Penelope N. Markham

Structural Basis of Multidrug Recognition by BmrR, a Transcription Activator of a Multidrug Transporter

Multidrug-efflux transporters demonstrate an unusual ability to recognize multiple structurally dissimilar toxins. A comparable ability to bind diverse hydrophobic cationic drugs is characteristic of the Bacillus subtilis transcription regulator BmrR, which upon drug binding activates expression of the multidrug transporter Bmr. Crystal structures of the multidrug-binding domain of BmrR (2.7 A resolution) and of its complex with the drug tetraphenylphosphonium (2.8 A resolution) revealed a drug-induced unfolding and relocation of an alpha helix, which exposes an internal drug-binding pocket. Tetraphenylphosphonium binding is mediated by stacking and van der Waals contacts with multiple hydrophobic residues of the pocket and by an electrostatic interaction between the positively charged drug and a buried glutamate residue, which is the key to cation selectivity. Similar binding principles may be used by other multidrug-binding proteins.

A structure-based mechanism for drug binding by multidrug transporters

Multidrug transporters bind chemically dissimilar, potentially cytotoxic compounds and remove them from the cell. How these transporters carry out either of these functions is unknown. On the basis of crystal structures of the multidrug-binding domain of the transcription activator BmrR and mutagenesis studies on the bacterial multidrug transporter MdfA, we propose a possible mechanism for the binding of cationic lipophilic drugs by multidrug transporters. The key element of this mechanism includes a conformational change in the transporter that exposes a buried charged residue in the substrate-binding pocket and allows access to this site by only those drugs that are its steric and electrostatic complements.

Efflux-mediated drug resistance in Gram-positive bacteria

Gram-positive bacteria express numerous membrane transporters that promote the efflux of various drugs, including many antibiotics, from the cell to the outer medium. Drug transporters can be specific to a particular drug, or can have broad specificity, as in so-called multidrug transporters. This broad specificity can be a consequence of the hydrophobic nature of transported molecules, as suggested by recent structural studies of soluble multidrug-binding proteins. Although the functions of drug transporters may involve both the protection of bacteria from outside toxins and the transport of natural metabolites, their clinical importance lies largely in providing Gram-positive pathogens with resistance to macrolides, tetracyclines and fluoroquinolones. A number of agents, discovered in recent years, that inhibit drug transporters can potentially be used to overcome efflux-associated antibiotic resistance.

Crime laboratory proficiency testing results, 1978-1991, II: Resolving questions of common origin.

A preceding article has examined the origins of crime laboratory proficiency testing and the performance of laboratories in the identification and classification of common types of physical evidence. Part II reviews laboratory proficiency in determining if two or more evidence samples shared a common source. Parts I and II together review the results of 175 separate tests issued to crime laboratories over the period 1978 to 1991. Laboratories perform best in determining the origin of finger and palm prints, metals, firearms (bullets and catridge cases), and footwear. Laboratories have moderate success in determining the source of bloodstains, questioned documents, toolmarks, and hair. A final category is of greater concern and includes those evidence categories where 10% or more of results disagree with manufacturers regarding the source of samples. This latter group includes paint, glass, fibers, and body fluid mixtures. The article concludes with a comparison of current findings with earlier LEAA study results, and a discussion of judicial and policy implications.

Crime laboratory proficiency testing results, 1978-1991, I: Identification and classification of physical evidence.

The proficiency testing of crime laboratories began in the mid-1970s and presently assumes an important role in quality assurance programs within most forensic laboratories. This article reviews the origins and early results of this testing program and also examines the progress of proficiency testing in allied scientific fields. Beginning in 1978, a fee-based crime laboratory proficiency testing program was launched and has grown to its present level involving almost 400 laboratories worldwide. This is the first of two articles that review the objectives, limitations and results of this testing from 1978 through 1991. Part I reviews the success of laboratories in the identification and classification of common evidence types: controlled substances, flammables, explosives, fibers, bloodstains, and hairs. Laboratories enjoy a high degree of success in identifying drugs and classifying (typing) bloodstains. They are moderately successful in identifying flammables, explosives, and fibers. Animal hair identification and human hair body location results are troublesome. The second paper will review the proficiency of crime laboratories in determining if two or more evidentiary samples shared a common origin.

IL-4 inhibits P-glycoprotein in normal and malignant NK cells

Patients presenting with a natural killer (NK) cell leukemia generally have a poor prognosis. NK cell tumors are generally resistant to numerous chemotherapeutic drugs and even combination chemotherapy usually results in only short term remissions. The drug resistance of NK cell leukemias may be at least partially explained by their expression of the multidrug resistant transporter, P-glycoprotein (Pgp). In this study, we demonstrate that the expression and function of Pgp activity on NK cells (leukemic and normal) can be reversed with IL-4.

Differential sensitivity of resting and IL-2 activated NK cells to R-verapamil

Natural killer (NK) cells are the first lymphoid population to reconstitute the peripheral blood compartment of immunologically compromised bone marrow transplant (BMT) recipients. Recent data suggest that, among patients transplanted for leukemia, NK cells can prevent or delay disease relapse by mediating a cytotoxic graft vs leukemia (GvL) response. Although the major mechanism by which NK cells mediate target cell lysis involves degranulation and release of cytolytic effector molecules (granzymes, proteoglycans, perforin), accumulating evidence suggests that NK cells possess additional pathways to mediate target cell killing. In fact, it is well recognized that recombinant cytokines such as IL-2 enhance the in vitro cytolytic activity of NK cells. In this study, we observed that the lytic activity mediated by resting and IL-2 activated NK cells against the same target cell appears to occur via two distinct pathways, as distinguished by their differential response to R-verapamil. Specifically, we observed that 25 microM R-verapamil inhibited the lytic activity of resting NK cells against K562 targets by approximately 50%. However, the lytic activity of IL-2 activated NK cells was unaffected by this concentration of R-verapamil. Additional studies suggested that the inhibitory effect of R-verapamil on NK cytotoxic activity was associated with its ability to prevent degranulation of cytotoxic granules. Specifically, R-verapamil inhibited BLT esterase release from resting but not IL-2 activated NK cells. These data suggest that IL-2 activated NK cells can promote target cell lysis by a pathway (possibly degranulation independent) distinct from that used by resting NK cells. We speculate that the target of R-verapamil on resting NK cells is P-glycoprotein (Pgp), an ABC transporter that we recently reported was expressed on NK cells and whose functional activity is known to be inhibited by R-verapamil.