The movement of proteins into and out of enamel was followed over time using a highly sensitive microprecipitation technique to quantify the amount of TCA-insoluble radioactivity present within small pieces of freeze-dried enamel and cells (enamel organ) dissected from the mandibular incisors of rats injected with L-[35S]-methionine. Conventional image processing techniques were also used to estimate the number of silver grains over enamel and cells in radioautographs of mandibular incisors from rats similarly injected with L-[methyl-3H]-methionine. Data from both techniques indicated that the average half-life for labeled proteins secreted into enamel was about 8.9 days. Typically, radioactive proteins accumulated in increasing amounts for 8 hours after which they were lost slowly up to 4 days and more rapidly thereafter when enamel formed during the secretory stage underwent maturation. The half-life for radioactive proteins in cells was only about 20.7 hours. No significant accumulation of radioactivity could be detected in the TCA-soluble or TCA-insoluble fractions of cells as enamel development proceeded. Results from this study suggest that radioautographs provide an accurate estimate of changes occurring to proteins in enamel and cells except at early time intervals (less than 1 hour) when a high percentage of total radioactivity is present within the TCA-soluble fraction of cells.

Animals↗

PubMed1990

Benzo(a)pyrene inhibits epidermal growth factor binding and receptor autophosphorylation in human placental cell cultures.

Studies investigated the effects of benzo(a)pyrene (BP) treatment on epidermal growth factor (EGF) receptor binding and kinase activity in human placental cell cultures. Specific binding of 125I-EGF to cells from early gestation placentae was significantly decreased by 37 and 60% following exposure to 1 and 10 microM BP, respectively, for 24 hr. In contrast, cells cultured from term placentae showed no inhibitory effect of either concentration of BP. Specific binding of 125I-labeled insulin and insulin-like growth factors-I and -II to early gestation cells was decreased only 15-18% at 10 microM BP, which indicates that loss of membrane receptors appears to be selective for EGF. Scatchard analysis of early gestation cells revealed that BP was associated with a dose-dependent loss in the number of high affinity EGF binding sites. Evidence from cross-linking and autophosphorylation experiments confirmed that the Mr 170,000 binding protein was decreased in a dose-dependent manner following BP treatment. In comparison, term placental cells exhibit a 26% loss of EGF receptor autophosphorylation without alteration in binding following exposure to 10 microM BP. Thus, early gestation cells exhibit a BP-related down-regulation of EGF receptors, whereas term placental cells show receptor desensitization. No adverse effect of BP treatment was observed on the incorporation of [35S] methionine into proteins secreted by early gestation cells. Further experiments compared the effects of BP with the related poly-cyclic compounds beta-naphthoflavone, alpha-naphthoflavone, and 3-methylcholanthrene. In early gestation cells, EGF binding and receptor autophosphorylation were measurably decreased at 10 microM concentrations of these polycyclic compounds, but to a lesser extent than observed with BP. In term placental cells, however, EGF binding was unchanged or increased, whereas receptor autophosphorylation was decreased 10-26%. Thus, exposure of term placental cells to these polycyclic compounds leads to a dissociation between EGF binding and receptor protein kinase activity. Finally, aryl hydrocarbon hydroxylase activity was induced 20- to 200-fold in early placental cells exposed to BP, beta-naphthoflavone, and 3-methylcholanthrene. In summary, the direct effects of BP and related compounds observed on placental EGF receptors may indicate altered function of EGF in the regulation of cell growth and differentiation in the human placenta.

Aryl Hydrocarbon Hydroxylases↗

PubMed1983

The effect of suboptimal concentrations of mitogens on the immunosuppressive action of azathioprine and prednisolone on human lymphocytes in vitro.

The interaction of two commonly used immunosuppressive agents, azathioprine (AZA) and prednisolone sodium succinate (PSS), with three mitogens, Concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) was studied. Using the endpoint of 50% inhibition of 3H-thymidine incorporation (ID50) as an indicator of cellular replication, the affect of suboptimal and optimal concentrations of mitogen on the immunosuppressive action of AZA or PSS on human lymphocyte replication in vitro was determined by isobolic analysis. The AZA ID50 for optimal concentrations of Con A (60 mcm/cc) was 242 +/- 21 ngm/cc (mean +/- SEM, n = 8), and at a suboptimal concentration of Con A (20 mcg/cc) it was 233 +/- 42, i.e., the AZA ID50 was independent of the concentration of Con A. This same independent interaction was found for PHA and PWM. However, at the optimal concentration of Con A (60 mcg/cc) the PSS ID50 of 523 +/- 55 ngm/cc was significantly higher (p less than 0.001) than the PSS ID at the suboptimal concentration of Con A (20 mcg/cc) which was 234 +/- 24 ngm/cc. A similar antagonistic interaction was found between PSS and either PHA or PWM. These findings emphasize the importance of accurately defining the testing parameters when assessing a drug's effect on lymphocyte proliferation. The data, along with other studies, support the hypothesis that drugs which directly interfere with DNA synthesis will not be affected by the strength of the signal for cellular replication, whereas those that modulate cellular replication are affected by the strength for lymphocyte replication.

Adult↗