Th. Wieland

Neue Anwendungen der Dünnschichtchromatographie

nehmen, dass die Ergebnisse der in der Tabel le zusammengeste l l ten Milchproben je nach der Zei t der E n t n a h m e anders ausgefaUen w~ren. Es war wahrscheinl ich, dass die in der Milch der stillenden Mii t te r v o r k o m m e n d e n Ant ik6rper auch im Blut se rum nachweisbar sind. Dass dies de r Fa l l ist, zeigt F igu r 2 a m Beispiel yon drei Mitittem, bei denen sowohl ]glut wie auch Milch auf Vorhandense in yon Kuhmi lchan t ikSrpern gepri i f t wurden . W i t haben also begr i inde ten Anlass zu der Annahme , dass ein grosser Tell der Ant ik6rper aus dem Blu t s t ammt .

Enhanced inhibition of virus DNA synthesis in hepatocytes by trifluorothymidine coupled to asialofetuin

The use of some drugs would be greatly improved by concentrating them in those cells where their pharmacological action is required. This might be obtained by conjugating the drug to amacromolecular vector which is selectively taken up by target cells. If the bond between the drug and the carrier is broken down in lysosomes, the drug should be released free and concentrated into the cells where it was transported by the carrier [ 1,2]. When inhibitors of DNA synthesis are employed as antiviral agents, their concentration in infected cells would allow a more effcient inhibition of virus replication accompanied by a lower toxicity for tissues containing proliferating cells, such as bone marrow and gut. In previous experiments two inhibitors of DNA synthesis, cytosine arabinoside and Wluorodeoxyuridine, were concentrated in liver Kupffer cells of mice by conjugation to albumin. The conjugates inhibited the replication of Ectromelia virus in these cells, whereas free drugs were ineffective [3]. It was shown that some glycoproteins, such as fetuin, after removal of sialic acid are selectively taken up by hepatocytes where they are digested in lysosomes [4-61. In the experiments reported here trifluorothymidine (F3T) was coupled to asialofetuin (AF) in order to concentrate the drug into hepatocytes, as a possible approach to the treatment of hepatitis caused by deoxyriboviruses. 2. Materials and methods

Stabilization of F-actin by phalloidin reversal of the destabilizing effect of cytochalasin B

The cyclic peptide phalloidin, a toxic component of the mushroom Amanita phalloides prevents the reduction by KI of the high viscosity of F-actin and inhibits the ATPase activity of F-actin during ultrasonic vibration [ 1 ] . Cytochalasin B (CB), a metabolite of the fungus Helminthosporium dematioideum, on the other hand obviously weakens the F-actin structure. This can be concluded from the fact that CB inhibits cellular functions that are believed to be linked to actin-like microfilaments (for reviews see [2] and [3 ] ) and that it decreases the visosity of F-actin [4] . This communication describes that phalloidin is able to antagonize the weakening effect, which CB exerts on F-actin structure.

Selective inhibition of ectromelia virus DNA synthesis in hepatocytes by adenine-9-β-D-arabinofuranoside (ara-A) and adenine-9-β-D-arabinofuranoside 5′-monophosphate (ara-AMP) conjugated to asialofetuin

L. FIUME, A. MATTIOLI, C. BUSI, P. G. BALBONI+, G. BARBANTIBRODANO+, J. De VRIES*, R. ALTMANN** and Th. WIELAND** Istituto di Patologia Generale, Via San Giclcomo 14, I-40126 Bologna, +Istituto di Microbiologz& Via L. Borsari 46, I-44100 Ferrara, Italy, *Medizinische Universi&Klinik, Abteilung Klinische Pharmakologie, Bergheimer Strasse 58, D-69 Heidelberg and **Max-Planck-Institut fir Medizinische Forschung, Abteilung Naturstoff-Chemie, Jahnstrasse 29, D-69 Heidelberg, FRG

The interaction of phalloidin. Some of its derivatives, and of other cyclic peptides with muscle actin as studied by viscosimetry.

Phalloidin (Ia), a component of the toxic green deathcap toadstool Amanita phalloides [ I] induces ir, vivo as well as in vitro the formation of filamentous structures in the cytoplasmic membrane fraction of (rat) liver as observed by electron microscopy [2]. The nature of these filaments as actin has been proven by their reaction with heavy meromyosin to give arrow head-like structures [3]. In contrast to F-actin, however, are the phalloidin-induced fibrils (Ph-filaments) resistant against 0.6 M KI. Consequently also F-actin from rabbit muscle was treated with the toxin and could so be transformed into a modified form (Ph-actin), which proved resistant against 0.6 M KI too. Ph-actin is also formed from G-actin by adding phalloidin to a solution of low ionic strength but sufficiently high ((0.7 mM) Mg2+ concentration. Since the process of polymerization of G-actin to F-actin (Phactin) can be followed more easily by viscosimetry we studied the interaction of G-actin with phalloidin (Ia), phallacidin (Ib), desmethylphalloin (Ic), its toxic and non-toxic sulfoxides (Id, Ie), the non-toxic derivatives desthiophalloidin (If) and secophalloidin (Ig) as well as a-amanitin and a soluble preparation of antamanide. Further the inhibiting effect of cytochalasin B (CB) on the formation of Ph-actin was investigated.

Phallotoxins bind to actins

The function of receptors can be ascribed to the fdamentous structures which appear with increased frequency in the cytoplasma fraction of rat liver after in vivo or in vitro intoxication with phalloidin [ 11. Using [3 H] desmethylphalloin we found that 3.6 mnoles of the phallotoxin are tightly bound to 1 mg of filamentous protein [2] . Shortly thereafter it was recognized that the phallotoxin induced filaments from liver @h-filaments) bind heavy meromyosin (of rabbit muscle) in an arrow head-like manner like muscle F-actin and so consist of an actin [3]. In contrast to F-actin, however, they are not depolymerized by 0.1 M potassium iodide. Consequently F-actin was also treated with phalloidin and thereupon lost its sensitivity against 0.1 M KI [ 31. The modified form, Ph-actin, is formed from G-actin in presence of phalloidin already in KCl free solution provided sufficient Mg’+ ions are present. The formation of analogous Ph-actins by several toxic derivatives of phalloidin, and the failure of their formation by nontoxic ones could be stated then by viscosimetry [4]. In the present communication we describe comparative quantitative studies on binding of 3 Hand I4 C-labelled phallotoxins to Ph-actin obtained by polymerization of G-actin in presence of the toxins and to Factin (which is transformed into a KI-resistant Phactin by the toxins).

Amaninamide, a new toxin of Amanita virosa mushrooms.

Amaninamide, a toxin closely related to the family of amatoxins, was found exclusively inAmanita virosa mushrooms. It differs from the well known toxin α-amanitin in that it lacks the 6′-hydroxyl group of the tryptophan unit, and from the toxin amanin found inAmanita phalloides by the presence of a carboxamide group instead of a carboxylic acid group.

Interaction of phallotoxins with actin.

Abstract Phalloidin (PHD) like its toxic relatives from the mushroom Amanita phalloides and like toxic derivatives obtained by chemical modifications combines with F-actin, thus stabilizing its polymer structure. Since there is no dissociation of F-actin to G-actin in presence of PHD F-actin filaments have been found in poisoned liver cells. The stabilization of F-actin from rabbit muscle by PHD has been established experimentally by its behavior towards several chemical and mechanical stresses. PHD-treated actin (Ph-actin) is not depolymerized by 0.6 M KI, it withstands the depolymerizing action of DNAase I, denuration by heating to 70°C for 3 min, and retards destruction by alkali (pH 12) about 30 fold. Ph-actin is also resistant to the hydrolyzing action of subtilisin. Phallotoxins prevent the ATPase activity of F-actin which normally is induced by ultrasonic vibration or by cytochalasin B around neutral pH values. Whereas on going to acidic pH without PHD the ultrasonic ATPase decreases and completely disappears at pH 3.5, PHD even under the same conditions preserves the structure to such an extent as to still retain ATPase activity. At all effects observed a ratio of 1 mol. PHD per 1 mol of G-actin seems sufficient for full protection. Phallotoxins are firmly bound to F-actin as observed by difference spectroscopy in the u.v. light. As to their stabilizing action either an allosteric effect enforcing the coherence between the actin units or crosslinking by bifunctionality of the phallotoxin molecules has been postulated.

Phalloidin counteracts the inhibitory effect of actin on deoxyribonuclease I

The naturally occurring inhibitor of pancreatic deoxyribonuclease I (DNase I) [l] has recently been found to be identical with actin [2]. From studies in our laboratories we know that the toadstool poison phalloidin accelerates the polymerization of actin [3,4] and stabilizes the microfilaments against 0.6 M KI, as well as against depolymerization under different conditions, e.g. by ATP [5], by ultrasonic vibration [6] or by cytochalasin B [7]. This prompted us to study the interaction of phalloidin with the system actin/DNase I.

Conjugates of adenine 9-alpha-D-arabinofuranoside monophosphate (ara-AMP) with lactosaminated homologous albumin are not immunogenic in the mouse.

Conjugates of adenine-9-β-D-arabinofuranoside (ara-A) or of ara-A monophosphate (ara-AMP) with asialofetuin or with heterologous lactosaminated serum albumin (L-SA) are strong antibody inducers. But ara-AMP conjugates prepared with homologous L-SA are not immunogenic, at least in mice.