Phenotypic heterogeneity for cells bearing surface immunoglobulin in human lymphoid lines.

Long-term cultures of human lymphoid lines, even after subcloning, were found to contain a mixture of surface immunoglobulin (SIg) positive and negative cells using three different techniques. Cell culture (TM) was mixed with anti-Ig antisera plus complement to lyse SIg positive cells. The remaining SIg negative viable cells were separated on a bovine serum albumin gradient, cultured, and sampled at different time points. Within 24–48 hr the same percentage of cells were SIg(+) as prior to treatment with antisera plus complement. Culturing the same cells with frequent additions of fresh or “spent” culture media failed to alter the ratio of Ig positive/ negative cells. Mechanisms for in vitro regulation of SIg are discussed.     

Effect of stabilizers on diaminobenzidine reactions of mitochondria.

The effect of various stabilizers--0.9% NaCl, 7.5% sucrose and 7.5% polyvinyl pyrrolidone (PVP) on reactions of mitochondria with fresh and photooxidized diaminobenzidine (DAB) was investigated. NaCl and PVP abolished the DAB staining of mitochondria incubated without exogenous cytochrome c; NaCl, however, was ineffective when exogenous cytochrome c or oxidized DAB were present in the incubation medium. PVP considerably decreased the intensity of all reactions performed in the presence of exogenous cytochrome c or oxidized DAB. The mechanism of the observed effect is not likely to involve osmotic protection of mitochondria; extraction of endogenous cytochrome c from fresh frozen tissue by NaCl and greatly increased viscosity of PVP-containing media seem to be most probable explanations.     

Synthesis of Gm allotypes by human tonsillar lymphocytes in culture: concordance with serum phenotype.

Isolated human tonsillar lymphocytes were cultured with pokeweed mitogen, phytohemagglutinin, and without mitogen for 9 to 28 days. IgK, Gm(a) and Gm(f) were then quantitated in the cell suspensions. In cultures of cells derived from persons whose blood was heterozygous for IgGl allotype antigens Gm(a+f+), approximately equal amounts of Gm(a) and Gm(f) were found. In cultures of cells of Gma or Gmf homozygotes, there was complete concordance between the Gm allotype antigens produced by the cultures and the donor's serum phenotype-with no instance, either at zero time or at culture termination in which a Gm antigen was detected which was absent from the donor's serum. It was concluded that in vitro genetic allotype synthesis in tonsillar lymphocytes during short-term culture mirrored accurately in vivo Gm expression. IgK and Gm antigen synthesis was highest in the flasks containing pokeweed mitogen although both phytohemagglutinin and no-mitogen control flasks showed, in certain experiments, proliferation and an increase in the Ig per viable cell. It was observed that no-mitogen flasks contained twice as much allotype antigen as did phytohemagglutinin flasks suggesting an inhibition of Ig synthesis associated with the mitogen. The tonsillar lymphocytes, under the experimental conditions employed, were shown by a radio-incorporation and immunoprecipitation technique to be synthesizing polyclonal Ig de novo, at the termination of the cultures.     

American and korean ginseng tissue cultures: Growth,chemical analysis,and plantlet production

Summary Roots, stems, or leaves of American (Panax quinquefolium) and Korean (Panax ginsing) ginseng were grown as callus or supension tissue cultures. Tissue cultures ofP. ginseng would occasionally form plantlets. The fundamental chemical composition, inorganic analysis, and saponin (panaquilin) content of American and Korean ginseng plants and tissue cultures were determined. The crude saponin content is very similar to, but approximately one-half (1.3%, fresh weight) of that present in ginseng roots. Two-dimensional thin layer chromatographic analysis revealed minor differences in the panaquilins present in American and Korean ginseng tissue cultures. The sapogenin, panaxadiol, was isolated from Korean ginseng callus.     

Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid: III. Formation of Long Single Strands During Recombination

Evidence was presented to support the hypothesis that long single strands appearing at late times (15 min after infection) are produced as a result of recombination and not as a continuous elongation during the replication process. The production of long strands does not depend on the multiplicity of infection, and the first long strands appear at the time when 20 to 50 phage equivalent units of deoxyribonucleic (DNA) are synthesized, and not earlier. The addition of chloramphenicol at 5 min, which prevents molecular recombination but allows replication of DNA, prevents the formation of long, single strands. Chloramphenicol added between 8 and 10 min after infection, a time at which molecular recombination is fully expressed and covalent repair of recombinant molecules is allowed, does not prevent formation of long single strands. Cutting of single-strand DNA with a limited amount of endonuclease I allows confirmation that the fast-sedimenting characteristic of intracellular denatured DNA is caused primarily by the length of the strands, and not by the formation of aggregates. The computer simulation of two recombination models indicates the feasibility of random breakage and rejoining of molecules in generating long concatenates.     

Standardization of Human Diploid Cell Cultivation

Human embryonic diploid lung fibroblasts grown in Eagle's medium were exposed continually to a variety of environmental conditions over a large number of passages to observe how these conditions affected the growth and longevity of these cells in vitro. The cells grew well at temperatures between 34 and 37 C and some cells could be adapted to grow at 40 C. Very limited growth occurred at 30 to 31 C; however, confluent monolayers of cells could be maintained for months at 30 C and still give rise to actively growing cultures. Increasing the amino acid concentration in Eagle's medium or the calf serum concentration above 10% had no effect on the growth rate or longevity. One per cent calf serum could not support prolonged active growth. Trypsin concentrations between 1 and 0.1% and crystalline trypsin at 50 μg/ml showed no influence on cell growth. Ethylenediaminetetraacetic acid treatment and scraping, however, destroyed many of the cells, and the survivors grew poorly. The clonal morphology varied with age. Young cells frequently gave rise to densely packed clones, whereas older cells gave rise to clones with widely scattered cells. The cloning efficiency was high when the cells were young but decreased rapidly with successive passage. It was relatively constant from the 7th to 20th passage at about 15%.