Multidrug-resistant human tumor cells overexpress the MDRI gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/Vol) ocyl 3-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCi fraction, enriched in P-glycoprotein but devoid of NaK-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P- Several previous reports suggest the association ofATPase activity with P-glycoprotein: (i) the ATP analog 8-azido-[32-P]ATP binds to P-glycoprotein (8); (ii) the immunoaffinity-purified protein exhibits a low level of ATPase activity (9); and (iii) in vitro mutagenesis of the consensus sequences of either or both ATP-binding domains of the MDR] cDNA fails to confer drug resistance in transfected cells that express the altered protein (10, 11). In addition, the ATP-dependent transport of vinblastine by membrane vesicles of multidrugresistant cells has been demonstrated (12). Thus, it has been proposed that P-glycoprotein catalyzes ATP-dependent efflux of drugs from resistant cells. These previous studies, however, do not conclusively prove that the two functions (i.e., ATP hydrolysis and drug transport) are directly mediated by P-glycoprotein. To establish that P-glycoprotein is an ATP-dependent multidrug transporter, purification and functional reconstitution into phospholipid vesicles is essential. We describe here partial purification and reconstitution of P-glycoprotein. The data suggest that P-glycoprotein exhibits a high level of substrate-stimulatable ATPase activity similar to other ion-transporting pumps. MATERIALS AND METHODSPreparation of Plasma Membrane Vesides. The multidrugresistant human carcinoma KB-V1 cells, a subclone of KB3-1, were grown in the presence of vinblastine (1 ,g/ml) to confluence (13). The membrane vesicles were prepared by nitrogen cavitation and sucrose density gradient centrifugation as described (12). The final pellet of vesicles was resuspended and stored in vesicle buffer (10 mM Tris HCl, pH 7.4/50 mM NaCl/250 mM sucrose/0.5 mM phenylmethylsulfonyl fluoride) at -700C.Solubilization of P-Glycoprotein. The solubilization of membrane proteins followed the protocols established earlier (14,15

In Drosophila, germ cells migrate in embryonic development from the lumen of the developing gut towards the overlying mesoderm, where they enter the gonads. The gene wunen is responsible for guiding the germ cells early in this process. Here we report that the protein Wunen has two properties that allow it to use repulsion to guide the germ cells. Wunen can transform a permissive cellular environment into a repulsive one, and is expressed in the gut in a pattern that guides germ cells towards the mesoderm. Wunen shows strong similarity to the enzyme type 2 phosphatidic acid phophatase (PAP2)4, suggesting that it is involved in lipid metabolism.

When the integrity of the skin got damaged, an endogenous electric field will be generated in the wound and a series of physiological reactions will be initiated to close the wound. The existence of the endogenous electric field of the wound has a promoting effect on all stages of wound healing. For wounds that cannot heal on their own, the exogenous electric field can assist the treatment. In this review, the effects of exogenous electrical stimulation on wound healing, such as the inflammation phase, blood flow, cell proliferation and migration, and the wound scarring is overviewed. This article also reviews the new electrical stimulation methods that have emerged in recent years, such as small power supplies, nanogenerators (NGs), and other physical, chemical or biological strategies. These new electrical stimulation methods and devices are safe, low‐cost, stable, and small in size. The challenge and perspective are discussed for the future trends of the electrical stimulation treatment in accelerating skin wound healing.