Immunochemical purification of probe-labeled plasma membrane proteins: an approach to the molecular anatomy of the cell surface

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labeling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80--100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37 degrees C but not at 41 degrees C, the expression of this component is related to the expression of the transformed phenotype.     

PSORALEN PLUS ULTRAVIOLET RADIATION-INDUCED INHIBITION OF DNA SYNTHESIS AND VIABILITY IN HUMAN LYMPHOID CELLS IN VITRO*

Abstract— 8-Methoxypsoralen (8-MOP) plus high intensity long wavelength ultraviolet radiation (UV-A) is presently being used to induce remissions of psoriasis and mycosis fungoides. Previous studies demonstrated inhibition of DNA synthesis in circulating leukocytes from some patients during this therapy. The present study is designed to determine whether conditions of 8-MOP concentration and UV-A exposure attained during therapy might be sufficient to result directly in decreased lymphoid cell DNA synthesis and viability in vitro. Tritiated thymidine (³HTdR) incorporation and cell growth in suspension culture following UV-A exposure alone or with therapeutic concentrations of 8-MOP was examined in peripheral blood lymphocytes and in Ebstein-Barr virus transformed human lymphoblas-toid cell lines. UV-A exposure alone induced a dose-dependent inhibition of HTdR incorporation in both types of lymphoid cells (3000 J/m² resulted in 77% of control ³HTdR incorporation). Pre-incubation with 0.1 μg/m/ 8-MOP before UV-A exposure induced a significantly greater inhibition of ³HTdR incorporation (3000 J/m² resulted in 61% of control ³HTdR incorporation). Greater inhibition of ³HTdR incorporation was observed by preincubation of the lymphoblastoid cells with 1.0μg/mC 8-MOP (3000 J/m² resulted in 41% of control) but not in the lymphocytes (3000 J/m² resulted in 63% of control). The concentration of viable lymphoblastoid cells did not decrease below the original concentration after the highest dose of UV-A alone (29,000 J/m²) but preincubation with 0.1 μg/mC 8-MOP resulted in 40% survival after 3000 J/m² (D₃₇ approximately 3000 J/m²) and preincubation with 1.0 μg/ 8-MOP resulted in 0.6% survival after 3000 J/,² (D₃₇ approximately 800 J/m²). This study suggests that the low doses of 8-MOP and UV-A received by patients' lymphocytes may be sufficient to explain the decreased DNA synthesis found in their circulating leucocytes. The long term consequences of such damage remain to be determined.     

EFFECT OF INTENSITY AND WAVELENGTH OF FLUORESCENT LIGHT ON CHROMOSOME DAMAGE IN CULTURED MOUSE CELLS

Abstract—A single 3- to 20-hr exposure of line NCTC 9266 mouse cells to cool-white fluorescent light (4.6 W/m²) produces chromatid breaks and exchanges. The effective wavelength is in the visible range and coincides with the mercury emission peak at 405 nm. Increasing light intensity from 4.6 W to 15.3 W/m² for 20 h causes a concomitant increase both in production of chromosome damage and formation of hydrogen peroxide (H₂O₂) in the serum-free medium. Cells washed free of medium and illuminated in saline for 3 h show chromosome damage to the same extent as cells illuminated in culture medium. Addition of catalase during the exposure period of 3 h eliminates the light-induced damage. We conclude that the light-induced chromatid breaks and exchanges result from H₂O₂ production within the cell and that exogenous catalase can enter the cell and prevent the damage.