38

Specialia

water-soluble c o m p o u n d s were dissolved in water, insoluble compounds were formulated as w e t t a b l e prowders. The concentrations used were 4000 and 1000 p p m of the active ingredients. Of the 7 compounds tested, 4-allyl-4-(3, 7-dimethyloctyl) morpholinium bromide and 1-allyl-l-(3,7-dimetbyl-2,6octadienyl)piperidinium bromide d e m o n s t r a t e d little or no growth r e t a r d a n t effect (Table I), whereas 1-allyl-1(3, 7-dimethyloctyl)piperidillium bromide, the corresponding chloride, and 1-propyl-l-(3, 7-dimethyloctyl)piperidinium iodide showed excellent g r o w t h r e t a r d a n t activities. T h e 1-allyl-1-(3, 7-dimethylnonyl)piperidinium bromide and t h e 1-ethyl-l-(3, 7-dimethyloctyl)piperidinium b r o m i d e were slightly less active .The growth r e t a r d a t i o n effect could be antagonized b y indole-3-acetic acid and gibberellic acid. As t h e 1-allyl-l-(3,7-dimethyloctyl)piperidinium bromide (ISO a p p r o v e d c o m m o n n a m e : p i p r o c t a n y l i u m bromide) was t h e m o s t active of the 7 compounds, its a c t i v i t y was further investigated in the greenhouse on 6 additional species : Vitis vini/era L. cv. Riesling • Sylv'aner, Euphorbia pulcherrima Wild. cv. P a u l Mikkelsen, Pachistachys lutea Nes. and Brassica napus L. cv. Rapol. Heights were recorded 4 weeks after t r e a t m e n t , e x c e p t with Euphorbia pulcherrima where the assessment was carried out 10 weeks after application. The effect on fruit ripening was tested on Lycopersicon escutentum cv. T i n y Tim. The plants were sprayed w h e n the first fruits t u r n e d red. The

EXPERIENTIA32/1

n u m b e r of ripe fruits was counted 3 weeks later. T h e latex-flow stimulation a c t i v i t y was investigated on Ficus elastica Roxb. The wide range of p l a n t g r o w t h r e g u l a t o r y a c t i v i t y of p i p r o c t a n y l i u m b r o m i d e is d e m o n s t r a t e d in Table If. G r o w t h r e t a r d a n t effects could be observed on Vitis vini/era, Euphorbia pulcherrima, Pachistachys lutea and Brassica napus. The c o m p o u n d accelerated fruit ripening on Lycopersicon esculentum and s t i m u l a t e d latex-flow on Ficus elastica. B o t h these effects were r e p o r t e d as being associated w i t h the p l a n t h o r m o n e e t h y l e n e b y WANG et a l ) . Of the q u a t e r n a r y a m m o n i u m derivatives which h a v e been synthesized and tested in our laboratories, a n u m b e r of c o m p o u n d s h a v e shown interesting p l a n t growth responses, b u t so far 1-allyl-l-(3,7-dimethyloctyl)piperidillium bromide is the m o s t promising. Therefore large scale greenhouse trials were initiated in commercial nurseries, especially w i t h pot varieties of Chrysanthemum mori/olium. The results o b t a i n e d w i t h a single application of 100-200 p p m c o m p a r e d f a v o u r a b l y w i t h those obtained from 2-3 applications of 3400 p p m of daminozide. There fore p i p r o c t a n y l i u m bromide will shortly be m a d e available, as A L D E N , for this use. Other uses, based on the results presented above, are currently under investigation. 5 C. Y. WANG, W. )~[. MELLI~NTHINand E. HANSEN, J. Proc. Am. hort. Soc. 97, 9 (1972).

Adenovirus Type 12 Infection of Defined Mouse-Human Hybrid Cell Clones K. K. BIRON and K. RASKA jU~r

Department o/ Microbiology, College o/ Medicine and Dentistry o/ New Jersey, Rutgers Medical School, Piscataway (New Jersey 0885d, USA), 15 August 7975. Summary. H u m a n adenovirus t y p e 12 does not m u l t i p l y in mouse cells; o n l y v i r a l T - a n t i g e n is detected. Mouseh u m a n cell h y b r i d clones containing h u m a n chromosomes A3, ]35, C7, C l l , C12, ])14, E17, F19 and F20, s u p p o r t synthesis of adenovirus ] ) N A and capsid antigens. R e c e n t advances in m a m m a l i a n somatic cell hybridization h a v e allowed the analysis of t h e mechanisms of host cell restriction to viral infections. The infection of mouseh u m a n cell hybrids w i t h poliovirus has d e m o n s t r a t e d t h a t t h e permissiveness for virus infection can be associated w i t h the h u m a n c h r o m o s o m e F19 coding for a cell surface receptor 1. Oncogenic adenovirus t y p e 12 multiplies in h u m a n cells b u t not in h a m s t e r or mouse ceils. I n h a m s t e r cells, an a b o r t i v e cycle is induced. The infected ceils synthesize T - a n t i g e n 2 and adenovirus-specific m R N A is transcribed, b u t synthesis of viral DNA, late m R N A , or viral capsid proteins c a n n o t be detected 8. W h e n h e t e r o k a r y o cytes of h a m s t e r and h u m a n cells were infected w i t h Ad 12, t h e synthesis of Ad12 D N A and late viral capsid proteins was d e m o n s t r a t e d in nuclei of h a m s t e r origin t h a t h a d been nonpermissive prior to cell fusion 4. Mouse cells h a v e been reported to be t o t a l l y nonpermissive to adenovirus t y p e 12 infection and unable to support synthesis of either T - a n t i g e n or viral capsid proteins ~. W e decided to s t u d y t h e infection of specific clones of m o u s e - h u m a n cell hybrids to determine whether the presence of certain h u m a n chromosomes in hybrid cells would m a k e t h e m permissive for adenovirus replication. C o n t r a r y to previous reports 5 we observed t h a t w h e n mouse cells (3T3 or L) are infected w i t h adenovirus

t y p e 12, the adenovirus-specific T - a n t i g e n can be dem o n s t r a t e d in t h e n u c l e i of infected ceils (Figure 1). No virus-specific D N A and late viral capsid proteins were detected, b u t the infected cells were killed b y t h e infection (Figure 2). The fraction of cells killed was in good correlation w i t h t h e f r e q u e n c y of T - a n t i g e n positive cells. As in t h e h a m s t e r cell system ~, w h e n s t a t i o n a r y 3T3 cells were infected, adenovirus t y p e 12 induced a round of cellular D N A synthesis (Table I). H y b r i d cells were m a d e b y h y b r i d i z a t i o n of C I - I D mouse cells deficient in t h y m i d i n e kinase to either t~OP-2 h u m a n fibroblasts 6 or a line of SV40-transformed h u m a n ceils derived from patients w i t h t h e L e s c h - N y h a n s y n d r o m e 7. Mass cultures of h y b r i d cells were cloned on

1 H. GREEN,New England J. Med. 290, 1018 (1974). W. A, STROHL, Virology 39, 642 (1969). a K. RASKA,JUN., and W. A. STROHL,Virology 47, 734 (1972). J. WEBER and S. MAK, Expl Cell Res. 74, 423 (1972). R. POLLACK,J. SALAS,R. WANG, T. KUSANOand H. GREE~, J. Cell Physiol. 77, 117 (1971). 6 F. RlCClOTIand F. H. RUDDL%Nature New Biol. 2d7, 180 (1973). C. I~r CROC~,A. J. GI•ARDI and H. KOP~OWSKr,Proc. natn. Aead. Set., USA 70, 3617 (1973).

15. 1. 1976

39

Specialia

Fig. 1. Adl2 T-antigen in mouse cells. Coverslips were fixed at 32 h after infection with 100 PFU/cell of Ad12 and stained by the indirect immunofluorescence procedure with serum from A d l 2 tumorbearing hamsters followed by fluoreseein-labeled rabbit anti-hamster globulin S. a) Infected 3T3 ceils; b) infected L cells.

Table I. Induction of DNA synthesis in stationary 3T3 ceils by adenovirus type 12 ir~fection

100

90

80

A A

~g o" oo frO

70

>

er"

60

oO

50

A INFECTED

40

IC

I

I0

I

I

30

I

I

50

l

I

70

I

I

90

TIME AFTER INFECTION (HOURS) Fig. 2. Killing of mouse cells by Adl2. The cells were infected wittl 100 PFU/eell of Adl2 and at times indicated the viability of cells was tested by trypan blue exclusion test. T-antigen was induced in 60 % of infected cells.

Time (h after infection)

Incorporation of ~H-thymidine/culture (cpm) Controls Infected

4 12 15 18 21 24 27 30 33

406 310 342 344 392 468 452 297 283

280 234 4,289 4,760 3,906 3,339 2,321 1,332 607

~Confluent cultures of 3T3 cells (6 • 105 cells/dish) were switched to medium supplemented with fetal calf serum reduced to 0.5%. 48 h later the cells were infected with Adl2 at input m.o.i, of 100 PFU/eell or mock-infected (controls). At times indicated the cultures were pulse-labeled with aH-thymidine (1 ~Ci/ml) for 60 min and then harvested, solubilized with SDS (0.5%), and precipitated with icecold triehloroacetic acid. Acid precipitable material was collected on Millipore filters, and radioactivity was determined in liquid scintillation spectrometer. Data represent the averages of 2 cultures. bHybridization of 120,000 c p m of D N A isolated from cells labeled from 18 to 22 h afterinfection to filterscontaining i0 ~g of Adl2 D N A failed to detect any radioactivity remaining on filters over that observed with i00,000 c p m of D N A isolated from rapidly growing uninfected cells; the host origin of the D N A is thus apparent.

40

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EXPERIENTIA 32[1

Table II. Infection of mouse cells and clones of mouse-human cell hybrids with adenovirus type 12 9 Cell clone

Human chromosomes present

Frequency of Adl2 DNA synthesis ~ T-antigen positive cells a Input DNA Fraction in (%) hybrid (cpm) (%)

Frequency of V-antigen positive cells f (%)

CI-1 D Hybrid C1 8 b Hybrid C1 84 c Hybr id C1 82 Hybrid Ct 15 ]3UDR

None B5, C6, C7, C l l , C12, D14, El7, F19, F20 A3, B5, C7, C l l , C12, El7, F19, F20 A3, B5, C7, C l l , C12, D14, It17, F19, F20, G22

65-82 67-80 65-80 84-92

88,141 13,869 78,826 12,377