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Comparison of mouse mammary tumors fixed with KMnO4 and with OsO4
DISCUSSION
AND
PRELIMINARY
REPORTS
501
cleoid is about 50 rnp in diameter and in many cases is joined to nucleoids of adjoining particles or cellular constituents by a rod about 18 rnp in diameter. The inner pair of membranes are fitted closely around the central nucleoid 10 rnp apart to produce a central portion with a diameter of about, 70 mp. The outer pair of membranes are more loosely fitted. They are separated from each other by about 10 rn+ The whole particle has an outside diameter of 9&120 mp or more depending upon separation between inner and outer membrane pairs. Intracellular particles are identical to the 70 mp-diameter central portion of the developHINAE KATO ing extracellular particles. Thus the OsO1Y. KANDA INOUE fixed particles are similar to those reported Institute for Virus Research in other publications (5, 6). Developing Kyoto L’niversity particles revealed by KMn04 fixation are Kyoto, Japan rluite different from those found in OSO~Received July 16, 1962 fixed tumors, just as most cellular components are different in appearance. KM&+fixed tumor acinar areas are shown in Figs. 2 and 3. PIToteespecially the triplet of parComparison of Mouse Mammary Tumors Fixed ticles in Fig. 2. These particles are 100-120 with KMn04 and with 0~04 111~in diameter and have a very dim outline Results of electron microscope studies of of a nuclear area about 60 rnp in diameter. mouse mammary tumors fixed with OsOd Like some of the OsO1-fixed particles, they are joined together by rods about 18 rnp in have been reported in numerous publications concerning the mammary tumor milk diameter. The appearance of intracellular agent. In this laboratory KMn04 has been particles has not been ascertained. If they found to give excellent fixation of membrane have the same appearance as the nuclear areas of the developing extracellular partisystems of one of these tumors. Extremely high contrast allows easy scanning and fo- cles, they will be difficult to discern from the cytoplasmic ground substance of the cusing with the electron microscope. A C3H mouse mammary tumor was pre- cell. The extracellular types of particles al,pared by fixation with 4% KMn04 for 2 pear in vacuoles and dark bodies in the cytoplasm of the cells, just as in Os04-fixed hours at room temperature, dehydration, and embedding in epoxy resin (1, 2). Other cells. One conclusion which can be made frum specimens were fixed in Dalt’on’s chromeosmium solution (3) for 30 minutes at O”, KMn04 and 0~0~ fixations of these mouse mammary tumors is that. developing extradehydrated, and embedded in methacrylate polymer. Sections both from KMn04 , and cellular particles which appear in chains from 0~04 fixation were post-stained with may not be just oddities. In fact, extrusion of chains of basic units may be the means of Millonig’s basic lead stain (4). production of such particles. Inner memWith both types of fixation extracellular particles were found in acinar areas of the branes which appear in the extracellular tumors, In Fig. 1 is shown a portion of an particles after OsOd fixation, but not after Os04-fixed cell bordering an acinar area. KMnO, fixation, may be nucleoprotein, Developing extracellular particles contain since 2-hour KMnOl fixation does not prea light central nucleoid bounded by two serve structures generally recognized as pairs of concentric membranes. The nu- nucleoprotein (7). Further studies of mouse REFERENCES
J. S., Nature 183, 1069-1070 (1959). 2. PORTERFIELD, J. S., Trans. Roy. Sot. Trop. Med. Nyg.53,458-466 (1959). 3. INOUE, KANDA Y., IWASAKI, T., and KATO, H., J. ZmmunoZ.87,337-341( 1961). 4. HEZIDERSON, J. R., Yale J. Bio2. Med. 33, 350358 ( 1961). 5. RHIM, J. S., Proc. Sot. Exptl. Biol. Med. 109, 887-889 (1962). 6. INOUE, KANDA Y., and Ocu~a, R., Virology 16, 205-207 (1962). 7. IWASAKI, T., and ISOUE, KAND.4 Y., Virology 15, 81-82 (1961). 8. DULBECCO, R., Proc. Natl. Acad. Sci. 38, 747752 (1952). 1. PORTEHFIELD,
FIG. 1. OsOAxed mammary tumor cell adjoining acinar area showing intracellular particles (ZP) and extracellular particles (EP). Magnification: X 33,000. FIGS. 2, 3. KMnOAxed tumor cells bordering acinar areas. Note especially the chain of extracellular particles (EP) in Fig. 2. Magnification : X 33,000. 502
DISCUSSION
AND
PRELIMINARY
REPORTS
503
ules at the surface of inclusions, and paler granules in the inclusion substance itself (presumably in the Feulgen-positive zone). He interpreted the osmiophile granules as elementary bodies of the virus, and the ACKNOWLEDGMENTS paler granules as developmental stages. In The author wishes to acknowledge the financial view of the inherent limitations of light miassistance provided by the United Fund of Austin croscopy it has seemed desirable to recxand Travis County, Cancrr Research Fund-Grant, amine the osmiophile granules. for a portion of this work. He is indebted to Mrs. Mature lymphocystis warts were obMary Lou Gammans and Mrs. Betty S. Gibson tained during the April spawning run of for their tcrhnical assistance. Stizostedion at, the New York State HatchREFERENCES ery at Constantia on Lake Oneida. Lym1. MOLLENIIAUER, H., J. Biophysic. Biochem. Cyphocystis cells were fixed in buffered ostol.6, 431-35 (1959). mium tetroxide and sectioned for electron 2. GLAUERT, A. M., and GLAUERT, R. H., J. Biomicroscopy. Virus particles are abundant in physic. Biochem. Cytol. 4, 191-94 (1958). the cytoplasm (Fig. l), especially in the 3. DALTON, A. J., Anat. Record 121, 281 (1955). inclusion zone, which is, howcrer, not 4. MILLONIG, G., J. Biophysic. Biochem. Cytol. sharply delimited. Virus was not seen in 11,73639 (1961). the nucleus, nor outside the giant ~11s. The 6. BERNHARD, iv., Came?" Rcseurch 20, 712-27 particles are completely embedded in the (1960). cytoplasmic matrix and are not signifi6. MOORE, D. H., “Tumors Induced by Viruses: cantly related to the endoplasmic reticulum. Ultrastructural Studies” (A. J. Dalton and F. Haguenau, eds.), pp. 113-150. Academic The latter is sparse, and ribosomes arc Press, New York, 1962. found scat,tered rat,her than restricted to 7’. IJcl~~, J. H., J. Riophysic. Biochem. Cytol. 2, the reticular membranes. Mitochondria are 799-801 (1956) also not closely related to the denser areas NORTOK G. MCDUFFIE, JR. of virus accumulation. The condition of Clayton Foundation Biochemical Institute mitochondria and cndoplasmic ret,iculum l,~'ti7,wsity of Texas might imply relatively slow metabolism and Austin 12, Texas synthesis, and it should be noted that the Received July 23, 1962 great cell size and virus quantity are the result, of months of growth. The present maFine Structure of Lymphocystis Virus of Fish terial includes no developmental stages. In Lymphocystis disease of the pike perch these Stixostedion cells virus part,icles have Stizostedion vitreum (Mitchill) is essen- not yet, been seen in crystalline array, tially similar to that of various other fresh- though in the sunfish (Lepomis; unpublished) there is occasionally an elegant water and marine fish species. Skin warts lattice. are composed of enormously hypertrophied The virus particles are large and polyconnective tissue cells, up t,o a millimeter in hedral (Figs. l-3). There is a sharply osdiameter. In each, the nucleus is propormiophile capsule (capsid) about 200 rnp in tionately enlarged and the cytoplasm condiameter (180-220 nip). The capsid is about tains a rich network of Feulgen-positive 12 mp thick, and there is a 12-rnp pale space inclusion bodies. The cells are heavily ensurrounding the ljO-mp nucleoid. The latter capsulated, but after a growth period of appears as a ball of osmiophile threads several months they slough out, presumably roughly 10 1~~ thick. The commonest capsid releasing virus. Weissenberg described the are hexagonal ; a frw smaller prodisease in European perches and flounders profiles (1) and has described developmental stages files are pentagons. These are compatible in Stizostedion (2). Virus was implicated in with sections of regular icosahedra. However capsomere arrangement is not clear the disease by transfer experiments (S-5) and by microscopy of the inclusion bodies. enough to verify the three-dimensional Weissenberg (6) showed osmiophile gran- form; and capsomere number is uncertain. mammary tumors fixed wit,h KMn04 should provide more information on relations hetween cellular membrane systems and the milk agent.
AND
PRELIMINARY
REPORTS
501
cleoid is about 50 rnp in diameter and in many cases is joined to nucleoids of adjoining particles or cellular constituents by a rod about 18 rnp in diameter. The inner pair of membranes are fitted closely around the central nucleoid 10 rnp apart to produce a central portion with a diameter of about, 70 mp. The outer pair of membranes are more loosely fitted. They are separated from each other by about 10 rn+ The whole particle has an outside diameter of 9&120 mp or more depending upon separation between inner and outer membrane pairs. Intracellular particles are identical to the 70 mp-diameter central portion of the developHINAE KATO ing extracellular particles. Thus the OsO1Y. KANDA INOUE fixed particles are similar to those reported Institute for Virus Research in other publications (5, 6). Developing Kyoto L’niversity particles revealed by KMn04 fixation are Kyoto, Japan rluite different from those found in OSO~Received July 16, 1962 fixed tumors, just as most cellular components are different in appearance. KM&+fixed tumor acinar areas are shown in Figs. 2 and 3. PIToteespecially the triplet of parComparison of Mouse Mammary Tumors Fixed ticles in Fig. 2. These particles are 100-120 with KMn04 and with 0~04 111~in diameter and have a very dim outline Results of electron microscope studies of of a nuclear area about 60 rnp in diameter. mouse mammary tumors fixed with OsOd Like some of the OsO1-fixed particles, they are joined together by rods about 18 rnp in have been reported in numerous publications concerning the mammary tumor milk diameter. The appearance of intracellular agent. In this laboratory KMn04 has been particles has not been ascertained. If they found to give excellent fixation of membrane have the same appearance as the nuclear areas of the developing extracellular partisystems of one of these tumors. Extremely high contrast allows easy scanning and fo- cles, they will be difficult to discern from the cytoplasmic ground substance of the cusing with the electron microscope. A C3H mouse mammary tumor was pre- cell. The extracellular types of particles al,pared by fixation with 4% KMn04 for 2 pear in vacuoles and dark bodies in the cytoplasm of the cells, just as in Os04-fixed hours at room temperature, dehydration, and embedding in epoxy resin (1, 2). Other cells. One conclusion which can be made frum specimens were fixed in Dalt’on’s chromeosmium solution (3) for 30 minutes at O”, KMn04 and 0~0~ fixations of these mouse mammary tumors is that. developing extradehydrated, and embedded in methacrylate polymer. Sections both from KMn04 , and cellular particles which appear in chains from 0~04 fixation were post-stained with may not be just oddities. In fact, extrusion of chains of basic units may be the means of Millonig’s basic lead stain (4). production of such particles. Inner memWith both types of fixation extracellular particles were found in acinar areas of the branes which appear in the extracellular tumors, In Fig. 1 is shown a portion of an particles after OsOd fixation, but not after Os04-fixed cell bordering an acinar area. KMnO, fixation, may be nucleoprotein, Developing extracellular particles contain since 2-hour KMnOl fixation does not prea light central nucleoid bounded by two serve structures generally recognized as pairs of concentric membranes. The nu- nucleoprotein (7). Further studies of mouse REFERENCES
J. S., Nature 183, 1069-1070 (1959). 2. PORTERFIELD, J. S., Trans. Roy. Sot. Trop. Med. Nyg.53,458-466 (1959). 3. INOUE, KANDA Y., IWASAKI, T., and KATO, H., J. ZmmunoZ.87,337-341( 1961). 4. HEZIDERSON, J. R., Yale J. Bio2. Med. 33, 350358 ( 1961). 5. RHIM, J. S., Proc. Sot. Exptl. Biol. Med. 109, 887-889 (1962). 6. INOUE, KANDA Y., and Ocu~a, R., Virology 16, 205-207 (1962). 7. IWASAKI, T., and ISOUE, KAND.4 Y., Virology 15, 81-82 (1961). 8. DULBECCO, R., Proc. Natl. Acad. Sci. 38, 747752 (1952). 1. PORTEHFIELD,
FIG. 1. OsOAxed mammary tumor cell adjoining acinar area showing intracellular particles (ZP) and extracellular particles (EP). Magnification: X 33,000. FIGS. 2, 3. KMnOAxed tumor cells bordering acinar areas. Note especially the chain of extracellular particles (EP) in Fig. 2. Magnification : X 33,000. 502
DISCUSSION
AND
PRELIMINARY
REPORTS
503
ules at the surface of inclusions, and paler granules in the inclusion substance itself (presumably in the Feulgen-positive zone). He interpreted the osmiophile granules as elementary bodies of the virus, and the ACKNOWLEDGMENTS paler granules as developmental stages. In The author wishes to acknowledge the financial view of the inherent limitations of light miassistance provided by the United Fund of Austin croscopy it has seemed desirable to recxand Travis County, Cancrr Research Fund-Grant, amine the osmiophile granules. for a portion of this work. He is indebted to Mrs. Mature lymphocystis warts were obMary Lou Gammans and Mrs. Betty S. Gibson tained during the April spawning run of for their tcrhnical assistance. Stizostedion at, the New York State HatchREFERENCES ery at Constantia on Lake Oneida. Lym1. MOLLENIIAUER, H., J. Biophysic. Biochem. Cyphocystis cells were fixed in buffered ostol.6, 431-35 (1959). mium tetroxide and sectioned for electron 2. GLAUERT, A. M., and GLAUERT, R. H., J. Biomicroscopy. Virus particles are abundant in physic. Biochem. Cytol. 4, 191-94 (1958). the cytoplasm (Fig. l), especially in the 3. DALTON, A. J., Anat. Record 121, 281 (1955). inclusion zone, which is, howcrer, not 4. MILLONIG, G., J. Biophysic. Biochem. Cytol. sharply delimited. Virus was not seen in 11,73639 (1961). the nucleus, nor outside the giant ~11s. The 6. BERNHARD, iv., Came?" Rcseurch 20, 712-27 particles are completely embedded in the (1960). cytoplasmic matrix and are not signifi6. MOORE, D. H., “Tumors Induced by Viruses: cantly related to the endoplasmic reticulum. Ultrastructural Studies” (A. J. Dalton and F. Haguenau, eds.), pp. 113-150. Academic The latter is sparse, and ribosomes arc Press, New York, 1962. found scat,tered rat,her than restricted to 7’. IJcl~~, J. H., J. Riophysic. Biochem. Cytol. 2, the reticular membranes. Mitochondria are 799-801 (1956) also not closely related to the denser areas NORTOK G. MCDUFFIE, JR. of virus accumulation. The condition of Clayton Foundation Biochemical Institute mitochondria and cndoplasmic ret,iculum l,~'ti7,wsity of Texas might imply relatively slow metabolism and Austin 12, Texas synthesis, and it should be noted that the Received July 23, 1962 great cell size and virus quantity are the result, of months of growth. The present maFine Structure of Lymphocystis Virus of Fish terial includes no developmental stages. In Lymphocystis disease of the pike perch these Stixostedion cells virus part,icles have Stizostedion vitreum (Mitchill) is essen- not yet, been seen in crystalline array, tially similar to that of various other fresh- though in the sunfish (Lepomis; unpublished) there is occasionally an elegant water and marine fish species. Skin warts lattice. are composed of enormously hypertrophied The virus particles are large and polyconnective tissue cells, up t,o a millimeter in hedral (Figs. l-3). There is a sharply osdiameter. In each, the nucleus is propormiophile capsule (capsid) about 200 rnp in tionately enlarged and the cytoplasm condiameter (180-220 nip). The capsid is about tains a rich network of Feulgen-positive 12 mp thick, and there is a 12-rnp pale space inclusion bodies. The cells are heavily ensurrounding the ljO-mp nucleoid. The latter capsulated, but after a growth period of appears as a ball of osmiophile threads several months they slough out, presumably roughly 10 1~~ thick. The commonest capsid releasing virus. Weissenberg described the are hexagonal ; a frw smaller prodisease in European perches and flounders profiles (1) and has described developmental stages files are pentagons. These are compatible in Stizostedion (2). Virus was implicated in with sections of regular icosahedra. However capsomere arrangement is not clear the disease by transfer experiments (S-5) and by microscopy of the inclusion bodies. enough to verify the three-dimensional Weissenberg (6) showed osmiophile gran- form; and capsomere number is uncertain. mammary tumors fixed wit,h KMn04 should provide more information on relations hetween cellular membrane systems and the milk agent.