Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium

Summary Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C₃H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.     

Scanning and transmission electron microscopy of three strains of Bifidobacterium.

Scanning and transmission electron microscopy was applied for a morphological study of three strains of Bifidobacterium grown on solid or liquid media. The pronounced pleomorphism of the cultures previously observed by light microscopy was confirmed. A possible sequence of the morphological events during transformation from one to another pleomorphic form is proposed for B. bifidum and B. longum. Ultrastructural differences such as the formation of extensive mesosomal complexes in B. longum and characteristic plasmalemma particles only observed in the B. bifidum mutant are described and discussed.     

Inhibitory effect of 2-deoxy-d-glucose on the formation of the cell wall in yeast protoplasts

2-Deoxy-d-glucose (2DG) acted as a competitive inhibitor of the synthesis of cell wall components in Saccharomyces cerevisiae protoplasts. The synthesis of fibrillar glucan cell wall component was inhibited at a glucose to 2DG ratio of 4:1 in the cultivating medium. The completion of the formation of cell wall by the synthesis of the amorphous mannan-protein cell wall component was inhibited at a glucose to 2DG ratio of about 20:1. The inhibition could be reversed by increasing the glucose to 2DG ratio in the nutrient medium. No incorporation of 2DG into fibrillar glucan cell wall component was observed.     

Repression of alpha 2-macroglobulin and stimulation of alpha 1-proteinase inhibitor synthesis in human mononuclear phagocytes by endotoxin

Mononuclear phagocytes are a bone-marrow-derived subgroup of white blood cells which circulate as monocytes and, after differentiation into macrophages, become resident in many tissues. By synthesizing the important proteinase inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor mononuclear phagocytes contribute to the control of proteolysis both in blood and tissues. Applying a culture system which enables human blood monocytes to differentiate into macrophages in vitro, synthesis of alpha 2-macroglobulin and alpha 1-proteinase inhibitor was studied. The normal course of monocyte-macrophage maturation is accompanied by a strong increase of specific alpha 2-macroglobulin synthesis and a concomitant slight decrease of alpha 1-proteinase inhibitor. alpha 2-Macroglobulin can be designated as a marker protein of the monocyte/macrophage differentiation. Endotoxin (Salmonella typhi) in a concentration as low as 100 ng/ml strongly represses alpha 2-macroglobulin synthesis both in monocytes and macrophages. Furthermore, endotoxin completely abolishes the induction of alpha 2-macroglobulin synthesis during the course of normal monocyte in vitro cultivation, indicating that endotoxin is a strong inhibitor of the monocyte-macrophage maturation. In contrast to alpha 2-macroglobulin, alpha 1-proteinase inhibitor synthesis is strongly stimulated by endotoxin in monocytes as well as in macrophages.     

On the origin of induced pancreatic islet tumors: a radioautographic study

Endocrine tumors of the pancreas are induced in a high percentage of young rats by injections of streptozotocin and nicotinamide (SZ/NA). Benign tumors first appear 20 to 36 weeks after drug injections. To determine the possible site of their origin, the incorporation of [3H]thymidine into islets, ducts, acini, microtumors, and gross tumors was examined by radioautography of histologic sections at 1 to 36 weeks after drug injection. Drug treatment led to early (1- to 6-week) increases in nuclear 3H labeling of exocrine pancreatic structures (ductal and acinar cells), which may involve DNA repair processes. A secondary increase in labeling of duct cells during the period of tumor emergence supports the assumption that SZ/NA-induced tumors are of ductal origin. Microtumors and gross tumors also exhibited markedly elevated rates of [3H]thymidine incorporation compared to control islets. Nontumorous islet tissue, which exhibited a gradual decrease in volume due to B-cell destruction by the drug injection, showed about 10-fold higher 3H labeling than islets of controls at all time points. The results suggest that in addition to ductal precursors, islets that survive SZ/NA-induced injury may also provide sites of focal endocrine cell differentiation to tumor tissue. Once established, both microtumors and gross tumors continue to grow by accelerated cell division.     

Determination of the density of cells from sedimentation studies at 1G

To obtain a possible correlation between cell density and cell size, the size of individual cells was measured under the microscope and their sedimentation velocity was measured; the density is obtained with Stoke's Law. Specifically, HeLa cells were sedimented in Joklik's medium at 30°C in a vertical glass tube with 2 mm×2 mm horizontal opening and cells observed with a horizontally aligned microscope. The overall mean density difference of HeLa cells at 30°C was 0.0316+–0.0044 g/cm³, resulting in a density of 1.0357 g/cm³ (with a density of 1.0040 g/cm³ for Joklik's medium at 30°C). Six size fractions had densities which were essentially the same within the errors of the mean densities of the fractions (from 0.0081 to 0.0202 g/cm³). The considerably varying deviations of individual densities from the mean suggested superimposed phenomena (see also Table I for microspheres of precise size). Careful observation in balancing countercurrent flow revealed microconvection over 5 to 15 m regions, most likely based on small thermal differences in the horizontal plane.     

Characterization of the gene products produced in minicells by pSM1, a derivative of R100

Summary At least ten polypeptides larger than 6 kilodaltons (K) are produced in minicells from the miniplasmid pSM1 in vivo. pSM1 (5804 bp) is a small derivative of the drug resistance plasmid R100 (ca. 90 kb) and carries the R100 essential replication region as well as some non-essential functions. Cloned restriction fragments of pSM1 and plasmids with deletions within pSM1 sequences were used to assign eight of the ten oberserved polypeptides to specific coding regions of pSM1. Two of these polypeptides were identified as RepA1 and RepA2, proteins encoded by the essential replication region of pSM1/R100. The nucleotide sequence consisting of 885 bp outside the essential replication region is presented here. This sequence contains an open reading frame,orf4, for a protein 22.9 K in size, and one of the pSM1-encoded polypeptides was identified as theorf4 gene product. Five additional polypeptides were shown to be the products of other open reading frames mapping outside the essential replication region. Specific functions have been assigned to four of these polypeptides and tentatively to the fifth.     

Stability of microsomal mono-oxygenase during incubations for the liver microsomal assay with S9 fractions of mouse liver under various inductions

Aminopyrine-N-demethylase and p-nitroanisole-O-demethylase activities were determined in incubation mixtures for the liver microsomal assay at time zero and after 1 h of incubation in the conditions for the mutagenic assay. The experiments were performed with the S9 liver fraction of mice in the basal state and induced with sodium phenobarbital, β-naphthoflavone or both. Lipid peroxidation was also determined.

The experiments were repeated with female mice and also in the presence of styrene, as an example of a xenobiotic substance. The activity of pNAD was much more stable than that of APD in all the conditions tested. The pattern of stability, however, was similar for the two activities: more stable than controls with S9 fractions from β-NF-induced mice, less stable than controls in PB-induced mice, intermediate between controls and PB-induced mice in those with combined induction by PB + βNF. Styrene 50 mM in the incubation mixtures led to a marked inactivation of enzymic activity, similar in all cases and reaching about 90% in 1 h. S9 fractions from female mice gave enzymes slightly more stable in almost all cases. Lipid peroxidation was appreciably more elevated in basal than in induced animals.

It was concluded that, for a mutagenesis test on an unknown xenobiotic, S9 fractions from mice following PB and β-NF induction are to be preferred from the point of view of activation.     

Specific antigenic relationships between the RNA-dependent DNA polymerases of avian reticuloendotheliosis viruses and mammalian type C retroviruses

Immunoglobulin G directed against the DNA polymerase of Rauscher murine leukemia virus (R-MuLV) could bind to 125I-labeled DNA polymerase of spleen necrosis virus (SNV), a member of the reticuloendotheliosis virus (REV) species. Competition radioimmunoassays showed the specificity of this cross-reaction. The antigenic determinants common to SNV and R-MuLV DNA polymerases were shared completely by the DNA polymerases of Gross MuLV, Moloney MuLV, RD 114 virus, REV-T, and duck infectious anemia virus. Baboon endogenous virus and chicken syncytial virus competed partially for antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. DNA polymerases of avian leukosis viruses, pheasant viruses, and mammalian type B and D retroviruses and particles with RNA-dependent DNA polymerase activity from the allantoic fluid of normal chicken eggs and from the medium of a goose cell culture did not compete for the antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. We also present data about a factor in normal mammalian immunoglobulin G that specifically inhibits the DNA polymerases of REV and mammalian type C retrovirus DNA polymerases.     

Successful treatment of experimental B virus (Herpesvirus simiae) infection with acyclovir.

The efficacy of the new nucleoside analogue acyclovir against B virus (Herpesvirus simiae) was investigated in rabbits and Vero cells infected with 2-136 and 0.3-1.0 TCD50 of the virus respectively. In the Vero cells 1 mg of acyclovir/1 reduced the yield of virus by 90%, which was slightly less than the effect on herpes simplex virus. Results in the rabbits varied with the interval between doses, duration of treatment, and delay before starting treatment. Acyclovir controlled an otherwise lethal infection when given not less than eight-hourly for 14 days. Withdrawing treatment after 9-10 days resulted in late-onset fatal disease in some rabbits. Treatment begun within 24 hours after infection gave complete protection, and rabbits first treated up to five days after infection showed a significant reduction in mortality (p less than 0.001). The plasma half life of acyclovir is twice as long in man as in rabbits and progression of the disease is much slower. Hence acyclovir may be useful for post-exposure prophylaxis against B virus infection in man and possibly also for treatment of the disease.