Partial characterization of C-type particles in a cell line (WR-9) derived from a rat epidermoid carcinoma of spontaneous origin.

A C-type virus continuously released from a cell line (WR-9) derived from a spontaneous epidermoid carcinoma was purified by means of large-scale tissue culture techniques and high-volume zonal centrifuges. With the use of relatively pure virus concentrates, partial characterization of the virus has been accomplished. Up to 60 liters of spent culture medium from relatively low virus-yielding cultures were processed at a time through the Model K ultracentrifuge in order to obtain quantities of virus sufficient for convenient Tween-ether extraction of the major polypeptide (30,000 daltons). This structural protein having group-specific reactivity was purified and isolated by isoelectric-focusing techniques. A UV absorption peak (A280) was found to be coincident with a major peak of radioacticity at pH 8.6, the isoelectric point (pI) for rat virus gs antigen previously reported by other investigators. Because species-specific (gs-1) and cross-reactive (gs-3) determinants coexist on this protein, fractions containing the group-specific antigen were identified on the basis of the mammalian interspecies determinant (gs-3), using antiserum prepared against Tween-ether-disrupted feline leukemia virus. At the same time, reactivity to the gs-1 determinants in identical fractions was observed in complement fixation and gel diffusion assays, using guinea pig antiserum known to contain principally antibodies to rat gs-1 determinants. Presently, the principal source of rat type C viral gs antigen is rat cell line MSB, which continuously releases a rat leukemia virus pseudotype of murine sarcoma virus. The WR-9 rat virus line may be of use in providing an additional source of C-type particles that are capable of yielding good gs reagents.

Effects of mercuric chloride on growth and morphology of selected strains of mercury-resistant bacteria.

A survey of the comparative cytological effects of growth in the presence of mercury by a group of mercury-resistant bacterial cultures and a characterization of the process of bacterial adaptation to Hg2+ ion was accomplished. Mercury resistance was found to be dependent upon the ability to volatilize mercury from the medium and upon the amount of mercury accumulated by the cells. The results indicate that most cultures which adapt to growth in the presence of HgCl2 exhibit extensive morphological abnormalities. Significant effects are delay in the onset of growth and cell division and numerous structural irregularities associated with cell wall and cytoplasmic membrane synthesis and function. A detailed analysis of the adaptation process and the resulting effects on morphology was performed on an Enterobacter sp. During the period preceding active multiplication, a selection for mercury-resistant mutants occurred. It was also demonstrated that growth commenced only at a specific threshold concentration of HG2+.

Viral polypeptide composition of Japanese encephalitis virus-infected cell membranes.

Chick embryo cells infected with Japanese encephalitis virus were separated into smooth and rough membrane fractions, as judged by electron microscopy. Polyacrylamide gel electrophoretic analysis of the membrane fractions showed them to contain all of the virus specific polypeptides; however, the relative proportions of the polypeptides varied among smooth and rough membranes. The relationship of these observations to current concepts of group B arbovirus morphogenesis is discussed.

Relationship between cell wall, cytoplasmic membrane, and bacterial motility.

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.

Motility tracks: technique for quantitative study of bacterial movement.

A method for recording movements of bacteria in time and space on a single photograph is described. Quantitative information on the behavior of various motile organisms may easily be obtained for comparative studies. The method possesses certain advantages over cinematography, and illustrations of applications of the technique are presented.

Growth of Mycobacterium lepraemurium in cultures of mouse peritoneal macrophages.

Successful growth of Mycobacterium lepraemurium was observed in cultures of mouse peritoneal macrophages. The optimal host cell maintenance medium was composed of 40% horse serum, 50% of the chemically defined medium NCTC 109, and 10% of a 1:5 dilution of beef embryo extract, supplemented with both liver extract and ferric nitrate. Multiplication of the bacilli was observed in 1 week and maximal growth in 6 to 7 weeks. All macrophages were filled with tens to hundreds of the organisms in cultures showing maximal growth. Glycerol caused an increase in the normal length of M. lepraemurium, without a corresponding increase in the number of the bacilli. Elongation of M. lepraemurium was observed 3 or 4 days after infection. Rapid and uniform growth of M. lepraemurium was achieved in serially transferred cultures (subcultures). The cumulative increase of the number of intracellular bacilli was 1.4 x 10(20)-fold in 14 transfers over a period of 68 weeks in one series, and 10(17)-fold in 12 transfers over a period of 56 weeks in another series. The generation time of M. lepraemurium was 7 days, a growth rate which approximates the fastest growth of the organisms in vivo. Organisms harvested from cultures at various stages of growth produced murine leprosy in mice, but showed no growth in bacteriological media. The present model offers an opportunity for studies on the host-parasite relationship without the complication of extracellular growth of the parasites.