Refined structures of mouse P-glycoprotein.

The recently determined C. elegans P-glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible misinterpretations in the latter model. To address this concern, we generated an experimental electron density map from single-wavelength anomalous dispersion phasing of an original mouse Pgp dataset to 3.8 Šresolution. The map exhibited significantly more detail compared to the original MAD map and revealed several regions of the structure that required de novo model building. The improved drug-free structure was refined to 3.8 Šresolution with a 9.4 and 8.1% decrease in R(work) and R(free), respectively, (R(work) = 21.2%, R(free) = 26.6%) and a significant improvement in protein geometry. The improved mouse Pgp model contains ∼95% of residues in the favorable Ramachandran region compared to only 57% for the original model. The registry of six transmembrane helices was corrected, revealing amino acid residues involved in drug binding that were previously unrecognized. Registry shifts (rotations and translations) for three transmembrane (TM)4 and TM5 and the addition of three N-terminal residues were necessary, and were validated with new mercury labeling and anomalous Fourier density. The corrected position of TM4, which forms the frame of a portal for drug entry, had backbone atoms shifted >6 Šfrom their original positions. The drug translocation pathway of mouse Pgp is 96% identical to human Pgp and is enriched in aromatic residues that likely play a collective role in allowing a high degree of polyspecific substrate recognition.

Enhanced rat islet function and survival in vitro using a biomimetic self-assembled nanomatrix gel.

Peptide amphiphile (PA) is a peptide-based biomaterial that can self-assemble into a nanostructured gel-like scaffold, mimicking the chemical and biological complexity of natural extracellular matrix. To evaluate the capacity of the PA scaffold to improve islet function and survival in vitro, rat islets were cultured in three different groups--(1) bare group: isolated rat islets cultured in a 12-well nontissue culture-treated plate; (2) insert group: isolated rat islets cultured in modified insert chambers; (3) nanomatrix group: isolated rat islets encapsulated within the PA nanomatrix gel and cultured in modified insert chambers. Over 14 days, both the bare and insert groups showed a marked decrease in insulin secretion, whereas the nanomatrix group maintained glucose-stimulated insulin secretion. Moreover, entire islets in the nanomatrix gel stained positive for dithizone up to 14 days, indicating better maintained glucose-stimulated insulin production. Fluorescein diacetate/propidium iodide staining results also verified necrosis in the bare and insert groups after 7 days, whereas the PA nanomatrix gel maintained islet viability after 14 days. Thus, these results demonstrate the potential of PAs as an intermediary scaffold for increasing the efficacy of pancreatic islet transplantation.

AMP-activated protein kinase attenuates nitric oxide-induced beta-cell death.

During the initial autoimmune response in type 1 diabetes, islets are exposed to a damaging mix of pro-inflammatory molecules that stimulate the production of nitric oxide by beta-cells. Nitric oxide causes extensive but reversible cellular damage. In response to nitric oxide, the cell activates pathways for functional recovery and adaptation as well as pathways that direct beta-cell death. The molecular events that dictate cellular fate following nitric oxide-induced damage are currently unknown. In this study, we provide evidence that AMPK plays a primary role controlling the response of beta-cells to nitric oxide-induced damage. AMPK is transiently activated by nitric oxide in insulinoma cells and rat islets following IL-1 treatment or by the exogenous addition of nitric oxide. Active AMPK promotes the functional recovery of beta-cell oxidative metabolism and abrogates the induction of pathways that mediate cell death such as caspase-3 activation following exposure to nitric oxide. Overall, these data show that nitric oxide activates AMPK and that active AMPK suppresses apoptotic signaling allowing the beta-cell to recover from nitric oxide-mediated cellular stress.

Human T cell clones specific for heterogeneous nuclear ribonucleoprotein A2 autoantigen from connective tissue disease patients assist in autoantibody production.

OBJECTIVE: To identify and characterize human T cells reactive with heterogeneous nuclear RNP A2 (hnRNP A2) antigen, and to determine the ability of hnRNP-reactive T cells to assist in the production of human autoantibodies. METHODS: T cells from patients with high serum levels of anti-hnRNP IgG autoantibody were stimulated with an hnRNP recombinant fusion protein, and the cells were cloned by limiting dilution. The surface phenotype and cytokine profiles of the T cells were examined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. T cell clones were cultured with highly purified autologous B cells, and the ability of T cells to enhance autoantibody production under a variety of conditions was measured by ELISA. RESULTS: Human T cells reactive with hnRNP antigen were cloned from 2 patients with systemic lupus erythematosus (SLE) and 1 patient with mixed connective tissue disease (MCTD). The T cells were CD4+ and had a Th1-like functional phenotype. In coculture in vitro with autologous B cells, T cell clones augmented anti-hnRNP autoantibody production and did so without the need for direct T cell-B cell contact. CONCLUSION: This study provides direct evidence for a role of anti-hnRNP-reactive T cells in autoantibody production in SLE and MCTD. These findings support the notion that hnRNP-reactive T cells play a role in the pathogenesis of these diseases.