Cell surface binding affinity of Simian virus 40 T-antigen

Simian virus 40-transformed cells are characterized by a virus-induced tumor transplantation antigen (SV40 TSTA) defined in vivo by the rejection of tumorigenic SV40-transformed cells in SV40-immunized mice and in vitro by SV40 tumor cell-specific cytotoxic T cells. Several experimental findings support the notion that SV40-infected and -transformed cells express SV40 large tumor antigen (TAg) or closely related antigens on the cell surface (surface T). In this report, evidence is presented for a cell surface binding affinity of SV40 TAg solubilized and extracted by disruption of SV40-transformed and SV40-infected cells in growth medium. Incubation of various transformed and nontransformed living monolayer cells in situ with these extracts led to a significant uptake of TAg to the cell surface (called “externally bound TAg”) up to two to five times higher amounts in comparison to native surface T on SV40-transformed cells. This was demonstrated by the highly sensitive ¹²⁵I-protein A assay using rabbit antisera directed against purified SV40 TAg. Serological analysis of TAg in cellular extracts and of externally bound TAg revealed no apparent differences suggesting the cell surface binding affinity as a new property of SV40 TAg. We interpret our results as an indication that this property enables purified TAg to initiate the cellular immune response necessary for the SV40-tumor rejection in mice.     

Model identification and estimation of organ-function parameters using radioactive tracers and the impulse-response function

Examination of the input-output events in functioning organs by the use of the impulse-response function (IRF) for a radioactive tracer is gaining more and more ground in nuclear medicine. This study summarizes the development of deconvolution analysis, laying special stress on the model-free approach. System linearity and time invariance are discussed, and means of eliminating noise in IRFs originating from the input and organ-time-activity curves are outlined. Typical IRFs are illustrated by flow diagrams, time-domain curves, and their representation by Laplace transforms. The cases of nondiffusible and diffusible tracers as well as parenchymally extracted and transported substances are discussed. Methods for the derivation of models and for the calculation of physiologically important parameters from theIRFs are suggested.At present, a guest scientist at the Institute for Medicine, Nuclear Research Center Jülich, Jülich, Federal Republic of Germany     

The pharmacokinetics of high-dose epirubicin and of the cardioprotector ADR-529 given together with cyclophosphamide, 5-fluorouracil,and tamoxifen in metastatic breast-cancer patients

A high-pressure liquid chromatographic method for determination of the bisdioxopiperazine derivative ADR-529 (ICRF-187), a compound proven effective in protection against anthracycline-induced cardiotoxicity, has been developed. The limit of quantitation was 5 ng/ml using a narrow-bore 5-m silica column and UV detection. The method was used for determination of pharmacokinetic profiles of ADR-529 after a 3-weekly i.v. administration of different doses of ADR-529 (600–1000 mg/m²) together with different doses of epirubicin (E, 60–100 mg/m²), fixed-dose cyclophosphamide (C, 600 mg/m²), fixed-dose 5-fluorouracil (F, 600 mg/m²), and daily administration of tamoxifen (T, 30 mg; CEF-T) in the treatment of patients with metastatic breast cancer. Pharmacokinetic parameters for epirubicin were also determined. The aim of the study was to determine (1) whether the pharmacokinetics of ADR-529 as part of a combination with CEF-T changes with increasing doses of ADR-529 and increasing doses of epirubicin and (2) whether the pharmacokinetics of epirubicin in the same combinations is altered with the administration of increasing doses of ADR-529. A total of 82 patients were included. A crossover study including 16 of the patients showed no significant difference in epirubicin pharmacokinetic parameters when epirubicin was given with or without concomitant administration of ADR-529. Apart from minor changes in the distributional half-lives, the pharmacokinetic parameters of epirubicin were not altered with increasing doses of ADR-529, nor were the pharmacokinetic parameters of ADR-529 itself. Escalating doses of epirubicin did not significantly alter the pharmacokinetic parameters of ADR-529 with the exception of a 30% increase in the terminal half-life and a decrease in total body clearance when the epirubicin dose was raised from 60 to 100 mg/m². We conclude that concomitant administration of ADR-529 does not alter the distribution and elimination of epirubicin in doses suitable for preventing the anthracycline-induced cardiotoxicity.     

Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists

The ATP-induced increase in tritium outflow from cultured chick sympathetic neurons prelabelled with [³H]-noradrenaline was investigated.Seven days-old dissociated cell cultures of embryonic paravertebral ganglia, loaded with [³H]-noradrenaline (0.05 M), were superfused in the presence of (+)-oxaprotiline and exposed to ATP, ATP-analogues, or 1,1-dimethyl-4-piperazinium (DMPP) for 2 min. ATP (3 LM-3 mM), 2-methylthio-ATP (3–100 M), as well as DMPP (10 and 100 M) induced a significant overflow of tritium. The EC₅₀-value of ATP was 20 M. Both the ATP-induced and the DMPP-induced tritium overflow was Ca²⁺-dependent and sensitive to tetrodotoxin (0.3 M) and -conotoxin (0.1 M); in addition, it was inhibited by the ₂-adrenoceptor agonist 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK-14,304; 1 M). The effects of ATP and DMPP were not additive. The ATP-induced as well as the DMPP-induced overflow of tritium was diminished by the P₂-purinoceptor antagonists suramin (300 M) and reactive blue 2 (3 M); in all 4 cases, the inhibition amouted to approximately 40%. The tritium overflow induced by ATP or DMPP was almost abolished by the nicotinic receptor antagonist mecamylamine (10 M) and markedly inhibited by hexamethonium (100 M). Neither ATP nor electrical stimulation caused an overflow of tritium from cultures loaded with [³H]-choline.The results suggest that ATP at molar concentrations induces noradrenaline release from cultured chick sympathetic neurons via an action on a subclass of the nicotinic cholinoceptor.     

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens

In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by α,β-methylene ATP (α,β-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by α,β-MeATP (3 μM), with IC₅₀-values ranging from 1.1 to 100 μM. Pyridoxalphosphate-6-azophenyl-2’,4’-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4’-diisothiocyanatostilbene-2,2’-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8’-[Carbonylbis(imino-3,1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate) (NF023), sura- min, pyridoxalphosphate-6-azophenyl-2’,5’-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34–43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6’-(1,1’-biphenyl-4,4’-diylbisazo)-bis-4-amino-5-hydroxy-naphtha-lene-1,3-disulphonate (NH01) and Evans blue even enhan- ced the response to ATP. For antagonists causing full or partial inhibition, the IC₅₀-values against ATP were close to those against α,β-MeATP. All antagonists attenuated the removal of ATP, with IC_25%-values ranging from 0.8 μM to >320 μM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X₁). Several observations, however, do not conform with this model, and the existence of multiple contraction-mediating receptors for ATP or multiple, pharmacologically distinct ecto-nucleotidases has to be considered. Received: 23 October 1998 / Accepted: 11 January 1999     

Tetrahydronorharmane (Tetrahydro-β-carboline), a physiologically occurring compound of indole metabolism

Summary In the present paper a sensitive method is described to measure tetrahydronorharmane (THN) in the urine of man and rats as well as in the forebrain of rats. The compound is extracted into diethyl ether, separated by thin layer chromatography (TLC), acetylated with radiolabelled acetic anhydride and further isolated by two-dimensional TLC development. The existence of THN in urine of man was proven by using chromatography with different solvent systems, cocristallisation, isotope dilution technique as well as mass-spectrometry. The amount of THN in the urine varied over a wide range.With the same method it was demonstrated that THN occurs also in the forebrain of rats. The concentration increases after loading with tryptamine.The findings are discussed in view of the hypothesis that THN acts as a compound modulating neuronal mechanism.     

Pattern discrimination with flickering stimuli

The spatial contrast at which Observers are able to discriminate between horizontal and vertical gratings in a 2AFC task shows the same dependence on spatial and temporal frequency as does 2AFC detection. We conclude that mechanisms carrying information about spatial contrast have their sensitivity to low spatial frequency sinusoidal gratings improved by flicker and that such mechanisms are likely to mediate the detection of low spatial-frequency gratings both at low and at high temporal frequencies.     

Multiple-dose pharmacokinetics of epirubicin at four different dose levels: studies in patients with metastatic breast cancer

Summary Pharmacokinetic analysis of epirubicin and its metabolites epirubicinol and 7-deoxy-13-dihydro-epirubicinol aglycone during the first and the fourth courses of treatment was performed in 78 patients with metastatic breast cancer. The patients were treated every 3 weeks with epirubicin given as 10-min i.v. infusions at four different dose levels: 40, 60, 90 and 135 mg/m². In most cases (76 of 78 cases), plasma concentration-time curves fitted to a three-compartmental pharmacokinetic model. The terminal half-life of epirubicin was independent of dose and duration of treatment. Large interindividual differences were demonstrated (meant _1/2, 21.6±7.9 h; range, 10.6–69 h;n=110). In two subjects, extremely long half-lives and high serum bilirubin concentrations indicated impaired liver function. No correlation was found between the half-life and levels of liver alanine aminotransferase (ALAT) or serum creatinine. The metabolite epirubicinol appeared quickly after epirubicin administration and its half-lives were shorter than that of the parent compound (meant _1/2, 18.1±4.8 h; range, 8.2–38.4 h;n=105).Formation of the aglycone metabolite was delayed and the half-life of this metabolite was shorter than that of epirubicin (meant _1/2, 13±4.6 h; range, 2.7–29 h;n=104). The AUC of epirubicin and the total AUC (drug and metabolites) were linearly proportional to the dose, with the former value constituting two-thirds of the latter. A correlation was found between AUC and the plasma concentration of epirubicin at two time points (2 and 24 h after administration). The proposed model was AUC=9.44×c ₂+62.5×c ₂₄+157.7 (r=0.953).This work was supported by the Lundbeck Foundation, the Michaelsen Foundation and Farmitalia Carlo Erba Ltd.