ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1972, p. 385-391Copyright @ 1972 American Society for Microbiology

Vol. 1, No. 5Printed in U.S.A.

Screening, Fermentation, Isolation, andCharacterization of Trypanomycin, a

New AntibioticW. FLECK, D. STRAUSS, C. SCHONFELD, W. JUNGSTAND, C. SEEBER, AND H. PRAUSER

Central Institute for Microbiology and Experimental Therapy, German Academy of Sciences at Berlin, Jena, andDepartment of Neurovirology, Nervous Clinic, Medical Academy of Erfurt,

Erfurt, German Democratic Republic

Received for publication 18 November 1971

A Streptomyces strain which is similar to S. diastatochromogenes (Krainsky)Waksman et Henrici 1948 sensu Hutter (1967) was found to produce a new anti-biotic designated trypanomycin. The red-pigment antibiotic, which has noveltrypanocidal activity in vitro and in vivo, was isolated from a C-, N-, and iron-containing culture of the strain IMET JA 10081/9 by extraction with organicsolvents, transfer into the aqueous phase, reextraction with organic solvents at pH6.8, precipitation by hydrocarbons, and purification by chromatographic methods.Trypanomycin has indicator properties. The main constituents of the antibioticmixture are readily soluble in water and are very stable in distilled water at roomtemperature (28 C) for 24 hr. The composition of the base oftrypanomycin A2 (melt-ing point, 175 to 183 C) corresponds to the empirical formula C41-42H5>66NO21-22 .

The absorption spectra in the ultraviolet and visible regions are very similar to thoseof the 4,5, 8-trioxyanthrachinones. Trypanomycin has strong antiprotozoal activity,e.g., against trypanosomes. The natural substance additionally inhibits the growthof gram-positive bacteria, stable as well as unstable L-forms of gram-negativebacteria, mycoplasmas, yeast protoplasts, and tumor cells in vitro. The LDw oftrypanomycin in mice was 60 mg/kg when administered intravenously and 31 mg/kgon intraperitoneal administration. If the antibiotic was added to cultures of animalor human cells in vitro, mitotic inhibition and chromosomal aberrations resulted.Trypanomycin differs in its biological activities and chromatographic behaviorfrom other anthracyclines, e.g., cinerubine A and B, daunomycin, adriamycin,nogalamycin, rutilantin, pyrromycin, cyclamycin, and ryemycin.

In the course of a screening program directedtoward the isolation and evaluation of new can-cerostatic antibiotics, we isolated from a soil aspecies of streptomycetes which was found toproduce the new antibiotic trypanomycin. Thisnew antibiotic proved to be of considerable in-terest because of it strong antiprotozoal activity,e.g., against trypanosomes. The antibiotic, whichrepresents a new type of the recently knownanthracyclines (1) with many distinctive charac-teristics, has been identified chemically as e-pyr-romycinone glycoside.

In this report, we present biological data rele-vant to the discovery and fermentation of theproducing strain, and describe the isolation andsome of the physicochemical and biological prop-erties of trypanomycin.

MATERIALS AND METHODSScreening. Among 7,000 strains, which were iso-

lated or deposited in the Institute for Microbiologyand Experimental Therapy (IMET) culture collectionfrom various geographical areas and which belong todifferent groups of Actinomycetales such as Strepto-myces, Nocardia, Pseudonocardia, Actinosporangiwn,Micromonospora, Microbispora, Microechinospora,Spirillospora, Micropolyspora, Chainia, Microellobo-spora, and Promicromonospora, we have found 290strains which produce red pigments with or withoutbiodynamic activity. Sixty of these strains produce redpigments with indicator character and different bio-dynamic and physicochemical properties. They hadbeen detected by means of a complex screening system(5).From mycelia or culture fluids of 30 strains, we

obtained red-pigment antibiotics which could bedivided into six groups according to phenomena in

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the applied screening models in vitro (3, 4, 7) and toresults and experiences with chemotherapy in animals.

In group 1 the red pigments are antibiotic com-plexes containing components which induce the Xprophages in lysogenic cells of Escherichia coli (7),components which interfere with the excision darkrepair mechanism in cells of E. colt (4), or both. Thecomplexes also contain components which inhibitthe multiplication of temperate phages in E. coli (3),but which are unable to disturb the regulationmechanism of the lysogenic state. Most pigments ofgroup 1 showed a strong activity against both leukemicdisease L1210 and the solid tumor sarcoma 180 inmice.

Pigments of groups 2 and 5 are also antibioticcomplexes containing components which inducespecific phenomena in the described "BIP" testmethod (3) and reduce the growth of sarcoma 180 inmice significantly. Biodynamic activity of pigments ingroups 3 and 6 was not observed in any screeningmodel in vitro or in vivo.Red pigments of group 4 were detected by means

of the BIP test method, but there is no indication ofany significant activity against experimental tumormodels in animals. However, several natural sub-stances of group 4 are antibiotic complexes of im-portance in the chemotherapy of protozoal diseases.Trypanomycin belongs to this last group. The pro-ducing strain was isolated from an Italian soil samplewhich was taken from a vineyard in the village Ana-capri on Capri and possesses the morphological andbiochemical properties of Streptomyces diastato-chromogenes (Krainsky) Waksman et Henrici 1948,sensu Hutter 1967 (8).

Fermentation. Trypanomycin was produced by theusual procedures of submerged fermentation. Thefirst cultures were grown on agar medium inoculatedwith spores. Then the organism was propagated inshaken flasks and fermentors ranging from 0.5 liter toseveral cubic meters. An inoculum of the strainIMET 10081/9 was prepared by growing the organismat 28 C in aerated 20-liter bottles for 48 hr in a mediumof the following composition (grams per liter): soy-bean meal, 15; glucose, 15; sodium chloride, 5;calcium carbonate, 1; and primary potassium phos-phate, 0.3. A 15-liter amount of inoculum was used toseed 400 liters of medium containing the followingingredients (grams per liter): soybean meal, 30;glucose, 30; calcium carbonate, 3; sodium chloride, 3;and iron chloride hydrate, 0.25. The fermentation wascarried out in a 700-liter V2A tank at 28 C with anair supply of 400 liters per min and at a stirring rateof 270 rev/min for 96 hr. The foam formation occur-ring was controlled by addition of small amounts ofsoybean oil. The maximal concentration of trypano-mycin was obtained after 4 days and reached morethan 100 mg/liter in the wild type. The resulting brothwas pink to orange-red to violet in color, dependingon the nature of the culture medium and its pH.

Analytical procedures. The first attempts to isolatetrypanomycin were extremely difficult because welacked any method of quantitative analysis. Initially,they were carried out by semiquantitative assessmentof the antiviral activity in vitro determined by the

"BIP" test method (3). The antibiotic activity wasobserved during fermentation by assaying samples ofcrude fermentation broth. The test organism wasE. coli C 600 multiply infected with free temperateX phages. The activity was determined by a micro-biological agar diffusion test in which the inhibitionof multiplication of temperate viruses in bacteria isused as a measure of antibiotic activity. Other sub-stances in the broth possess antibacterial but no anti-viral activity. Because trypanomycin contains severalconstituents, the quantitative determination of theconstituents was possible only by use of paper or thin-layer chromatography for separation. After separa-tion, the constituents of trypanomycin could be de-termined by colorimetric or microbiological methods.Paper chromatography and thin-layer chromatographyturned out to be equally successful.

Paper chromatography with trypanomycin A2 wascarried out on Schleicher & Schuell No. 2043b paperwith the use of ascending development. Several sol-vents and solvent mixtures saturated with water wereused: butanol, water-saturated, RF 0.65; methanol,RF 0.9; acetone, 50%, RF 0.48; acetone-benzol-water,12:3:2, RF 0.87; and, as the round-filter chromatog-raphy system, butanol-methanol-water, 4:1:2, RF 0.7.Active substances were detected bioautographically onBIP test plates of tryptopeptone-agar seeded in thetop layer with E. coli C 600 and free X- phage (multi-plicity, 50).

For thin-layer chromatography, the absorbent wasMerck silica gel D in layers of 0.25 mm. The absorbentwas not impregnated, and the developing solvent wasthe upper phase of the system chloroform-methanol-acetone-water (50:5:15:10, v/v). As the constituentsof trypanomycin are colored and grouped in a num-ber of small areas, it also was possible to examinethem directly. The colored zones were scraped offand eluted. Each constituent was determined byspectrophotometry at 290 nm, in comparison withstandards chromatographed in parallel.To facilitate the identification of trypanomycin, the

aglycone was isolated by a method used for someanthracyclines. Acid hydrolysis of trypanomycin withsulfuric acid on a boiling-water bath yielded theaglycone and sugars.

Thin-layer chromatography could be used for dif-ferentiation of trypanomycin A2 from cinerubin Aand B (2). Comparative chromatography of the twoanthracycline antibiotics was performed on silica gelD plates with a chloroform-methanol-acetone-water(50:5:15:10) solvent mixture used for developmentof plates. The red pigment antibiotics differ signifi-cantly in RF values (Table 1).

Isolation. The active substances were at first ex-tracted from the culture broth as a crude base con-taining a mixture of trypanomycin constituents andother substances. The 400 liters of harvested mashwere filtered, and, after separation of the myceliawith unchanged acidity, the filtrate of about 360 literswas extracted with 90 liters of butanol. The mycelia(36 kg, fresh weight) were extracted three times with72 liters of methanol. The combined extracts wereconcentrated to one-fifteenth of the initial volume, andwere completely freed from organic solvent by azeo-

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TABLE 1. Physicoclhemical properties of trypanomycin A2 and trypanomycinionie A2

Property Trypanomycin A2 Trypanomycinone A2

Empirical formula ....... ....... C41-42H52 5602,NC24HN2024010Analytical results (%)CalculatedC........... 54.53 61.10H.... 6.05 4.63O (difference) ... 37.86 34.27N..1........ 1.56

FoundC...... 54.49 61.20H........... 6.11 4.60O (difference) 37.84 34.20N........... 1.56

Melting point (C) 175-183 182-187Molecular weight

Calculated.................... 884-926 470-472Found ........................ 910 4 20 (Rast) 470 (nile, M+ peak)

TABLE 2. Ultraviolet and visible absorption spectraof trypanornycin A2 and trypanomcinonoe A2

Trypanomycin A2 Trypanomycinone A2

Absorption Absorptionpeak (nm) E'%icm peak (nm) El icm

260a 183290 102 282b 541470 75 465 74484 80 480 220495 105 500 330510c 520 216529 534 190

a Determined in methanol.bDetermined in chloroform.c Shoulder.

tropic distillation. The 30 liters of aqueous suspensionobtained were twice extracted with half the volume ofchloroform. The chloroform extract was concen-trated to one-tenth of its volume, and the antibioticwas precipitated by addition of a threefold volume ofpetroleum ether. The constituents of this crude basewere then fractionated and purified by columnchromatography methods, with the use of cellulosepowder saturated with phosphate buffer, pH 5.4.Trypanomycin A2 was eluted by n-butanol, buffer-saturated. The active yellow-orange colored fractionswere combined and evaporated, and the residue wasrecrystallized from water-free n-butanol. The orange-red crystals were filtered off and dried.

RESULTS AND DISCUSSION

Physicochemical properties. The crude base oftrypanomycin was isolated as a complex ofamorphous, red-brown substances which dis-solve readily in chloroform, acetone, and dimethyl

formamide, less readily in alcohols, esters, andbenzene, and sparingly in water. Four biologicallyactive constituents of crude base designatedA1 to A4 are readily soluble in water. The mainconstituent in broth is trypanomycin A2.

In the solid state, the mixture of trypanomycinconstituents is very stable. It can be kept forseveral years at a temperature of 18 to 22 C with-out any loss of activity. As shown by in vitroexperiments with microbiological methods (agardiffusion technique), the aqueous solution oftrypanomycin does not lose any activity withinmore than 8 weeks at 0 or 30 C. A 1 mg/ml solu-tion in sterile distilled water was very stable atroom temperature (28 C) for 24 hr.The composition of the base of trypanomycin

A2 corresponds to the empirical formulaC41-52H52-56NO21-22 (Table 1). Trypanomycin A2(melting point, 175 to 183 C) has an absorptionspectrum in the ultraviolet (UV) and visibleregions (Table 2) which is very similar to those ofthe 4,5,8-trioxyanthrachinones (1). The anti-biotic is a chromoglycoside formed by a tetra-cyclic quinoid aglycone (trypanomycinone A2)from the e-pyrromycinone type and three notyet identified sugars. After acid hydrolysis(0.1 N hydrochloric acid methanolic solution,90 C, 30 min), the aglycone and the sugars wereobtained. The sugar moiety was chromatographedon silica gel (thin-layer chromatography) andpaper sheets, and showed three spots with sugarreagents, two ofthem with aniline phtalate (reduc-ing sugars) and one by diphenylamine only. Oneof the first is an amino sugar. Trypanomycin A2,the main constituent of the crude base, is a light-red substance and can serve as an indicator,being red-orange in acid solution with yellow-

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TABLE 3. RF values determined by thin-layer and paper chromatography

Substance Material Systema RF

Cinerubin A and B Silica gel D 2 0.94 and 0.98Trypanomycin A2 Silica gel D 2 0.46e-Pyrromycinone Paper 2043b 1 0.80Trypanomycinone A2 Paper 2043 1 0.77e-Pyrromycinone Paper 2045 1 0.83Trypanomycinone A, Paper 2045 1 0.77

a System 1: benzene-cyclohexane, 2:1; paper saturated with formamide-acetone (4:1). System 2:chloroform-acetone-methanol-water, 200:20:50:50 (organic phase).

I Obtained from Schleicher & Schuell Co.

TABLE 4. In vitro anltimicrobial spectrum oftrypantomycina

MinimalTest organism inhibitory

concn (ssg/ml)

Bacillus subtilis ATCC 6633. 0.3B. globifer OHI1 .. 0.6B. mycoides DG 756 ..0.6Sarcinta lutea SG 125 .......... 0.8Staphylococcus aureus SG 511 0.3S. aureus 209P .........0...........0.3S. aureus UV-2 mutant (Gauze) O 0.05Nocardia sp. IMET 133.. . 0.2Chromobacterium sp. 1-S ............ 0.1Caryophanont latum (Peshkov) 0.3C. latum (Robinow) .. 0.3Streptomyces hygroscopiclis JA6669-6......0.............. O.05

Mycobacterium smegmatis SG 987 1.5M. phlei SG346 ..................... 1.0Escherichia coli mutabile SG 458 >50.0Serratia marcescens SG 621 >50.0Aerobacter aerogenies SG 117 >50.0Proteus vulgaris SG 2............... >50.0P. morganii SG 464 ............ >50.0Pseudomonas aerugintosa SG 137..... > 50. 0Saccharomyces cerevisiae ............ > 50.0Kloeckera brevis .. >50.0Schizosaccharomyces pombe ......... >50.0S. pombe protoplasts ..20.0Fusarium culmorum JP 15 .. 25.0F. solani JP 20... ...... .... ... > 50. 0Scopulariopsis sp. JP 25 ....... .. > 50. 0Peniicillium notatum JP 42. 3.0

a The agar diffusion test was used.

orange fluorescence in UV light and blue-violetin alkaline solution. The antibiotic belongs tothe group of anthracyclines and to the subgroupof anthracyclines with an aglycone of the E-pyr-romycinone type, as shown by its UV spectra inconcentrated sulfuric acid, the chemistry of whichhas been reviewed by Brockmann (1). Cinerubin,pyrromycin, rutilantin, tavromycetin, cycla-mycin, and ryemycin belong to the same sub-

TABLE 5. In vitro susceptibility of L-fornis, sphero-plasts, antd mutanits of gram-iiegative bacteria

to trypaliomycint

Test organism Minimal inhibitoryconcn (Mg)

Proteus mirabilis VI, normalform....... >50.0

P. mirabilis L VI, stable L-form. 6.5P. mirabilis D52, normal form. >50.0P. mirabilis LD52, stable L-form 6.5Escherichia coli K-12 (7r),

normal form ............... >50.0E. coli K-12 (7r), unstable

L-form .................... 8.0E. coli K-12 AB 1157/AS 19 5.0

TABLE 6. In vitro activity of trypanomycin againistMycoplasma

Mlinimal inhibitoryconcn (jug/ml)

Antibiotic OrganismBacterio- Bacteri-static cidal

Trypano- M. galliseplicum 0.76 19.0mycin M. hyorhiniis 3.8 19.0

Tylosin M. gallisepticiuim 0.15 95.0M. hyorhin1is 0.15 >476.0

group, but all of them are derivatives of theE-pyrromycinone.

Acid hydrolysis of the minor constituents ofthe crude base of trypanomycin gave the sameaglycone as the main constituent trypanomycinA2. Comparative chromatography of trypano-mycinone A2 and a reference sample of e-pyrromy-cinone showed that the RF values are different(16). Trypanomycin differs in its biological ac-tivities and chromatographic behavior fromcinerubin A and B (Table 3). Thus, trypanomycinis a new antibiotic from biological, chemical,and physical points of view.

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Biological properties. Trypanomycin was activein vitro against gram-positive bacteria (Table 4).The most susceptible organisms were Strepto-myces hygroscopicus, a UV-2 mutant fromStaphylococcus aureus 209P, Chromobacteriumsp. 1-S, Caryophanon latum, staphylococci, andBacillus subtilis. Its inhibitory action againstgram-negative bacteria, yeasts, and filamentousfungi was slight or nonexistent. Trypanomycinalso inhibited the growth of protoplasts ofSchizosaccharomyces pombe, which were usedas a wall-free model in the screening for newantibiotics. Normal cells ofS. pombe were not sus-ceptible in vitro to high concentrations oftrypanomycin; however, the protoplasts were sus-ceptible to 20 ,ug in the agar diffusion test (12).

Because high concentrations of trypanomycin

TABLE 7. In vitro antiprotozoal spectrumof trypanomycin

Test organism Minimal inhibitoryconcn (ug/ml)

NonpathogenicEuglena gracilis................. 100.00Polytoma uvella . ............... 1.25Paramecium caudatum........... 1.25Tetrahymena vorax............. 5.00

PathogenicEntamoeba invadens............. 50.00E. histolytica ................... 10.00Trichomonas vaginalis........... 50.00Leishmania brasiliensis.......... 12.5Trypanosoma equiperdum........ 5O .

T. gambiense ............5.......O.T. cruzi........................ 12.5

a Produced killing after 6 hr.

TABLE 8. In vivo trypanocidal activity oftrypanomycin

Individual Survival Prolonged No. ofdose (mg/ time period of Nimal20-g mouse) (days) survival (%) anmls

-a 6.1 142.0 19.5 217.4 61.0 12.8 108.4 50.5 10.5 70.9 60.25 6.3 3.1 6

a Control.

TABLE 9. Subacute LDw (mouse) of trypanomycin

Mode of injection LD&o (mg/kg)

Intravenous. 60.0Intraperitoneal ................. 31.0

inhibited the growth of intact cells of gram-negative bacteria only, the in vitro susceptibilityof stable and unstable L-forms such as sphero-plasts of gram-negative bacteria was determined.It can be seen in Table 5 that the antibioticinhibited the growth only of L-forms (14, 17)and mutants of E. coli permeable to actinomycin(15). An important bactericidal activity oftrypanomycin against two strains of Myco-plasma was observed by use of a method devisedby P. Kramer (unpublished data). In Table 6 itcan be seen that trypanomycin inhibited thegrowth of Mycoplasma in vitro to a higher degreethan did the macrolide antibiotic tylosin underthe test conditions used.

Antiprotozoal activity. Not only fresh fer-mentation broths of the strain JA 10081 or themutant strain JA 10081/9 but also the isolatedantibiotic inhibited the growth of nonpathogenicand pathogenic protozoa in vitro (Table 7).Trypanosoma gambiense (cause of sleeping sick-ness) showed in vivo (mouse) sensitivity totrypanomycin also. Female mice (AB-Kol strain)7 to 9 weeks old were infected subcutaneouslywith T. gambiense. Beginning the second day, theanimals received each day intravenous injectionsof trypanomycin in various concentrations untilthe fifth day. It can be seen in Table 8 that theantibiotic, in a dosage range of 1.0 to 2.0 mg/20-g mouse (daily individual doses) resulted in asignificant prolongation of the period of survivalof the treated animals. The acute LD50 of trypano-mycin depended upon the mode of administration(Table 9). There was a difference between in-fected and healthy animals in their sensitivity totrypanomycin, but its cause is unknown.

In further investigations, the susceptibility totrypanomycin of several species of Trypanosomawas checked. It could be shown that trypano-mycin was also active against T. rhodesiense,T. congolense, and T. vivax, but the therapeuticeffect in vivo against these species was temporaryor only moderate in comparison with the effectagainst T. gambiense and T. equiperdum.

Antitumor activity. Trypanomycin was shownto have an inhibitory action on the growth oftumor cells in vitro. An in vitro test was used forthe determination of the cytotoxicity of trypano-mycin to a permanent growing suspension cultureof Ehrlich ascites carcinoma cells (6). Cells ofEhrlich ascites carcinoma adapted to suspensionculture were maintained in a medium described byKarzel (10). The results show that cell divisionwas completely eliminated if 5 X 105 tumor cellsper ml of culture broth were exposed to 0.1 MAg oftrypanomycin for 20 hr. After this time, Ehrlichascites carcinoma cells retransplanted in NMRImice could not grow in vivo.

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FIG. 1. Representation2 of mitotic index from treated HeLa cells (solid line) and untreated cells (control, dashedline). The concentrationt of trypaniomycin was 0.32 j.g/ml. The nuimbers oni the ordiniate represent the mitoticindex; those on the abscissa represent the time of treatment in hours.

The cytotoxicity of trypanomycin was testedby tube cultures of FL, PN (human kidney cellline), and HeLa cells. The inoculum was 105cells/ml, and the time of observation was 3 to 5days. No cytotoxic effect could be shown aftertreatment with trypanomycin in concentrations of0.16 to 0.32 ,ug per ml of medium. But concen-trations of >0.32 j,g/ml showed cytotoxic effectson monolayer cultures of these cell lines.The antitumor activity of trypanomycin

against leukemia L1210 (mice) and solid sarcoma180 (mice) was not significant. The screeningmethods used in vivo were described by Jung-stand (9).

The cell division of Ehrlich ascites carcinomacells was completely inhibited by concentrationsof 0.1 ,ug or more of trypanomycin per ml after24 hr. Cell counts were performed on replicatecultures in duplicate; the cells were cultivated insuspension form and enumerated, after appro-priate dilution, with a "TuR" ZG1 electronic cellcounter (produced by VEB Transformatoren undRontgenwerk, Dresden, German DemocraticRepublic). No Ehrlich ascites carcinoma cellsunderwent division after exposure to 0.1 ,ug oftrypanomycin/ml for 24 hr when the method ofFleck and Hertwig (6) was used. This suggested acomplete loss of reproductive capacity.To determine the influence of trypanomycin

on the mitotic index of HeLa cells, the methoddescribed by Penso (11) was used. HeLa cellswere grown in lactalbumin hydrolysate mediumsupplied with 5% Parker medium, 10% calfserum, 2%O NaHCO3 (stock solution 0.75%), andkanamycin (50 Ag/ml). HeLa cells in cover slipculture were treated with trypanomycin (0.32 ug/ml), fixed after Carnoy for increasing intervals oftime (4 to 96 hr), and stained with "Kernechtrot"(13).

Figure 1 represents the mitotic index with 95%confidence intervals of HeLa cells based on acount of 6,000 stained cells. Trypanomycincaused some decrease in the number of mitoticfigures during the period (96 hr) studied. A great

number of mitoses showed various abnormalitiessuch as multipolar division and disorientedchromosome patterns. The most characteristicchanges in cell morphology induced by trypano-mycin may be chromosomal fragmentation andstar metaphase in dividing cells, and nuclearfragmentation and nucleolar pyknosis in inter-phase cells. Anomalies of the anaphase and ir-regular arrangement of fragmented chromosomesalong the spindle filaments were general andshowed no preponderance over any of the drugs.A trypanocidal effectiveness has hitherto not

been reported for red pigments with indicatorproperties from streptomycetes. Therefore, it issurprising that the antibiotic trypanomycin ac-cording to the group of deoxyribonucleic acid-intercalating anthracyclines has a strong trypano-cidal activity in vitro and in vivo.

Further investigations with trypanomycinshowed an inhibitory action against Eimeriatenella in chickens. Therefore, determination ofthe coccidiostatic activity of trypanomycin is inprogress.

ACKNOWLEDGMENTS

We thank K. Wohirabe for the determination of the LDro inmice, Prof. Dr. Zahner of the University of Tubingen for provid-ing a reference sample of cinerubine, and J. Balanova and Dr.Dobias from the Department of Microbiology of the Institute ofBiology, SAV-Bratislava, for some tests with Leishmania brasili-eatse and Trypanosoma cruzi in vitro. We are grateful to ErikaBaumann, Peter Grosse, and Roswitha Mattern for valuabletechnical assistance.

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3. Fleck, W. 1968. Eine neue mikrobiologische Screening-Methode fUr die Suche nach potentiellen Carcinostatica undVirostatica mit Wirkung im Nucleinsaure-Stoffwechsel. Z.AlIg. Mikrobiol. 8(2) :1 39-144.

4. Fleck, W. 1969. E. coli-Zellen mit intaktem und defektemDunkelreparaturmechanismus fiir Strukturschaden ander DNA als Screening-Modell fur die Suche nach poten-tiellen Carcinostatica. Z. Alig. Mikrobiol. 9(3):217-221.

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