- Email: [email protected]
Effect of mercury-containing preservatives on agar gel diffusion precipitin reactions with several plant viruses
376
SHORT
COMMUNICATIONS
6. BAUM, S. G., REICH, P. R., HYBNER, C. R., ROWE, W. P., and WEISSMAN, S. M., Proc. Natl. Acad. Rci. U.S. 56, 1509-1515 (1966). 6. HENRY, P. H., BLACK, P. H., LEVIN, M. J., and WEISSMAN, S. M. In preparation. 7. REICH, P. R.,BLAcK, P. H., and WEISSMAN, S. M., Proc. ,vatl. Acad. Sci. U.S. 56, 78--85 (1966). 8. LACY, SR. S., and GREEN, M., J. Gen. Viral. 1, 413318 (1967). STEPHEN G. BACM WILLIAM H. WIESE Laboratory of Viral Diseases National Institute of Allergy and Infectious Diseases Bethesda,
Maryland
20014
PAUL R. REICH Metabolism Branch 1Vational Cancer Institute Bethesda, Maryland ZOO14 Accepted January 2, 1968
Effect
of
Mercury-Containing
atives on Agar
Gel Diffusion
ci pi tin Reactions
PreservPre-
with Several
Plant Viruses The mercury-containing compounds Merthiolate (sodium ethyl mercury-thiosalicylate) and Cialit (sodium 2-ethyl mercury mercapto-benzoxazol-5-carbonate) are frequently used as preservatives in serological agar gel diffusion tests. Recently, adverse effects of Merthiolate in these tests were demonstrated with poliovirus (I), bean pod mottle virus (2), foot-and-mouth disease virus, and an antigen associated with foot-and-mouth disease virus infection (3). An inhibition of precipitin line formation was observed with the top component of bean pod mottle virus and also with the middle and bottom components after repeated freezing and thawing (2). Likewise, t,he precipitating activity of the antigen associated with virus infection in foot-andmouth disease was markedly reduced, whereas in the case of foot-and-mouth disease virus itself two precipitin lines developed instead of one. This was due to partial degradation of the 140 S virus to 12 S protein subunits. Sodium azide at a concentration of 0.5 % had no such effects (3).
In this laboratory, similar observations were made with several plant viruses which hithert,o had not been known t’o be affected by h4erthiolat’e or Cialit. The effect of the mercurials proved to be dependent upon the pH of the agar and upon certain additives to the virus preparations. In Table 1, the results of an experiment are shown in which the development of precipitin lines of several plant viruses with their respective antisera was studied in 0.85% Difco Special AgarNoble containing 0.85 % sodium chloride. The pH of the agar was adjusted to 6.0, 6.5, 7.0, 7.5, or 8.0 with phosphate buffers at a final concentration of 0.05 LV; 0.01% Cialit or Merthiolate or 0.5 % sodium azide was added as preservative. In cont,rol plates 110 preservative was added. The diameter of the reactant wells was 2 mm; the distance between the wells measured 3.5 mm. Crude sap of virus-infected plants or partially purified virus preparations which had been kept frozen for varying lengths of time were placed into the antigen wells. In some cases, 0.2 “/(I sodium sulfite and 0.2 % ascorbic acid were added to the preparations. Preliminary experimentas were performed in agar with no added preservative to find out which antisera dilutions yielded the sharpest precipitin lines wit#h their respective antigens. These dilut#ions were used in all plates regardless of whether preservatives were present or not. The format#ion of precipitin lines by several spherical plant viruses was prevented by the presence of Cialit or Mert,hiolate (Table 1). With some viruses this effect, was observed at all pH values tested (tomato ringspot virus and crude sap of Nicot&a tabacum I,. var. Samsun containing Belladonna mottle virus isolate 1). With other viruses, a distinct dependence of the effect upon the pH of t’he agar was observed (crude sap of Set&u italica Pal. Beauv. containing Dactylis virus II, purified preparations of fanleaf virus, tomat,o blackring virus, and St. 10, an unidentified grape vine virus). With increasing pH an increasing number of viruses was affected. The fact that t’he precipitation of some viruses by their respective antisera \vas impaired, whereas other syst’ems remained unaffected (carnation ringspot virus, beet
ON THE
+
+ -
-
+
-
+
+ -
-
TABLE
1
7.5
60
l(l)1
6.5
7.5
AGAR
GEL
1 1 111
1 1 111 1 11 1 1 1 1 1 1 1 11
8.0
11 1 (1) 1 (1) 1
11 11 11
11 11 11 11 11 11 11 11 11 11 11 11 11
7.0
0.5% Sodium Azide PH
IN THE
fanleaf virus, infected isolates 1 and 3.
Setaria
italica
0 1
6.5
2 2
2 2
otherwise.
0 0
0 0
0 0 111 2 3
2 2
2 2
0 0
0 0
0 0 0 0
1 0 0 0 0
1 0 1 0 1
0 0 1
7.5
0 0 1 1 111 1 1
7.0
1 1111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11111 1 0 0 1 1
1
0 1
6.0
II,
WITH
0 1 1 1 1 1 1 1 1 1 1 1 1
6.5
infected
0 0 1 2 3
0 1 1 1 1 1 0 0 0 0 1 0 0
7.0
0.01% Cialit PH
0 0 0 0 11 2 3 2 3
0 1 1 1 1 1 1 1 1 1 1 1 1
6.0
TEST”
virus
8.0
DIFFUSION
0.01% Merthiolate PH
DOUBLE
in the case of Dactylis
wells, 3.5 mm. of time where used unless stated
11 11 11 1 11
8.0
11111 11111 11111 11111 11111 reactant lengths 0.05 M.
7.0
11 11 11 11 11 11 11 11 11 11 11 11 11
6.5
11111 11111 11111 11111 11111 11111 11111 11111 11111 11111 11111 11111 11111
6.0
PH’
No preservative
NUMBER OF PRECIPITIN LINES SEVERAL PLANT VIRUSES
a Diameter of the reactant wells, 2 mm; distance between the b Purified preparations which had been kept frozen for varying c Adjusted with phosphate buffers to a final concentration of nodosa. d Unidentified ringspot virus from Scrophularia e Unidentified grape vine virus, / Crude sap from infected Chenopodium quinoa in the case of tiana labacum var. Samsun in the case of Belladonna mottle virus g (1) = Weak indication of a precipitin line.
Tomato ringspot virus Tomato blackring virus Scr Vd Carnation ringspot virus St. 1@ Beet ringspot virus Fanleaf virus Fanleaf virus Fanleaf virus! Fanleaf virusf Arabis mosaic virus Dactylis virus 11’ Dactylis virus II’ Belladonna mottle virus’ Isolate 1 Isolate 1 Isolate 3 Potato virus Xg Potato virus X@
PRESERVATIVES
Presence or absence of 0.2% ascorbic acid and 0.2% sodium sulfite
OF VARIOUS
Virusb
INFLUENCE
0 0 1 2 2
0 0 1 1 0 1 0 0 0 0 1 0 0
8.0
Nico-
0 0 1 2 2
0 0 1 1 0 1 0 0 0 0 1 0 0
7.5
378
SHORT
COMMUNICATIONS
ringspot’ virus, and an unidentified ringspot virus from Xaophularia nodosa I,.) suggest#edthat the action of the mercurials was on the virus rather than on the antibody. This view was strengthened by some observations on the cross reactions of isolates 1 and 3 of Belladonna mottle virus (4) with t,heir respective antisera. As seen in Table 1, the reaction of Belladonna mottle virus isolate 1 in crude sap of N. tabacunz var. Samsun with its homologous antiserum was strongly affected by mercurials, whereas t#he reaction of Belladonna mottle virus isolate 3 with its homologous antiserum appeared not to be affect,ed. The cross react’ion between isolate 1 wit’h the antiserum against’ isolat,e 3 was affected whereas the cross reaction of isolate 3 with the antiserum against, isolate 1 was not affected. Thus, the mercurials acted on the virus, not on t,he antibody. Wit,h t,he majority of the viruses affected, the inhibitory act,ion of Cialit became apparent at somewhat lovver pH values t,han t,hat#of Merthiolate. With the isolate St. 10, only Cialit was effect,ive and only at pH 7..5 and 8.0. In the case of Merthiolate the critical hydrogen ion concentrat#ion for the inhibitory action could be lowered by the addition of 0.2 %, ascorbic acid and 0.2 7, sodium sulfite. An apparent shift of sensitivit’y toward the more alkaline range could also be achieved by using larger reactant wells and shorter distances between them. Thus, with t,omato ringspot virus, despite the presence of mercurials, well-defined precipitin lines were observed at, pH 6.0 lvhen reactant wells with a diameter of 4 mm and a dist,ance of 2 mm were used. At pH 6.5, however, no lines formed in the presence of mercurials. A similar shift’ of the sensitivit! toward the more alkaline range was observed with crude sap of Xetaria italica in fected with Dactylis virus II. In these cases part of t#he virus probably inactivated the inhibit,or and the remainder reactled with t,he aruiserum. This concemration effect, probably also explains why purified preparations of Belladonna mottle virus 1 yielded intense precipitat,ion lines at neutral pH in the presence of mercurials. The pH-dependent effect of mercurials is responsible for the reported (5) failure t’o obtain precipitin lines with tomato ringspot, virus and
fanleaf virus at higher pH values, when wells with a diameter of 4 mm were used at a distance of 2 mm. With potfat’ virus X a different effect of mercurials was observed. In addition t,o a sharp line close to the antigen well, one or two intense but more or less diffuse lines were observed nearer t’o the antiserum well. A photograph, where this diffuse line is also visible, has been published by van Regenmort,el (6). Occasionally, a weak indicat’ion of t,his line was also observed in the presence of 0.5 ‘% sodium azide, but never in the absence of a preservative. With disease virus (J), i.e., the virus becomes partially degraded. On t,he ot’her hand, it cannot! be excluded that, under the influence of mercurials some impurit#y is released from the surface of the virus. This is not ver\ likely, however, since many different. purification methods were tried without success in efforts to obtain virus preparations that would not yield the diffuse line under t,he influence of mercurials. It is of imerest that only part’ of our ant,isera against potato virus X gave rise to t’he format’ion of the diffuse line or lines. The abilit,y to form diffuse lines developed during prolonged immunization (Y’), but, the tit,er against t#he possible breakdown product, never exceeded 1: 16, lvhereas t’he titer against potato virus X ranged between 1: 512 and 1:4096. Individual differences occurred between the ant isera from different rabbits, and no strict correlation between the two t,it’ers w-as found. This could indicate that the subunit of potat,o virus X is antigenically different from t,hc intact virus. I>uring prolonged immunization some virus may be degraded and thus give rise t80 antibodies which are able to react with the subunits or with other breakdown products. It has been reported (8) that antisera against the intact turnip yellow mosaic virus are unable to react with the protein subunits and vice versa. This might, explain the failure to obtain precipitin lines with
SHORT COMMUNICATIONS several spherical viruses under the influence of mercurials. Further investigations are necessary to determine whether the mercurials exert their effect in all cases by breaking up the quaternary structure (3, 9, IO), or whether in some cases a direct interaction with sulfhydryl-containing antigenic determinants may occur. For practical purposes the use of azide, or of no preservative at all, rather than the use of mercury-containing preservatives is recommended. ACKNOWLEDGMENTS We like to express our gratitude to Professor Bercks and Miss Gertrud Querfurth for their interest and helpful discussions, to the Deutsche Forschungsgemeinschaft and the Alexander von Humboldt-Ptiftung for financial support, and to Miss Ingeborg Thierer for technical assistance. REFERENCES 1. LE BOUVIER, G. L., Brit.
2. :: 5. 6. 7. 8. 9. 10.
J. Exptl.
Pathol.
40,
605620 (1959). BANCROFT, J. B., Virology 16, 419-427 (1962). COWAN, K. M., J. ImmunoE. 97,647-653 (1966). PAUL, H. L., BODE, O., JANKULOWA, M., and BRANDES, J., Phytopathol. 2. in press. BERCKS, R., and STELLMACH, G., Phytopathol. Z. 56, 288-296 (1966). VAN REGENMORTEL, M. H. V., Advan. Virus Res. 12, 207-271 (1966). KOENIG, R., and BERCKS, R., Phytopathol. Z. in press. RAPPAPORT, I., SIEGEL, A., and HASELKORN, R., Virology 25, 325328 (1965). PHILIPSON, L., Arch. Ges. Virusforsch. 17,472480 (1965). KAPER, J. M., and HOWWING, C., Arch. Bio-
them. 96, 125-138 (1962). RENATE KOENIG MARGARITA JANKULOWA Biologische Bundesanstalt fiir Land-und Forstwirtschaft Institut fiir Virusserologie Braunschweig, Germany Accepted December 6, 1967
Surface formed
Layer
in Tumor Cells Trans-
by Adeno-12
and SV40
Viruses The loss of contact inhibition shown by cultured cells after transformation with an oncogenic virus has been correlated with
379
changes of cell surface properties. By the demonstration of an augmented intensity of the Hale’s reaction in comparison to that of normal cells, Defendi and Gasic (1) found an increased content of surface acid mucopolysaccharides on cells transformed by polyoma virus (PV). PV-transformed cells were later shown to possess an increased electrophoretic mobility (2). Both the enhanced reaction with Hale’s stain and the altered electrophoretic mobility are reduced by neuraminidase treatment, which suggests that the surface modifications are due to an increase in sialic acid-containing substances (1, 2). A study of surface mucopolysaccharides in cells transformed by other oncogenic viruses would determine whether the increase in these substances is restricted cells or whether it to polyoma transformed is a more generalized phenomenon, present also in other transformation systems. The development of the ruthenium red technique for the visualization of surface mucopolysaccharides (3) enabled us to detect at the ultrastructural level the presence of such substances in normal hamster embryo cells and in hamster cells transformed by adenovirus type 12 (Ad 12). Hamster cells derived from tumors induced in vivo by the inoculation of Ad 12 and SV40 viruses were also studied. In the present study the following cell types were studied: (a) normal fibroblasts derived from Chinese hamster embryos; (b) Chinese hamster embryo cells transformed in vitro by adenovirus 12 (4) ; (c) cultured transformed cells derived from a tumor induced in vivo by the inoculation of Ad 12 into a newborn Syrian hamster; and (d) transformed Syrian hamster cells derived from an in vivo SV40-induced tumor. Three different passages from each cell type were studied. Cells were fixed in the prescription bottles with a 2.5 % glutaraldehyde-cacodylate buffer solution at pH 7.3 for 1 hour at 4”. After a 30.minute rinse in cacodylate buffer, postfixation was carried out with 2% osmium
tetroxide
in cacodylate
buffer
at
room temperature for 3 hours. Ruthenium red was added to both fixatives at a concentration of 50 mg/lOO ml. After in increasing concentrations
dehydration of acetone,
SHORT
COMMUNICATIONS
6. BAUM, S. G., REICH, P. R., HYBNER, C. R., ROWE, W. P., and WEISSMAN, S. M., Proc. Natl. Acad. Rci. U.S. 56, 1509-1515 (1966). 6. HENRY, P. H., BLACK, P. H., LEVIN, M. J., and WEISSMAN, S. M. In preparation. 7. REICH, P. R.,BLAcK, P. H., and WEISSMAN, S. M., Proc. ,vatl. Acad. Sci. U.S. 56, 78--85 (1966). 8. LACY, SR. S., and GREEN, M., J. Gen. Viral. 1, 413318 (1967). STEPHEN G. BACM WILLIAM H. WIESE Laboratory of Viral Diseases National Institute of Allergy and Infectious Diseases Bethesda,
Maryland
20014
PAUL R. REICH Metabolism Branch 1Vational Cancer Institute Bethesda, Maryland ZOO14 Accepted January 2, 1968
Effect
of
Mercury-Containing
atives on Agar
Gel Diffusion
ci pi tin Reactions
PreservPre-
with Several
Plant Viruses The mercury-containing compounds Merthiolate (sodium ethyl mercury-thiosalicylate) and Cialit (sodium 2-ethyl mercury mercapto-benzoxazol-5-carbonate) are frequently used as preservatives in serological agar gel diffusion tests. Recently, adverse effects of Merthiolate in these tests were demonstrated with poliovirus (I), bean pod mottle virus (2), foot-and-mouth disease virus, and an antigen associated with foot-and-mouth disease virus infection (3). An inhibition of precipitin line formation was observed with the top component of bean pod mottle virus and also with the middle and bottom components after repeated freezing and thawing (2). Likewise, t,he precipitating activity of the antigen associated with virus infection in foot-andmouth disease was markedly reduced, whereas in the case of foot-and-mouth disease virus itself two precipitin lines developed instead of one. This was due to partial degradation of the 140 S virus to 12 S protein subunits. Sodium azide at a concentration of 0.5 % had no such effects (3).
In this laboratory, similar observations were made with several plant viruses which hithert,o had not been known t’o be affected by h4erthiolat’e or Cialit. The effect of the mercurials proved to be dependent upon the pH of the agar and upon certain additives to the virus preparations. In Table 1, the results of an experiment are shown in which the development of precipitin lines of several plant viruses with their respective antisera was studied in 0.85% Difco Special AgarNoble containing 0.85 % sodium chloride. The pH of the agar was adjusted to 6.0, 6.5, 7.0, 7.5, or 8.0 with phosphate buffers at a final concentration of 0.05 LV; 0.01% Cialit or Merthiolate or 0.5 % sodium azide was added as preservative. In cont,rol plates 110 preservative was added. The diameter of the reactant wells was 2 mm; the distance between the wells measured 3.5 mm. Crude sap of virus-infected plants or partially purified virus preparations which had been kept frozen for varying lengths of time were placed into the antigen wells. In some cases, 0.2 “/(I sodium sulfite and 0.2 % ascorbic acid were added to the preparations. Preliminary experimentas were performed in agar with no added preservative to find out which antisera dilutions yielded the sharpest precipitin lines wit#h their respective antigens. These dilut#ions were used in all plates regardless of whether preservatives were present or not. The format#ion of precipitin lines by several spherical plant viruses was prevented by the presence of Cialit or Mert,hiolate (Table 1). With some viruses this effect, was observed at all pH values tested (tomato ringspot virus and crude sap of Nicot&a tabacum I,. var. Samsun containing Belladonna mottle virus isolate 1). With other viruses, a distinct dependence of the effect upon the pH of t’he agar was observed (crude sap of Set&u italica Pal. Beauv. containing Dactylis virus II, purified preparations of fanleaf virus, tomat,o blackring virus, and St. 10, an unidentified grape vine virus). With increasing pH an increasing number of viruses was affected. The fact that t’he precipitation of some viruses by their respective antisera \vas impaired, whereas other syst’ems remained unaffected (carnation ringspot virus, beet
ON THE
+
+ -
-
+
-
+
+ -
-
TABLE
1
7.5
60
l(l)1
6.5
7.5
AGAR
GEL
1 1 111
1 1 111 1 11 1 1 1 1 1 1 1 11
8.0
11 1 (1) 1 (1) 1
11 11 11
11 11 11 11 11 11 11 11 11 11 11 11 11
7.0
0.5% Sodium Azide PH
IN THE
fanleaf virus, infected isolates 1 and 3.
Setaria
italica
0 1
6.5
2 2
2 2
otherwise.
0 0
0 0
0 0 111 2 3
2 2
2 2
0 0
0 0
0 0 0 0
1 0 0 0 0
1 0 1 0 1
0 0 1
7.5
0 0 1 1 111 1 1
7.0
1 1111 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11111 1 0 0 1 1
1
0 1
6.0
II,
WITH
0 1 1 1 1 1 1 1 1 1 1 1 1
6.5
infected
0 0 1 2 3
0 1 1 1 1 1 0 0 0 0 1 0 0
7.0
0.01% Cialit PH
0 0 0 0 11 2 3 2 3
0 1 1 1 1 1 1 1 1 1 1 1 1
6.0
TEST”
virus
8.0
DIFFUSION
0.01% Merthiolate PH
DOUBLE
in the case of Dactylis
wells, 3.5 mm. of time where used unless stated
11 11 11 1 11
8.0
11111 11111 11111 11111 11111 reactant lengths 0.05 M.
7.0
11 11 11 11 11 11 11 11 11 11 11 11 11
6.5
11111 11111 11111 11111 11111 11111 11111 11111 11111 11111 11111 11111 11111
6.0
PH’
No preservative
NUMBER OF PRECIPITIN LINES SEVERAL PLANT VIRUSES
a Diameter of the reactant wells, 2 mm; distance between the b Purified preparations which had been kept frozen for varying c Adjusted with phosphate buffers to a final concentration of nodosa. d Unidentified ringspot virus from Scrophularia e Unidentified grape vine virus, / Crude sap from infected Chenopodium quinoa in the case of tiana labacum var. Samsun in the case of Belladonna mottle virus g (1) = Weak indication of a precipitin line.
Tomato ringspot virus Tomato blackring virus Scr Vd Carnation ringspot virus St. 1@ Beet ringspot virus Fanleaf virus Fanleaf virus Fanleaf virus! Fanleaf virusf Arabis mosaic virus Dactylis virus 11’ Dactylis virus II’ Belladonna mottle virus’ Isolate 1 Isolate 1 Isolate 3 Potato virus Xg Potato virus X@
PRESERVATIVES
Presence or absence of 0.2% ascorbic acid and 0.2% sodium sulfite
OF VARIOUS
Virusb
INFLUENCE
0 0 1 2 2
0 0 1 1 0 1 0 0 0 0 1 0 0
8.0
Nico-
0 0 1 2 2
0 0 1 1 0 1 0 0 0 0 1 0 0
7.5
378
SHORT
COMMUNICATIONS
ringspot’ virus, and an unidentified ringspot virus from Xaophularia nodosa I,.) suggest#edthat the action of the mercurials was on the virus rather than on the antibody. This view was strengthened by some observations on the cross reactions of isolates 1 and 3 of Belladonna mottle virus (4) with t,heir respective antisera. As seen in Table 1, the reaction of Belladonna mottle virus isolate 1 in crude sap of N. tabacunz var. Samsun with its homologous antiserum was strongly affected by mercurials, whereas t#he reaction of Belladonna mottle virus isolate 3 with its homologous antiserum appeared not to be affect,ed. The cross react’ion between isolate 1 wit’h the antiserum against’ isolat,e 3 was affected whereas the cross reaction of isolate 3 with the antiserum against, isolate 1 was not affected. Thus, the mercurials acted on the virus, not on t,he antibody. Wit,h t,he majority of the viruses affected, the inhibitory act,ion of Cialit became apparent at somewhat lovver pH values t,han t,hat#of Merthiolate. With the isolate St. 10, only Cialit was effect,ive and only at pH 7..5 and 8.0. In the case of Merthiolate the critical hydrogen ion concentrat#ion for the inhibitory action could be lowered by the addition of 0.2 %, ascorbic acid and 0.2 7, sodium sulfite. An apparent shift of sensitivit’y toward the more alkaline range could also be achieved by using larger reactant wells and shorter distances between them. Thus, with t,omato ringspot virus, despite the presence of mercurials, well-defined precipitin lines were observed at, pH 6.0 lvhen reactant wells with a diameter of 4 mm and a dist,ance of 2 mm were used. At pH 6.5, however, no lines formed in the presence of mercurials. A similar shift’ of the sensitivit! toward the more alkaline range was observed with crude sap of Xetaria italica in fected with Dactylis virus II. In these cases part of t#he virus probably inactivated the inhibit,or and the remainder reactled with t,he aruiserum. This concemration effect, probably also explains why purified preparations of Belladonna mottle virus 1 yielded intense precipitat,ion lines at neutral pH in the presence of mercurials. The pH-dependent effect of mercurials is responsible for the reported (5) failure t’o obtain precipitin lines with tomato ringspot, virus and
fanleaf virus at higher pH values, when wells with a diameter of 4 mm were used at a distance of 2 mm. With potfat’ virus X a different effect of mercurials was observed. In addition t,o a sharp line close to the antigen well, one or two intense but more or less diffuse lines were observed nearer t’o the antiserum well. A photograph, where this diffuse line is also visible, has been published by van Regenmort,el (6). Occasionally, a weak indicat’ion of t,his line was also observed in the presence of 0.5 ‘% sodium azide, but never in the absence of a preservative. Wit
SHORT COMMUNICATIONS several spherical viruses under the influence of mercurials. Further investigations are necessary to determine whether the mercurials exert their effect in all cases by breaking up the quaternary structure (3, 9, IO), or whether in some cases a direct interaction with sulfhydryl-containing antigenic determinants may occur. For practical purposes the use of azide, or of no preservative at all, rather than the use of mercury-containing preservatives is recommended. ACKNOWLEDGMENTS We like to express our gratitude to Professor Bercks and Miss Gertrud Querfurth for their interest and helpful discussions, to the Deutsche Forschungsgemeinschaft and the Alexander von Humboldt-Ptiftung for financial support, and to Miss Ingeborg Thierer for technical assistance. REFERENCES 1. LE BOUVIER, G. L., Brit.
2. :: 5. 6. 7. 8. 9. 10.
J. Exptl.
Pathol.
40,
605620 (1959). BANCROFT, J. B., Virology 16, 419-427 (1962). COWAN, K. M., J. ImmunoE. 97,647-653 (1966). PAUL, H. L., BODE, O., JANKULOWA, M., and BRANDES, J., Phytopathol. 2. in press. BERCKS, R., and STELLMACH, G., Phytopathol. Z. 56, 288-296 (1966). VAN REGENMORTEL, M. H. V., Advan. Virus Res. 12, 207-271 (1966). KOENIG, R., and BERCKS, R., Phytopathol. Z. in press. RAPPAPORT, I., SIEGEL, A., and HASELKORN, R., Virology 25, 325328 (1965). PHILIPSON, L., Arch. Ges. Virusforsch. 17,472480 (1965). KAPER, J. M., and HOWWING, C., Arch. Bio-
them. 96, 125-138 (1962). RENATE KOENIG MARGARITA JANKULOWA Biologische Bundesanstalt fiir Land-und Forstwirtschaft Institut fiir Virusserologie Braunschweig, Germany Accepted December 6, 1967
Surface formed
Layer
in Tumor Cells Trans-
by Adeno-12
and SV40
Viruses The loss of contact inhibition shown by cultured cells after transformation with an oncogenic virus has been correlated with
379
changes of cell surface properties. By the demonstration of an augmented intensity of the Hale’s reaction in comparison to that of normal cells, Defendi and Gasic (1) found an increased content of surface acid mucopolysaccharides on cells transformed by polyoma virus (PV). PV-transformed cells were later shown to possess an increased electrophoretic mobility (2). Both the enhanced reaction with Hale’s stain and the altered electrophoretic mobility are reduced by neuraminidase treatment, which suggests that the surface modifications are due to an increase in sialic acid-containing substances (1, 2). A study of surface mucopolysaccharides in cells transformed by other oncogenic viruses would determine whether the increase in these substances is restricted cells or whether it to polyoma transformed is a more generalized phenomenon, present also in other transformation systems. The development of the ruthenium red technique for the visualization of surface mucopolysaccharides (3) enabled us to detect at the ultrastructural level the presence of such substances in normal hamster embryo cells and in hamster cells transformed by adenovirus type 12 (Ad 12). Hamster cells derived from tumors induced in vivo by the inoculation of Ad 12 and SV40 viruses were also studied. In the present study the following cell types were studied: (a) normal fibroblasts derived from Chinese hamster embryos; (b) Chinese hamster embryo cells transformed in vitro by adenovirus 12 (4) ; (c) cultured transformed cells derived from a tumor induced in vivo by the inoculation of Ad 12 into a newborn Syrian hamster; and (d) transformed Syrian hamster cells derived from an in vivo SV40-induced tumor. Three different passages from each cell type were studied. Cells were fixed in the prescription bottles with a 2.5 % glutaraldehyde-cacodylate buffer solution at pH 7.3 for 1 hour at 4”. After a 30.minute rinse in cacodylate buffer, postfixation was carried out with 2% osmium
tetroxide
in cacodylate
buffer
at
room temperature for 3 hours. Ruthenium red was added to both fixatives at a concentration of 50 mg/lOO ml. After in increasing concentrations
dehydration of acetone,