The effects of anthracycline drugs on the conformational distribution of mouse P-glycoprotein explains their transport rate differences.

P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The Km of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (KDs) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a "closed" conformation, while DOX shifts Pgp to an "intermediate" conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.

The conformation and dynamics of P-glycoprotein in a lipid bilayer investigated by atomic force microscopy.

The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed "small" and "large" protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these "small" and "large" protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80 ŠAFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the "small" and "large" protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.

IL-17 promotes Type 1 T cell response through modulating dendritic cell function in acute allograft rejection.

IL-17 is a cytokine that produced by various type of cell. Previous studies have been shown that IL-17 plays a critical role in the pathogenesis of different diseases. However, few studies have addressed the source and mechanism of IL-17 in the development of allograft rejection response. In this study, we present that the IL-17 expression reaches the strongest response at the early stage of cardiac allograft rejection, and was elevated earlier than DC maturation. The IL-17 is predominantly produced by CD3(+) T cells, whereas CD11c, CD11b, and NK1.1 positive cells rarely expressed IL-17. It is worth noting that blockade of endogenous IL-17 activity suppressed DC maturation, decreased inflammatory cytokines and impaired Th1 immune response during acute allograft rejection. Furthermore, adoptive transfer with DCs from IL-17-treated mice had a significant longer allograft survival time and decreased number of IFN-γ produced by T cells. Consistently, in an in vitro experiment, recombinant IL-17 significantly up-regulate co-stimulatory molecules of bone marrow derived dendritic cells (BMDCs), and IL-17-treated BMDCs show that an increased capacity to enhance T cell function was also observed. In conclusion, our data provide clear evidence that the early elevated level of IL-17 contributes to allograft rejection through modulating dendritic cell function.

Eosinophilic fascitis: a rare fibrosing disorder.

Eosinophilic fasciitis is a rare systemic disease characterized by inflammatory thickening of the skin and underlying fascia, peripheral eosinophilia, elevated inflammatory markers and typical biopsy findings. Internal organ involvement is rare unlike in other fibrosing disorders. Though this is usually a benign disease, it is often difficult to diagnose and the course may also be variable. Glucocorticoids are the mainstay of therapy, but in many cases steroid sparing immunosuppressive drugs are required particularly if the diseases has protracted course. The author reports a typical case of this rare disorder which could well be a first report from Nepal.

Behçet's disease: an account of three cases.

Behçet's disease is a chronic relapsing inflammatory condition affecting multiple organ-systems of the body. Patient's with this disease present to different specialists with wide spectrum of clinical features of varying severity. Worldwide, this disease is clustered around certain parts of Asia, previously known as 'Silk route'. Here we present three cases of Behçet's disease who presented to our clinic with ocular, dermatological, and articular manifestations.

Effect of carbon nanotubes on the isotropic to nematic and the nematic to smectic- A phase transitions in liquid crystal and carbon nanotubes composites.

A high-resolution ac-calorimetric study on the weakly first-order isotropic to nematic (I -N) and the continuous nematic to smectic-A (N -SmA) phase transitions of the liquid crystal octyl-cyanobiphenyl (8CB) doped with well-dispersed multiwall carbon nanotubes (CNTs) as a function of CNT concentrations is reported. Thermal scans were performed for all samples having CNT weight fraction from Φ(w) = 0.0005 to 0.0060 over a wide temperature range well above and below the two transitions in pure 8CB. Both the I -N and the N -SmA transitions evolve in character and have their transition temperatures qualitatively offset by ∼ 1.10 K lower as compared to that in pure 8CB for all 8CB+CNTs samples. The enthalpy change associated with each phase transition is essentially the same as that of pure 8CB and remains unchanged with increasing Φ(w). However, there is an evidence that the thermal transport properties of the composites differ from the pure LC upon cooling below a Φ(w)-dependent temperature within the nematic phase. In addition, a new C(p) feature is resolved for intermediate Φ(w) samples that appears to be correlated to this onset temperature.

Evolution of the isotropic to nematic phase transition in binary mixtures of octylcyanobiphenyl and n-hexane.

High-resolution calorimetry has been performed to study the effect of dilution by a nonmesogenic, low-molecular weight solvent (linear n-hexane) on the isotropic-nematic (I-N) phase transition in the liquid crystal (LC) octylcyanobiphenyl (8CB) as a function of n-hexane concentration. Numerous temperature scans were performed without continuous mixing for pure 8CB and all mixture samples of n-hexane mole fraction ranging from x(hex)=0.02 to 0.12. The I-N specific heat peak remains first-order for all samples and shifts toward lower temperature nonlinearly while the two-phase I+N coexistence width broadens linearly with increasing x(hex). Multiple heating and cooling scans are reproducible and indicate phase separation, if it occurs, must be on very short length scales and is reversible. These results may be a consequence of a competition between random dilution effects and the tendency to phase separate. It is shown that solvent dilution of a LC, if miscible and depending on solvent structure, can lead to a controlled altering of the intermolecular potentials and softening of the LC viscoelastic properties.