Carol E. Bennett

Trace metal levels in commercially prepared tissue culture media

Four trace elements, lead, copper, tin and zinc, in addition to certain electrolytes, were measured in 11 commercially prepared tissue culture media. Glass media bottles and plastic tissue culture dishes and flasks were treated with a HCl acid solution to determine the amounts of trace metals leached from their surfaces. Zinc, lead and copper were detected in all media. Tin was detected only in RPMI Medium 1640, fetal bovine serum, minimum essential medium and penicillin-streptomycin. It is possible that a major cause of variability in tissue culture experimental results may be due to effects on growth caused by fluctuation in trace element contamination from batch to batch. Variability in establishing primary cultures of corneal endothelial cells was traced to high lead levels in commercially prepared tissue culture media. A strong case is made for continued diligent efforts to expand analytical horizons and our definition of substances in culture media.

Corneal endothelium: A modified method for cultivation

SummaryA modified method for establishing cultures of rabbit corneal cells is described. The new technique utilized a Lucite disc in combination with a Tygon ring for growth of pure cell cultures and was compared with an explant method for growing cells. Each method provided adequate cell cultures for biochemical or ultrastructure studies of rabbit corneal cells, but the ring and disc method described here allowed the isolation of specific cell types without the interference of stromal cell contamination.

DNA and protein synthesis in normal and transformed lymphocytes exposed to abrin

The dose dependent effects of abrin, a toxic D-galactose binding plant lectin, on 3H-TdR and 14C-leucine uptake are studied in normal and Epstein Barr Virus (EBV) transformed lymphocyte cultures. Results show that while abrin is highly toxic to both the DNA and protein synthesis in EBV lymphocytes, some toxicity to the normal cells is also seen. It is postulated that lymphocyte DNA synthesis is affected by ribosomal shutdown induced by the abrin.

Studies on the Toxicity and Binding Kinetics of Abrin in Normal and Epstein Barr Virus-Transformed Lymphocyte Culture-I: Experimental Results – 3

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of sites bound/EBV-lymphocyte needed to exert a biological influence upon the cell DNA synthesis lies between 59,264 and 370,000 sites/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 and 360,000 sites/cells.

On the dynamics of abrin binding to receptor sites in normal and Epstein Barr Virus transformed lymphocyte cell cultures.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA an protein synthesis of normal and Epstein Barr Virus transformed lymphocytes has been investigated. Activation, stimulation, and relative toxicity factor indices are studied, as well as possible relationships between DNA and protein synthesis rates, as measured by simultaneous tritiated thymidine (3H-TdR) and 14C-leucine uptake. Studies of the two new indices, the metabolic self and cross coupling indices lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin receptor site concentrations. This prediction is supported by SEM morphological differences. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of receptors bound-EBV-lymphocyte needed to exert a biological influence lies in the interval 59,264 receptors/cell to 370.040 receptors/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 receptors/cell and 360,000 receptors/cell.

Studies on the Toxicity and Binding Kinetics of Abrin in Normal and Epstein-Barr Virus-Transformed Lymphocyte Culture-I

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein-Barr virus (EBV)-transformed lymphocytes have been previously investigated. Using data on EBV-transformed lymphocyte cell density as a function of both time and dose of abrin, the authors introduced the concept of self- and cross-coupling metabolic variables as a means of understanding how abrin affected DNA and protein uptake. In this paper, the self-coupling constant is studied in more detail and the relationship between DNA and protein synthesis is further expanded. We find that there is a significant linear relationship between DNA and protein synthesis in normal lymphocyte culture as measured by abrin interaction in the culture. We further find that there is a much stronger relationship between these variables in EBV-transformed lymphocyte culture. This relationship is further examined, and possible analytic equations are expressed.

Carbohydrate metabolism of lymphocytes: Modified methodology and comparisons of diabetics with non-diabetics

Changes in the hexose monophosphate shunt (HMPS) and Krebs cycle activity during lymphocyte blast transformation are reported in 50 patients with diabetes mellitus and 50 non-diabetics. A modified technique using 12 × 75 mm sterile tubes and micropipette tips stuffed with filter paper was used. The filter paper, soaked with hyamine hydroxide, absorbed radioactively labeled CO2 produced from [14C]-labeled glucose incorporated by cells. [14C]CO2 from glucose labeled at the C-1 position measured the activity of the HMPS. [14C]CO2 from glucose labeled at the C-2 position measured the HMPS activity associated with the feedback of pentose sugars. [14C]-labeled CO2 from glucose labeled at the C-6 position was used to measure Krebs cycle activity. A statistically significant decrease in HMPS activity was found in diabetic cells exposed to the mitogens phytohemagglutinin-P (PHA-P), concanavalin-A (CON-A) and pokeweed mitogen (PWM) (P < 0.01). This decrease in HMPS activity and its relation to lymphocyte blast transformation may be related to the increased incidence of infection known to occur in patients with diabetes mellitus.