs then showed marked accumulations of radioactivity in neurons. Direct intraspinal injection of free [125I]IgG, on the other hand, failed to produce heavy neuronal labeling.

Perfusion of the cervical spinal cord in situ of adult rats using a perfluorocarbon emulsion.

A method was developed for the perfusion of the cervical spinal cord of adult rats through the supplying arteries. A medium containing perfluorotributylamine as an oxygen carrier was used. The electrophysiological responsiveness was continuously monitored; pO2, pCO2, pH, Na+ and K+ were intermittently measured in the medium. The perfused cord maintained its responsiveness for more than 5 h. No changes in its structure became apparent in electron micrographs.

False-positive EMIT indication of opiates and methadone in a doxylamine intoxication.

The EMIT-dau for methadone and the EMIT-ST for opiates (both EMITs from Syva-Merck) gave false-positive results when applied to urine samples of a patient suffering from a moderate doxylamine monointoxication. The error was not detected in routine TLC. Gas chromatography and mass spectrometry unequivocally indicated the presence in urine and plasma of doxylamine and its metabolites and the definite absence of methadone. Opiates could not be detected with the Abuscreen RIA (Roche), which is a more sensitive method than EMIT-ST.

Pharmacokinetic parameters for thallium (I)-ions in man.

The pharmacokinetics of Tl+ were studied in 9 patients who underwent myocardial scintigraphy with 201Tl+. The time course of the 201Tl+ concentration fitted to an open two-compartment model. The following values were calculated: k12 = 0.164 +/- 0.025 min-1, k21 = 0.011 +/- 0.004 min-1, k10 = 0.005 +/- 0.004 min-1, t1/2(lambda 1) = 3.92 +/- 0.54 min, t 1/2(lambda 2) = 3108 +/- 896 min, CL = 0.08 +/- 0.03 l/min, V1 = 0.26 +/- 0.13 l/kg, Vz = 4.36 +/- 0.65 l/kg, Vss = 4.23 +/- 0.67 l/kg. Using previously published dialysance values the influence of hemodialysis on Tl+ elimination was calculated. Hemodialysis in Tl+ intoxications should be moderately effective.

Calculation of pharmacokinetic parameters from hemodialysis or hemoperfusion data--a mathematical treatise.

A method is outlined to estimate from hemodialysis or hemoperfusion data, respectively, the rate constants, the distribution volume in the beta-phase, and other pharmacokinetic parameters of toxic substances. All calculations are based on the assumption that the usual linear open two-compartment system is also the appropriate model in a particular intoxication. No prior knowledge of the time of application or of the dose of the toxic substance is needed. The pharmacokinetic parameters may be helpful to estimate the benefit of hemodialysis or hemoperfusion, respectively, in future intoxications.