J. W. Beard

Determination of Small Quantities of Nitrogen in Serological Precipitates and Other Biological Materials.

Summary A simple and accurate colorimetric method is described for the routine determination of quantities of nitrogen in the range from 5 to 80 y. The method, which is a modification of the direct-nesslerization procedure of Koch and McMeekin, has been developed especially for the analysis of serological precipitates, but is adapted readily to the analysis of protein solutions.

Counts of Virus Particles by Sedimentation on Agar and Electron Micrography.

Summary A method has been devised for counting virus particles in electron micrographs of the virus sedimented on an agar surface and removed quantitatively in a collodion pseudoreplica. With influenza virus A (PR8 strain) as test virus, the accuracy of the counts, under conditions practical for routine studies, was the same as that observed with the spray technic or by sedimentation of the agent on collodion. In common with the latter method, sedimentation on agar is applicable to materials of relatively low virus content and, since salt diffuses into the agar, to viruses harmed either by drying in the presence of salt or by salt-free water. The usefulness of this procedure is limited to the study of virus particles which can be recognized either because of characteristic morphology or of other properties that can be related quantitatively to virus behavior.

Ultrastructure of viruses of myeloblastosis and erythroblastosis isolated from plasma of leukemic chickens.

Summary Ultrathin sections of the pellets formed by ultracentrifugation of plasmas from birds with myeloblastosis and erythroblastosis have been examined by electron microscopy. In the material from both diseases there were observed structures of similar appearance represented by images of round or oval shape consisting centrally of a dense core of about 40 mμ diameter. These were surrounded by a less dense material and were limited by an outer membrane-like structure with a total particle diameter of about 80 mμ. In many instances the limiting membrane appeared to be double. The ultrastructure of these agents of the avian leukemic diseases was very similar, if not identical, to that of the viruses of other chicken neoplasms. No particles of these characteristics were obtained from the plasmas of apparently healthy chickens.

Study of the Papilloma Virus Protein with the Electron Microscope.

Information relative to the physical character of the papilloma virus protein 1 has been obtained by means of ultracentrifugation, 2 electrophoresis, 3 , 4 diffusion and viscosity. 2 Recently, more direct studies of this animal virus have been undertaken with the electron microscope. In the present paper are described the preliminary observations dealing chiefly with data gathered with this instrument relative to the appearance and size of the virus. Papilloma virus protein isolated in 0.05 M phosphate buffer pH 6.5 by previously described ultracentrifugal procedures was diluted with water to concentrations of the order of 0.05 to 0.5 mg per cc. The salt concentration in the final solutions was approximately 0.005 M. Preparations for study were made by pipetting the solutions onto collodion membranes supported on 200-mesh wire gauze. Free fluid was removed with the pipette, and the resulting thin film allowed to dry in the air. For comparison, examinations have been made also of mixtures of papilloma and tobacco mosaic viruses† the latter in concentration of about 0.4 mg per cc of the mixture. The essential findings are illustrated in Figs. 1 and 2. In Fig. 1 are shown the results obtained with papilloma virus alone. Repeated micrographs of 3 different batches of the purified protein have resulted consistently in observation of the circular images shown in Fig. 1. When the protein concentration was kept in the region of approximately 0.1 mg per cc, the images were for the most part single, though grouping was frequent as seen in Fig. 1. In preparations of higher concentration, large groups of particles occurred and fewer single images were seen. A regular arrangement of images in the groups was not observed.

Converted-Cell Focus Formation in Culture by Strain MC29 Avian Leukosis Virus.

Summary Infection of chick embryo cell monolayers in high multiplicity with strain MC29 leukosis virus in conventional tissue culture induces rapid, massive morphological conversion within 4 to 7 days. Treatment of the cells in appropriately lower inoculation multiplicities results in the occurrence of infectious centers distinguished as foci of converted cells. When the infected cells are overlaid with agar, the number of foci occurring in 7 days is closely proportional to virus dose. This relationship is applicable to bioassay of strain MC29 leukosis virus with reproducibility and precision of results compatible with practical quantitative studies on the agent. The bioassay procedure and the rapid massive culture conversion make available a unique avian virus-tumor system for studies on cell-virus interrelationships in the induction of neoplasia.

Inhibition by toyocamycin of RNA synthesis in mammalian cells and in normal and avian tumor virus-infected chick embryo cells

Abstract 1. The adenosine analog toyocamycin selectively inhibits synthesis of rRNA in mammalian cells, blocking the conversion of the 45-S precursor molecule. In contrast, toyocamycin inhibition in chick embryo cells is less specific, and some rRNA synthesis continues even at relatively high concentrations of toyocamycin. 2. In untreated chick cells rRNA formation was like that in L cells. A broad range of heterogeneous chick cell nuclear RNAs were synthesized most rapidly, followed by the rRNA precursor of about 45 S. This was cleaved to forms of about 38 S, 32 S and, finally 28 S and 18 S. 3. The similarity of normal pathways suggests that the differences in species response to toyocamycin probably reflect different enzyme sensitivities. The inhibitory effect in chick cells appears to be on both synthesis and conversion of the 45-S precursor RNA. 4. The toyocamycin-resistant nucleolar RNA formed in chick cells infected with MC29 virus above that in noninfected cells was principally rRNA precursor with no unequivocal evidence of virus-specific RNA. 5. The differential thermal phenol procedure for separate extraction of nuclear and cytoplasmic RNAs was modified for application to chick cells.

Rabbit papilloma and vaccinia viruses and T2 bacteriophage of E. coli in shadow electron micrographs.

Summary Electron micrographs of the rabbit papilloma and vaccinia viruses and the T2 bacteriophage of E. coli were obtained with the metal-shadowing technic. The papilloma virus dried in vacuo for application of the metal appeared spheroidal in shape, flattened especially at the region of contact with the film. The vaccinia virus was greatly flattened and showed the presence of a denser internal material bulging beneath the surface. The bacteriophage was a tadpole-shaped structure with a headpiece of well-rounded contours and a stubby tailpiece. The findings were discussed with respect to their bearing on the shape of the viruses in the wet state.

Lipids of the BAI strain A avian tumor virus and of the myeloblast host cell

Abstract Lipids of the BAI strain A virus and of the myeloblast host cell and nuclear, microsomal and external membrane cell fractions were analyzed by thin-layer chromatography. Total virus lipids consisted of 34% cholesterol, 5.3% neutral fat and 61% phospholipid. Of the latter, the principal components were sphingomyelin, 16; phosphatidyl ethanolamine, 21; and lecithin, 11%, respectively. This constitution of the BAI strain A virus was proportionally similar to that of the influenza agent. Virus lipids were qualitatively similar to those of the whole host cell and the nuclear, microsomal, and cell membrane fractions. Marked quantitative differences were observed between the virus and host cell components, particularly with respect to the cell membrane fraction in which the low content of cholesterol, 9%, and 30% neutral fat were the reverse of the virus with 34 and 5.3% of these constituents. Major differences observed also with the phospholipids were, again, greater in the comparison between the virus and the cell membrane. The findings were interpreted to indicate the improbability of major cell membrane contribution to the virus lipids. Consideration of the analytical data and correlation with the ultrastructural aspects of BAI strain A budding from the myeloblast surface suggest that the virus lipids are specifically assembled in the process of virus synthesis not only from the external cell membrane but also principally from contiguous and possibly other cytoplasmic structures.

Morphology of Characteristic Particles Associated with Avian Erythroblastosis.

Summary A particulate component, which may be the etiological virus, was isolated by ultracentrifugation of the blood plasma of chickens with erythroblastosis. The particles were of spheroidal shape and variable size, averaging approximately 102 mμ diameter. Micrographs of unshadowed preparations showed circular images of an appearance suggesting that the particles consisted predominantly of a watery, low-electron contrast, gellike material surrounding a small relatively dense internal structure. The findings were compared with those obtained in the analogous studies of particles representing the virus of myeloblastosis and that of one form of lymphomatosis.

Dephosphorylation of Adenosine Triphosphate by Concentrates of the Virus of Avian Erythromyeloblastic Leucosis.

Summary A highly potent capacity for the dephosphorylation of adenosine triphosphate has been observed with preparations containing the virus of avian erythromyeloblastic leucosis. This enzymatic reaction occurred with the filtered plasma of birds diseased with the virus and with virus concentrates obtained from the plasma by ultracentrifugal procedures. The partition of enzymatic activity in these preparations has closely paralleled the distribution of virus infectivity of analogous materials measured in other work by titration of the virus in susceptible host chicks. No evidence of the reaction was obtained with the plasma from normal chickens. These experiments demonstrate the specific relationship of the dephosphorylation of adenosine triphosphate with avian erythromyeloblastic leucosis and indicate that the activity is a property of the etiological virus.