Specific protein domains and amino acids responsible for the apparent capacity of P-glycoprotein (mdr) to recognize and transport a large group of structurally unrelated drugs have not been identified. We have introduced a single Ser -* Phe substitution within the predicted TM11 domain of mdrl (position 941) and mdr3 (position 939) and analyzed the effect of these substitutions on the drug-resistance profiles of these two proteins. Mutations at this residue drastically altered the overall degree of drug resistance conveyed by mdrl and mdr3. The modulating effect of this mutation on mdrl and mdr3 varied for the drugs tested: it was very strong for colchicine and adriamycin and moderate for vinbLastne. For mdrl, the Ser'4' -+ Phe'4' substitution produced a unique mutant protein that retained the capacity to confer vinblastine resistance but lost the ability to confer adriamycin and colchicine resistance. These results strongly suggest that the predicted TM11 domain of proteins encoded by mdr and mdrlike genes plays an important role in the recognition and transport of their specific substrates.Multidrug resistance is caused by the amplification and overexpression ofa small gene family, designated mdr orpgp (1), which is composed of two members in humans, MDR] and MDR2 (2, 3), and three members in rodents, mdrl (mdrlb), mdr2, and mdr3 (mdrla) (4)(5)(6)(7), that code for membrane P-glycoproteins (P-gps). P-gp has been shown to bind ATP (8) and drug analogs (9, 10) and has ATPase activity (11). It is believed to function as an ATP-dependent drug efflux pump to reduce intracellular drug accumulation in resistant cells (1,12). Sequence analyses of mdr gene cDNA clones predict polypeptides composed of 12 transmembrane (TM) domains and two nucleotide binding (NB) sites. P-gps are formed by two symmetrical and sequence homologous halves that share a common ancestral origin with a large group of bacterial transport proteins (13). The mdr gene family is part of a larger family of mdr-like genes encoding sequencehomologous proteins sharing similar predicted secondary structures and proposed membrane-associated transport functions. The pfmdrl gene of Plasmodium falciparum (14) associated with chloroquine (CLQ) resistance, the yeast STE-6 gene responsible for export of the a mating pheromone in Saccharomyces cerevisiae (15), and the CFTR gene in which mutations cause cystic fibrosis in humans (16) form part of this family. The regions of strongest sequence homology among these proteins overlap the predicted NB sites, which are believed to underlie a common functional aspect of transport (17). The protein segments and residues implicated in substrate binding and transport have not been precisely identified. Despite a very high degree of sequence homology (between 75% and 85% identity), striking functional differences have been detected between individual mdr genes. Mouse mdrl (18) and mdr3 (6, 19) and human MDR] (20) can confer multidrug resistance in transfection experiments, whereas mouse mdr2 (5) and human MDR2 (3...

The substitution of a single serine to phenylalanine residue within the predicted transmembrane domain 11 of P-glycoproteins (P-gps) encoded by mouse mdr1 (Ser941, 1S;Phe941, 1F) or mdr3 (Ser939, 3S; Phe939, 3F) strongly modulates both the overall activity and substrate specificity of the two P-gps. In cell clones expressing either wild-type (1S, 3S) or mutant P-gps (1F, 3F), we show that the modulating effect of the mutation on the levels of adriamycin (ADM) resistance detected in drug cytotoxicity assays is paralleled by a similar modulation of the intracellular accumulation and extracellular efflux of radiolabeled adriamycin ([14C]ADM) from preloaded cells. Cytofluorescence studies with ADM on live cells produce similar results and demonstrate strong nuclear ADM accumulation only in drug-sensitive LR cells and in the 1F expressing cells, with little if any accumulation in 1S, 3S, or 3F expressing cells. Drug cytotoxicity and drug transport assays carried out in the presence of verapamil or progesterone suggest that the Ser to Phe substitution also reduces the capacity of these two reversal agents to modulate P-gp activity. Labeling experiments with the photoactivatable P-gp ligands iodoarylazidoprazosin and azidopine indicate a strong reduction in binding of these photoactivatable probes to the mutant P-gps (1F, 3F) as compared to their wild-type counterparts (1S,3S). These results indicate that the studied mutations in TM11 reduce drug transport by decreasing initial drug binding to P-gp. This phenotype is opposite to that of a mutation near TM3 in human MDR1 (pst 185), where decreased drug transport is associated with increased drug binding and decreased drug release from P-gp [Safa, A. R., Stern, R. K., Choi, K., Agresti, M., Tamai, I., Metha, N. D., & Roninson, I. B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7225-7229].

The possibility that simple lipophilic cations such as tetraphenylphosphonium (TPA+), triphenylmethylphosphonium (TPMP+), and diphenyldimethylphosphonium (DDP+) are substrates for the multidrug-resistance transport protein, P-glycoprotein, was tested. Hamster cells transfected with and overexpressing mouse mdr1 or mouse mdr3 exhibit high levels of resistance to TPP+ and TPA+ (20-fold) and somewhat lower levels of resistance to TPMP+ and DDP+ (3-12-fold). Transfected cell clones expressing mdr1 or mdr3 mutants with decreased activity against drugs of the MDR spectrum (e.g., Vinca alkaloids and anthracyclines) also show reduced resistance to lipophilic cations. Studies with radiolabeled TPP+ and TPA+ demonstrate that increased resistance to cytotoxic concentrations of these lipophilic cations is correlated quantitatively with a decrease in intracellular accumulation in mdr1- and mdr3-transfected cells. This decreased intracellular accumulation is shown to be strictly dependent on intact intracellular nucleotide triphosphate pools and is reversed by verapamil, a known competitive inhibitor of P-glycoprotein. Taken together, these results demonstrate that lipophilic cations are a new class of substrates for P-glycoprotein and can be used to study its mechanism of action in homologous and heterologous systems.

Abstract:The monoclonal antibody 21E7-B12

Several hybridoma cell lines producing murine monoclonal antibodies (mAbs) directed to the Clostridium tyrobutyricum outer cell wall have been established and characterized. Whole bacteria, crude extract of cell wall, and polysaccharide fraction of crude extract have been used as immunogens. The immunizations were performed either in vivo or in vitro after priming in vivo. Amongst the clones obtained, six hybridoma cell lines were selected. Four mAbs recognized only the immunizing strain (ATCC 25755), while two mAbs recognized all the C. tyrobutyricum tested strains. Three mAbs were IgM, one IgG3, and two IgG1 isotypes. The antigens (proteins or polysaccharides) recognized by these mAbs have been characterized by Western Blot. These mAbs could be used for an early detection of C. tyrobutyricum in milk.