Bacteriophage T4 mutants which propagate on E. coli K12 but not on E. coli B

Article

Reference

Bacteriophage T4 mutants which propagate on E. coli K12 but not on E. coli B

GEORGOPOULOS, Costa Panos, et al.

Abstract

We have isolated and characterized 2 mutants of coliphage T4 which are able to propagate on E.coli K12 but not on E. coli B. We have assigned the mutations to genes 8 and 53, both structural genes. The products of genes 8 and 53 are found in the baseplate.

GEORGOPOULOS, Costa Panos, et al . Bacteriophage T4 mutants which propagate on E. coli K12 but not on E. coli B. Experientia , 1977, vol. 33, p. 1157-1161

Available at:

http://archive-ouverte.unige.ch/unige:127853

Disclaimer: layout of this document may differ from the published version.

1 / 1

l j. 9. 1977 Specialia

T"bk 4. Comparison of the anlipolio effects of dichloropyrimidines pyri111idine in MNCTD** ID 95• .. ID 95/MNCTD

. .1:rE•

^{medium (iJ.g/ml) (µg/ml)}

!,-l·Dicltloropyrimidines 5 2.1 0.42

1,6-Di.ch loropyrimidine 20 4.6 0.23

u-:ll el hyl-2,4-dichloro-

p)Timidine 20 9.2 0.46

2-:\ mi110-4,6-dichloro-

pyrimidine 150 31.5 0.21

•Amino acid free Eagle's MEM. ••Maximum non-cytotoxic dose.

••• ~!inimum dose producing 95% inhibition on virus growth.

s

R. Dulbecco and M. Vogt,]. exp. l\iccl. 99, 167 (1954).

Moreover, as already observed for 2-amino-4,6-dichloro- pyrimidinc, th.c antiviral effect of the dichloropyrimi- dines is antagonized, in amino-acid-free-medium, by glu- taminc and cysteine, but not by pyrimidine precursors of nucleic acids. ln complete media, containing glutamine and cysteine (or cystine) in the amino acid supplement, the antiviral cifect is potentiated by 2-mercaptoethanol (table 3).

The limited number of compounds tested so far does not permit any conclusion on structure activity relation- ship inside the dichloropyrimidine group. As shown in table 4, it can only be said, at present, that the 4,6 po- sitions of Chlorine atoms in the pyrimidine ring are to be preferred to the 2,4 (2,6) positions in that the former enhance the therapeutic index of the molecule.

·nacteriophage T4 mutants which propagate on E. coli K12 but not on E. coli B

C. Georgopoulos, M. Georgiou, G. Selzer and H. Eisen ¹

TJ.Jfmrtement de Biologie Moltfculafre, Universite de Gencve, 30, quai Ernest-Anserinet,

CH- 1211 Summary.'vVe Geneve 4 (Switzerland). have isolated and characterized 2 mutants of coliphage T4 which are able to propagate on E.coli K12 14 Febrttary.J.,Jl.g

----

but not on E. coli B. \Ve have assigned the mutations Lo genes 8 and 53, both st ructural genes. The products of genes 8 and 53 are found in the baseplate.

In an effort to understand the bacterial functions which are necessary for proper phage development, we have isolated and characterized many bacterial mutants unable to propagate bacteriophage .l.. vVe have found 2 classes of such mutants which block ). DN /\.. replication ^{2 • 3 ,} another 2 classes which block ). RN A transcription 4• 5 and a fifth class which affects the morphogenesis of several phages including }., T4 and T5 ^6• In the present report, we have extended our studies of host-phage interactions and show that one can isolate T4 mutants which discriminate be- tween 2 naturally occurring hosts, E. coli K12 and E.coli B.

1\Iaterials and methods. The E. coli BR sup- (called B) and E.coli K12 W3101 ^sup~ (called Kt2) were the bacterial hosts. In order to isolate T4 mutants which propagate on Kl.2 but not on B, a nitrosoguanidine mutagenized stock of bacteriophage T4rI was absorbed to Kl 2 cells and the iniected cells plated on a mixed bacterial lawn of K12 and B. Vve anticipated that derivatives of T4rI which grow on Kl2 but not on B would form small, turbid plaques on this mixed bacterial lawn as opposed to the

Table 1

Phage

T4rI T·trl No. 4 T-lrl No. 20

e.o.p.* on

B K12

1.0 1.0

2.0 x 10-• 1.0 3.0 x 10--• 1.0

e.o.t.•• on

B Kl2

1.0 !.O 0.4 1.0 0.3 1.0

Phage yield*** on

B 1{12

GS 1.1 1.2

95 29 27

*e.o. p., the efficiency of plating, denotes the number of plaques pro- duced hy a phage strain on a given bac terial host relati\c to the n11n1bcr on l<12; ••c.o.t., the efficiency o[ tr;rnsmission, denotes the prubahilily that an iu[edecl bacterial host will produce at least one viable phage progeny; '**phage yield denotes the average number of 'iable phage progeny released per infectious ccnter.

--

large, clear plaques made by the parent strain. Plating efficiency of transfer and phage yield were as previously described ^2. Complementation tests were done in liquid by infecting the non-permissive Bsup- bacteria with 5 phage of each type per bacterium, and allowing the culture to lyse at 37°C.

Results and discussion. 2 T4rl derivatives, called No. 4 and No. 20, were isolated as being able to propagate on Kl2 bacteria but not on B bacteria. The frequency of occurrence after nitrosoguanidine mutagenesis was ap- proximately 5

x

10-^{4 •} Table 1 shows that the growth of the 2 mutants is slightly depressed on the K12 host, but is severdy inhibited on B (regardless of whether they are sup- or sup+).

Preliminary experiments showed that both T4 No. 4 or T4 No. 20 infected bacteria lysed after 20 min of growth at 37 °C, indicating that the early events of infection as well as cell lysis functions occur normally during the abortivP. infection, and that the failure to yield phage results from a block at the level of phage morphogenesis.

Su bscq uen tl y we tested by spot complementation on Bsup - T ·+ No. 4 and T4 No. 20 against amber mutations in all T4 late genes. Vile found that T4 No. 4 comple- mented phage mutants in all genes except gene 8 and that T4 No. 20 did not complement phage mutants in gene 53 (table 2). From recombination data obtained on the K12 sup+ host, it appears that the T4 No. 4 mutation is very

Supported by grant No 3.519.75 from the Fonds National Sui,;•;c de la Recherche Scicntifique.

2 C. P. Georgopoulos and I. Hcrskowitz, it1: The Bacteriophage Lambda, p. 553. Ed. A. D. Hershey. Cold Spring Harbor Lab- oratory New York 1971.

3 C. P. Georgopoulos, Molec. gen. Genet. in press 1977.

•f C. P. Gcor;<opoulos, Proc. nat. Aca<l. Sci. USA 68, 2977 (1971).

5 F. Keppel, C. P. Geoq-:oponlo,; and H. Eisen, Biochirnic 56, 1503 (1974).

6 C. l'. Gcoq;opoulos, R. VI. Hendrix, S. R. Casjcns and A. D.

Kaiser, J. molec. Biol. 76, 45 (1973).

.i

i

I

I

·I _:

•'

_!

·I

·!

_!

-,(

, .

_ ....,.,:;.,'_.,r ... ;~',._._ ;.,. r ..:..L•U:::.r.:..'!;; .. ,;_.,_...,. ___ J. ,:..; ~-.. ~ •• O~~-,J.. ~ -~··1-... ~ ·, .... -·31,-~.:!.;.•.:,,.."K!H~·J,_-... _

1158

^Specialia EXl' l"<!E>< 1 IA 3j .,

Table 2

Phage Yield of phage Yield of Wt

in com- ph age in

plemcnta tion test .. recombination test•••

T4 No. 4 5 x lO'

T4No.20 6x10'

T4 am H626* 4 X 10⁶

T4 am H28* 2xl0⁶

T4 No. 4+T4 am H626 4 x 10' T4 No. 4+T4 am H28 5 x 10⁹ T4 No. 4+T4 No. 20 3 x 10⁹ T4 No.20+T4 am H626 2 x 10⁹ T4 No.20+T4 am H28 7 x 10' T4 am

H626+ T4 am H28 9 x 10⁹

l.2 2.8 3.4 2.5 0.005 4.1

•am H626 is located in gene 8; am H28 is located in gene 53; **the crosses were performed on the non-permissive strain 13 sup-. The progeny phage yield was assayed on the permissive strain K12 sup•;

•••the crosses were performed 011 the permissive strain K12 sup+.

Total phage progeny was assayed or K12 sup+ and wild type recom- binant phage on B sup-. The value given is the percentage of wild type phage present in the burst.

7 Y. Kikuchi and J. ^King,

J.

molec. Biol. 99, 645 (1975).

8 T. Takano and T. Kakefuda, Nature New Biol. 239, 34 (1972).

9 C. P. Georgopoulos, R. W. Hendrix, A. D. Kaiser and W. B.

Wood, Nature New Biol. 239, 38 {1972).

10 A. Coppo, A. Manzi, J. F. Pulitzer and H. Takahashi, J. ^molcc.

Biol. 76, 61 (1973).

11

J.

F. Pulitzer and M. Yanagida, Virology 45, 539 (1971).

12 H. R. Revel, R. Herrmann and R.

J.

Bishop, Virology 72, 255 {1976).

closely linked lo amT·MG2 (0.005% rcco111bina tion) :>.!: ' th at the T4 No. 20 mntaticm is very clniwly linhc! _1"

::un1-J28 (0.005% recombina tion). The 8 anc\ 53 gene pr ...

ducts of phage T4 arc known to be components "f . · . outer wedges of the bacteriophage basepla te structur· .. ·_-,-

\Ve have considered 2 types of explanation to acconnt [ .. ; the existence of the T4 No . 't and T4 No. 20 mutati;,_{11 ,} The first type suggests th<tt i.he structural gene prrJdU( : , of genes 8 and 53, induced by T4 No. 4 a nd T·I _::-.;," ^~, respectively, arc different. in the 2 hosts. T his could i,,.

due to differences in the trauslalional appar;i tus of h: ₁ ^~ and B cells as, for example, in the specificity oi t l: .- tRNA. The second type of explanation derives r"rom nn-- vious observations of host involvement in T4 pl·;a~ ..

morphogenesis 8 - 10, and in T4 tail fibre function 11, " · 1;

suggests that the 8 and 53 a ltered gene products are ai.;, to interact effectively with a Kl 2 host component ;11 .

valved in baseplate assembly, but cannot d o so with t h : corresponding B component. The elegant electron mien,.

graphs of Simon ^13, which show that T4 base.plate form;,.

tion takes place at or near the bacterial m embrane, suti·

port such an explanation. It is interesting to note ^th~1t the T4 mutant HL626, originally thought t o be an amLv:r mutation in gene 60, has b een shown ¹⁴ to be analoguus ^l11 T4 No. 4 and T4 No. 20 in that it grows on all l\12 ~train>;

tested but not on all B sup- or B sup+ strains tested . Th"

gene 60 product is thought to associate with the bacteri;d membrane to promote proper phage DNA replicatic)n 1;.

13 L. D. Simon, Virology 38, 285 (1969).

14 C. D. Yegian, M. Mueller, G. Selzer, V. Russo and F. St;d;J, Virology 46, 900 (1971).

15 S. Mufti and H. Bernstein,

J.

Virol. 14, 860 {1974).

Heterogeneity of HeLa cell DNA as evidenced by CsCl density gradient centrifugation

M. Durante

1.

Anna Covi and L. Barsanti

Centro Nucleare CAMEN, S. Piero a Grado, I-56100 Pisa (Italy), 3 March 1977

Summary. HeLa cell DNA was analyzcd through CsCl density gradient centrifugation and thermal denatur.1ti•>11.

Neutral centrifugation without ionic treatment allowed the ,resolution o! a main peak with dens ity 1.699 g/ml and •if .l satellites DNAs with densities 1.683, 1. 710 and 1. 720. First d erivatives of m elting curves showed the. presence o i ·I I J::\ .\

families, whose G+C content calculated from Tm values oorrespo.ndc<l alm<1st exactly to the G + C content expC'Ct-·¹! from the previously densities. The extraction method seems parlicular.ly suitable for quantitative separation of 11:-..'.\

classes.

The existence of the heterogeneity of human DNA was demonstrated by Cornea et al.^2-', who showed the pres- ence of satellite DNAs after fractionation in Ag+-Cs2S04

density gradients. In order to obtain quantitative yields of all human DNA components, a method has been developed whose application in plant DNA extraction has showed remarkable results 5 • 6• This paper presents the results obtained in the resolution and fractionation of HeLa DNA components using neutral CsCl density gradients.

Materials and methods. DNA was obtained from whole cells suspended in a solution containing 5 · 10-² M Tris buffer, pH 7.8, 8 · 10-² M EDTA, 4% Na-dodecylsarcosi- nate and lysed by gentle stirring for 1 hat room tempera- ture. NaCl was added to a 2 M final concentration and the solution was stirred for 1-2 h. After dialysis at 4 °C against 1

x

SSC (0.15 M NaCl and 0.015 M trisodium citrate). RNase (100 µg/ml preheated at 90°C for 10 min)

and ix-amylase (150 µg/ml) were added directly in th e·

dialysis tube. After incubation at 37 °C for 1 h, pr(lll:t~<·

(500 µg/ml, self digested for 2 h a.t 37 °C) was adlkd :1nc!

allowed to act for 2 h at 37 °C. The solution was fr,_.::

centrifuged at 18,000 rpm for 15 min at 4°C : to the s11p«r · natant CsCl was added to a refractive index of 1.4H'"'·

The CsCl solution containing the DNA was ccntri!U.!"'' to equilibrium in

a.

30 rotor of a, 5pinco L 2-65

n

ll l1r .. · centrifuge at 20°C and 25,000 rpm or in a 40 rot .. r ,1t

2 3 4 5 6

Present address: Istituto di Gcnetica dell'Unive"ita, \ 'ia \I.,'.·

!c(ltli l/A, 56100 Pisa, ltalv.

G. Cornco , E. Ginelli and I·:: Polli, J. molec. Biol. 2J, 619 'I ' <•~

G. Cornco, E . Ginelli a.l\d E. Polli,

J.

molec. Biol. 33, 331 1 I'-·"

G. Corn«o, E. Ginclliand E. Polli,

J.

molec. Biol. ./8, 319 !i'•:•'

J. Grisnrd and E. Guillc, Prep. Diochem. 3, 83 (J 973).

R. Parenti, E . Guille, ] . Gris vard, ~!. Durante, \!. Bui.<t:i ,,,,-1 L. Giori;i, Nature New Biol. 246, 237 (1973) .

( 1

I

15. 9. 1977 Specialia

1159

JJ,000 rpm alternatively. Gradients were fractionated under continuous scanning at 257 nm in a LKB Uvi- cord II.

Fractions containing DNA were extensively dialyzed

;igai nst 1

x

SSC in Ultrahiilsen UH 100 membranes I ;chleicher-Schiill) and further characterized. Analytical

; 11tracentrifogation was performetl according to Schild- kraut et al.⁷ in a Spinco E ultracentrifuge for 20 h at

H,770 rpm at 25°C. The DNA amounts per analytical cdl ranged from 3 to 5 µg; DNA from Bacillus su bblis

Denaturation derived cnrve Peak No. Tm (⁰C) Percent

I II

!II JV

82.5 85.8 90.8 95

(G+Cl

31.18 39.23 51.42 61.67

•Relative importance.

b

Bottom Top

Analytical ultracentrifugalion Density Percent RI%*

(g/ml) (G+C)

1.683-4 l.699 1.710 1.720

25.2 39.4 51.02 61.22

~---Density

10 76 12 2

Fig. 1. Preparative (a) and analytical (b) CsCI density gradients of whole HeLa cells DNA.

II I

I::

_'' ~

+/ _'

f ^' ' ¹⁰

•• ^{I :}

^'

!100

1/

~ r' _x11-

E ₇₅ ^{I I} Ill

., ""

0 ^I

l

.. _'

z: ^I

u

..

^{50 I}

.

' ' ^{' ~./} ., ^{I, \}

..

_a.

^,, _,,

_\

> I,' ^I

x 25 _' ' ~

... !

IV

' . .

^,

.

^'

^..

80 85 D 0 9 5

Temperature •c

l'iJl. 2. Ther111al dcnaturatiou profile of 1-IcLa cells DNA in 1 X SSC ( ·) and d ri,·alive 111clti11;.: Clll'\'C (--·-) obtained by graphical <li£- for~1 .tiati11n u~i ng- 1 dcgr<:>c in tervals.

phage

tfic

(buoyant density 1.742 g/ml) was added as a standard. UV photographs were taken and photoplatcs :xarnincd i11

a

joyce-J.ocbl recording microdensitometer.

DN J\ molecular weight was determined according to Eigner and Uotys.

Thermal denaturation was carried out in a UNICAM SP 800 spectrophotometer equipped with a SP ⁸⁷⁶ tem- perature conlro1 program mer. Temp raturc increase was 0.5 ° ·/min <ind melting curves were recorded directly by a Phllips , ' -Y recorder PM 8120.

Results and discussion. The estimate of the mol.wt of our D A . amplcs gives va lues greater than 10¹ daltons.

Curves of NA distribution arc given in iigure l. D~ A satellite components witl1 a buoyant density of 1.683 and 1. 710 are present in all prepara ions, while t he heavy satellite (a 1. 720) is not a lways pre ent. ·n1e light satellite cle<irly accounts for more than_ 1

%

a previously reported ^{2 •} It is wor I nocing t hat its ab orbance spectru m is shifted towards higher wavelengths with a maximum at 270 nm.

Such a property could be tentatively attributed to the presence of complexes with divalent ions ^{9 •} On the other hand, Cornco et

ru.

⁴ observed hat repetitive human satellite D ¹As d1splny peculiar properties related to ano- maJQuS binding witl1 Ag + a nd Hg++ ions. More recently Si<>soef( et a.1. 10 made evident the prcsenc-e of divalent ions complexed with satellite DNAs extracted from several sources. This heterogeneity of HeLa cell DNA was con- firmed from denaturation profiles (figure 2). The Lit (or width of thermal transition) is 7.55 °C and the thermal denaturation curve is polyphasic. First derivatives of rnclti11g curves show the presence of 4 major DNA families (solid arrows) whereas other 2 (dashed arrows) are not clearly d istingu ishnble.

In order to establish a possible correlation between the DNA denaturation classes and the density gradient com- ponents, we report in lh table the G+C percent calcu- lated both from the Tm of each family ¹¹ and from the buoyant dcnsilies^{7 •} The relative importance or percent of the DN components were calculated by determination of area of the separate components from the microden. ito- meter tracing. A clear correspondence was found between derivative classes and CsCl gradient fractions, with the exception of family 1: on the other hand, if the assump- tion of a cation-light satellite complex. is true, the high Tm value could be attributed to ^<Ul increased thermal stability of the complex.

7 C. L. Schildkraut, J. Marmur and P. Doty,

J.

molec. Biol. 4, 430 (1962).

8 j. Eigner :md P. Do ty , J. 111iil c. 13101. 12, 5·19 (1965).

9 T. Ya11rn11 and N. Duvi<ls1111,

J.

Am. chem. Soc. 76, 2599 (1961).

10 I. Siss cff, J. C1·isv11r<l and H .• 11ill6, C. r. Acad. Sci. Paris 280, 2389 (J 97.5).

11 C. L. Schildkrallt and S. Lifson, Biopolymcrs .J, 195 (1965).

'

a_

"

n

..

/

I

,:

· ·~ ·~ _ _ ... ., .... ,_;j,R;,,,'-~ •.. :.::.-_;.i,o<l·~ii~~ ... i .._~.,..._; ... ,~;·~.~~;:b..,..,. -~~,-~,;._.::

1160

^Spccialia EXl'EJU!C>;TJA 3l ,

Comparative studies on the trichomonacidal activity of 5-nitroimidazole-derivatives

in mice infected s.c. or intravaginally with T. vaginalis¹

]. G. Meingassner

Sandoz Forsch1mgsinstitut, Bnmne1's/rasse 59, A-7235 Wien (Austria), 23 February 1977,

51,mmary. The trich<imunacidal c!Cicacy of mcf:ronidal.l>lc, tini<lazolc , nimnra r.ole and (ll"nidazolc w as slucl ied in rn 1, ,_

infected s.c., or for comparison p11rpo!>cs intravaginally. ln both tci;t systcm.s, lhc drugs rovenlcd nearly Lhc.: snm<: <.rdcr of of(icacy, whereby \"he compound:; showcJ a marked ^dccn~ase of activity when analyl.Cd in s.c. infected mice.

In a previous paper it was shown that ovariectomizecl and estrogcn- prnmedicatcd rats could be infected with Tri- chomonas vaginalis iutravaginally with more reliability when the animals were infected first wit h Candida. albi- cans topically2 • This double infection, which, with certain modifications, was found to be applicable to mice also, turned out to be a further useful model for chemothera- peutic tests.

The efficacy of metronidazolc, tinidazole, nimorazole and ornidazole was studied in the established model: mouse - T . vaginalis/s.c. infeclion :~nd for comparison purposes in this new testing techn krue described in detail subse- quently.

Methods. 1. S.c. iniection. Fema le NMHl mice weighing 10-13 g were s.c. i11fec1"ed with 4· X 10 ⁵ T. vaginal is L1 2, in each shaved :flank. The trichom.on ads from an over-night culture in CACH-mcdim11³ were concentrated by cc.ntri- fugation and resuspendcd. iu 0.15- 0_2 ml supernatant. per inoculum. For eacl1 experiment, samples or the ax cn ic stock culture (which was transferred 23 times after iso- lation before storage in liquid nitrogen) were thawed aud passaged 4 times before infection. Systemic treatment was started orally 2 h after the infection and was repeated at 18 and 24 h p.i. The experiments were terminated 6 days after infection. The absence of lesions and micro- scopically detectable motile trichomonacls were used as the criteria by which the activity was measured. The compounds were dissolved in 10% DMS0/0.2% CMC stock solutions followed by a further Zfolcl dilution with 0.2% CMC solution only. The drugs were given in volumes of 0.1 ml per 10 g b.wt to give concentrations of 50- 0.4 mg/kg.

2. Intravaginal infection. NMRI mice 25- 30 g in weight were pretreated once with 40 mg/kg estradiolundecylatc (Progynon Depot®, Schering AG) in 0.2 ml sesame oil 3 days before infection. The estrogen was administered in two equal doses s.c. a nd i.p. The inoculnm per mouse con- sisted of 0.05 ml GACH-medium contai.ning approxi- mately 1x10⁵ T. vaginalis I.I 2 which was grown as out-

Efficacy of several 5-nitroimidazole-derivativcs against topical and ectopical infections with T . vaginalis in mice

Substance

Metronidazo\e Tinidazole Nimorazole Ornidazole

Dosage (mg/kgx 3•)

ED60•

ED60

ED60

ED60

Model: T. vagii1alis inoculation site intra vaginally• s.c. c

3.71 (2.76- 4.99) 10.95 (9.10-13.18) 1.41 (1.02-1.95) 7.50 (6.19-9.10) 5.62 (4.15-7.61) 33.95 (30.62-37.73) 4.57 (3.G0-:-5.82 10.59 (9.07-12.37)

•2, 18 and 24 h p.i. orally. •3 x9 animals at each dosage level, mean infection rate of untreated control groups 94%. '3 X (j animals at each dosage level, infection rate of untreated control gr0l1ps 100% . •ED,0

according to Spearman-Kurbcr : figures in brackcls are 95 % con- fidence limits.

lined above, a nd Ca11di<la <i lbicans. 1000 JU Na-pcntt!lli:, G and. J OOO µg slrnptomyc in sulfalc per ml inoculum "' ., ..

also added. Candida a lbicans .d l H was grown in :--\H.

broth for 24 h at 30 "C rtnd stored with 5% J):\fS(l in Ii q u id nitr<>{{e n~. (The conccnl'ratio11 o-f

C.

al bicans i₁₁

t'.•

SA.B-culture ,,,as about 3.6x10⁷/ ml.) T he tlm\\ ed w."i- werc acl<lcd to lhe concenl-ralcd flugellates in 11 r~11^,. ,.1

1 :80 v/v inm1ccliatcl)' before the infect ion o r lltc anii11;\i.

T he ''agina l cavily was stuffed with a plug of 'JI' •m.:•

(Spongosl.a n ®, l?errosan) to avoid loss of the i1wc11 l11rn The therapeutic method and the preparation of the c• ,rn . pounds were the same as used in s.c. infected mice. TL.- estimation of vaginal infection was based on c11it111.ti findings. The vagina of each infected animal was ri11 ,,•·d 4 clays p.i. with culture medium which was transferrc·d '"

tubes containing CA CH-medium and a final concL·n 1 r.;.

tion of 1000 IU Na-peniciJlin G, 1000 µg/ml strcptu1ll\ cm snlfate and 10 IU nyslatin/ml. The seeded tnbcs wL·r •: 111 .

cubatcd at 37 °C and after 48 h observed for motile: t ri

chomonads ou an inverted microscope. The efficacy "f t h1·

compounds was determined by the presencc/absenc•: •.J hichomona<.1::: in the tubes.

Results and discussion. The results of the compar;1 t "'"

chemotherapeutic studies are summarized in the L.i>ll".

The data presented indicate that all compouncls tl'>t<-d are systemically active against T. vaginalis in mice· ;n - fected intravaginally. By comparison the cc•mpound, showed a marked decrease in efficacy when analyzL"d 11:

s.c. infected mice, although the evaluation was based ""

native observations only and not on cultural find1'."'' which arc more reliable. ln both test systems, the tin;~,

revealed n early the same order of efficacy.

The compounds tested are registered drugs for the t r•·.<l · ment of trichomoniasis in man. Therefore an activ!t\' "' mouse models, which are comn10nly used for scrc·•·!0< 11;•

programmes, was expected. The different degrees ol •.- 1!:·

cacy in both models may be explained by the d1fft r .. :;:

locations of the trichomonads in the animal host as ··1 ·• ;;

as by the altered physiological conditions of t!1c l'S\r. c: •-r\

treatment. The effect of mixed T. vaginalis/Cancllc!:. ::;

fections on their drug sensitivity requires further st1:•'.:.-- However, it has been observed that the degree of di !•: ".·' of nimorazole differs far less from that of mctn,nid • · :, and ornidazole in intravaginally infected mice th.\r> ''' ectopically infected animals; the activity of nin11•r:_1:

.:c

ascertained in ectopicallv infected animals is only :i :· ·::

1/ 3 of that of metronida~ole, which is in agreement "' ,·.!.

I am grateful to Dr H. Mieth for helpful discussion; an•! i. ' ' "

criticism of the manuscript and Miss B. Ruschizka ·"'d \'' ;_, Wasserma nn for technical assistance.

2 ]. G. Mcingassncr, A. Georgopoulos and M. Patoschka, Tr·- ;~:·.r mcd. Parasit. 26, 395 (1975).

J

·w.

A. Miillrr and C. Gottschalk, Angew. Parnsit. 11 , \ /0 : l '·

4 A. Georgopoulos, Mykosen, in press.

1'~.x., __ £4(,t•J:! -J: ZCICJJUiZ :w;a>AL!I • :tw«ll...-. - - - - - ... ;;:;:_-'\"(;':'"i:'""

I _I

I

I

I

'

-

1 I l

< ' · 9. 1977 Spccialia

1161

. , . results of ·ameri et al. ^5, a fact which was a Ltrilrntccl . ·: :htJ ph.annacoki11ctical pe.cul iarilics of the dru •s in mice ... i rais. The aulh rs reportc I t hat the acti,·e -frnction o{

~·:. · rnnidazo le in mouse urine was more than twice that

··; : .. rnorazl.lle, whereas in rats and man the opp site is : .: . .'.: Th~"· thus considered iha.l the m1Jusc was nut µa rti-

· ~:: ·. ~h· s1;itablc for assessi ng the thcrnpeu ic properties of

·. '.: ... r ",₁z >le in human infections. This suppnsilion to some

~ ".:.":. :::II contr;idicls tlw present as well as previous results

6,

... ,,·.:· the compounds show similar activity to mctronida-

.>.-

ⁱⁿ rats as well as in mice, if they are infected intra-

: • • \~1^{:~ ally.} The degree of systemic efficacy of nimorazole

,., the experimental animal docs not correbte exactly

. _., ;th the activity in the urine.

Tinichwle was the most effective drug in both models a,nd

·howctl a clear superiority in th new one. These results con:ela tc closely wit! the dosage of tinidazole used in practice, which arc I wer tha.n those of the other drugs⁷ recomllJendcd f r a 7-uay·courne treatment of tricho- monim;i!i. Th se Findings underline the suitability of the intravaginal test systems.

5 l. de C:trneri, A. Cantone, A. Eman uc.li, P. N. Giraldi, W. Logc-

11i:u111, L. Longo, G. i\!ci11ardi, G. ^~lonti, G. Nannini, G. Tosolini an.<t G. \7i ta, Progr. Antimicrob. Anticanc. Chemolher. 1, 149

(l!l7Q) .

6 J. G. Meiugassner and H. Mieth, Arzneimittel-Forsch. (Drug Res.) 27, 638 (1977) .

7 Dosage specification as given by the manufacturers.

Effects of 5-bromodeoxyuridine and 2-aminopurine on antheridium differentiation

j n

Anemia phyllitidis L.

J ! . Schraudolf

. ! i.t. A l/ar.meine Botanih (Biol. I I) der Universiti.it Ulm, Obei-er Eselsberg, D-7900 Ulm (Federal Republic of Germany),

·"' .f 11 1111(/Y')' 1977

' :1m111(1ry. Both 5-BUdR ancl '2-Ap cause AG{fC transition dnring DN replication or transcript ion. However their . i1,•ct on differentiation of n.n thcridia in the fern Anemia. phyllitidis differ totally. Since 5-BdU causes pattern simpli-

.. . 11 ion, __ \p leads to suppression of correlative cell in tcrnctions. This results indicate different targets for this muta-

·~· nic compound in J\ncmia-Di: A.

! ;ibbcrellins substitute for the native nnth,eridiogens of ::1e'fern Anemia phyllitidis ^{1• ~·.} Addition of lhese phyto- i·.• ·rmones to culture media causes lhe premature diffcrcn- lidtion of male sexual organ (antheridia)3. AILhough the :1urmonal induction of cell d ifCcrcn t iation is not blocked hr inhibitors u( prote in a nd 11ucleic acid synthesis, 5- 1•1•11ml)deoxynridine (I3UdR} has a shiking inf!nencc on t hl· ma nifasta t ion oi th e a n therid ial pattern ^{4 •} lclentica.l

.. 1fccts a r obtained with 5-iodocleoxyuridine and 5- iiromodcoxycyti line. Addition of these a na logue to the

111l11rc medium (2

x

IQ - G M- 5 x 10 M) gives rise to the :ur n1:1ticm of si mplified se:•oi aJ orgarts, \.Vitti bigl1 inhibitor

• :unccntrations, or after long limes o[ applicntion, the .. ntheridia finally resemble vegetative chloronemata.

fl'i"ure 1 a , b). As in various animal cells^{5 ,} the inhibition

b

of cell differentiation precedes the inhibiting effect on rate of cell division.

The presence of BUdR in total DNA of prothallia of Anemia phyllitidis fed with UC-labelled BUdR was proved by Koop6 • Since these effects of BUdR on anther- 1 :\ly thank nrc due to Miss C. Stich! for tt::chnica.l assistance

nnd Dr P. i\l:icnico.l lor checking the English version of the manu- script. S11pportcd br the Deutsche Forschu11gsi;emeinschaft.

2 H. SchraucloH, Ulol. Zcntr. 8 1, 731 (1962).

3 11. '•:hraud If, Plant Cell. Physiol. 7, 277 (1966).

4 J L S ·hr:wrlolf, l)kmt:i 68, 335 (1966) . 5 H. S hra11dolf, Plan ta 74, 1'?3 (1967) .

6 W. j. Hutter, R L. Plctcd, P. W. J\lorri.s, Anu . Rev. Biochem.' 42, 60 1 (1973).

Fi;:-. I. Effect or n d R on ;ui-

lh ridium dlffcrcntialion of light

{{1'0W11 proth;1llia of Ancmia phyl-

lllidis. n) Control; ^{10 - ~} r:{rnl Gibb

A~; h) z x 10-~ M n llR; io- s gf

1111 G ibb A, . 10 d; ?.Q °C; con linu- uw; ligh t (1,2 x 10³ erg cm- 2 sec-L),

....