Structural basis for recognition of transcriptional terminator structures by ProQ/FinO domain RNA chaperones.

The ProQ/FinO family of RNA binding proteins mediate sRNA-directed gene regulation throughout gram-negative bacteria. Here, we investigate the structural basis for RNA recognition by ProQ/FinO proteins, through the crystal structure of the ProQ/FinO domain of the Legionella pneumophila DNA uptake regulator, RocC, bound to the transcriptional terminator of its primary partner, the sRNA RocR. The structure reveals specific recognition of the 3' nucleotide of the terminator by a conserved pocket involving a ß-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif. Structure-guided mutagenesis reveals key RNA contact residues that are critical for RocC/RocR to repress the uptake of environmental DNA in L. pneumophila. Structural analysis and RNA binding studies reveal that other ProQ/FinO domains also recognize related transcriptional terminators with different specificities for the length of the 3' ssRNA tail.

Cyanidin-3-O-Glucoside improves the viability of human islet cells treated with amylin or Aß1-42 in vitro.

Islet transplantation is being considered as an alternative treatment for type 1 diabetes. Despite recent progress, transplant recipients continue to experience progressive loss of insulin independence. Cyanidin-3-O-Glucoside (C3G) has shown to be protective against damage that may lead to post-transplant islet loss. In this study, human islets cultured with or without C3G were treated with human amylin, Aß1-42, H2O2, or rapamycin to mimic stresses encountered in the post-transplant environment. Samples of these islets were collected and assayed to determine C3G's effect on cell viability and function, reactive oxygen species (ROS), oxidative stress, amyloid formation, and the presence of inflammatory as well as autophagic markers. C3G treatment of human islets exposed to either amylin or Aß1-42 increased cell viability (p<0.01) and inhibited amyloid formation (p<0.01). A reduction in ROS and an increase in HO-1 gene expression as well as in vitro islet function were also observed in C3G-treated islets exposed to amylin or Aß1-42, although not significantly. Additionally, treatment with C3G resulted in a significant reduction in the protein expression of inflammatory markers IL-1ß and NLRP3 (p<0.01) as well as an increase in LC3 autophagic marker (p<0.05) in human islets treated with amylin, Aß1-42, rapamycin, or H2O2. Thus, C3G appears to have a multi-faceted protective effect on human islets in vitro, possibly through its anti-oxidant property and alteration of inflammatory as well as autophagic pathways.

Early immune mechanisms of neonatal porcine islet xenograft rejection.

BACKGROUND: Neonatal pigs have the potential to provide an inexhaustible source of islets for the treatment of type 1 diabetes. However, the immunological barriers to islet xenotransplantation still need to be overcome. A better understanding of the xeno-specific immune responses that are involved in neonatal porcine islet (NPI) xenotransplant rejection will help to facilitate the identification of new targets for anti-rejection therapies, and thus enable more specific targeting of the immune cells and molecules involved. METHODS: In this study, we examined the early events of NPI xenograft rejection in the absence of autoimmunity using an immune-competent B6 mouse transplant model. Immune cells were identified by immunohistochemistry and immune molecules were identified by reverse transcription-PCR and flow cytometry assays. RESULTS: Our results demonstrated that early events in NPI xenograft rejection are characterized by initial infiltration of innate immune cells such as macrophages (M1) and neutrophils. CONCLUSIONS: Targeting these cells, which appear early in the rejection process, may provide an opportunity to abort the rejection process prior to activation of T cells. One strategy could be the blockade of chemotactic signals associated with preferential recruitment of immune cells into the graft site. Collectively, our studies demonstrated that early recruitment of immune cells into graft site is controlled by chemotactic activities and suggest a potential target to prevent the early infiltration of immune cells within the graft. Our findings in this study will have significance in improving NPI xenograft acceptance and induce long-term xenograft survival.

Protective effect of cyanidin-3-O-glucoside on neonatal porcine islets.

Oxidative stress is a major cause of islet injury and dysfunction during isolation and transplantation procedures. Cyanidin-3-O-glucoside (C3G), which is present in various fruits and vegetables especially in Chinese bayberry, shows a potent antioxidant property. In this study, we determined whether C3G could protect neonatal porcine islets (NPI) from reactive oxygen species (H2O2)-induced injury in vitro and promote the function of NPI in diabetic mice. We found that C3G had no deleterious effect on NPI and that C3G protected NPI from damage induced by H2O2 Significantly higher hemeoxygenase-1 (HO1) gene expression was detected in C3G-treated NPI compared to untreated islets before and after transplantation (P < 0.05). Western blot analysis showed a significant increase in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K/Akt) proteins in C3G-treated NPI compared to untreated islets. C3G induced the nuclear translocation of nuclear erythroid 2-related factor 2 (NRF2) and the significant elevation of HO1 protein. Recipients of C3G-treated NPI with or without C3G-supplemented drinking water achieved normoglycemia earlier compared to recipients of untreated islets. Mice that received C3G-treated islets with or without C3G-supplemented water displayed significantly lower blood glucose levels at 5-10 weeks post-transplantation compared to mice that received untreated islets. Mice that received C3G-treated NPI and C3G-supplemented drinking water had significantly (P < 0.05) lower blood glucose levels at 7 and 8 weeks post-transplantation compared to mice that received C3G-treated islets. These findings suggest that C3G has a beneficial effect on NPI through the activation of ERK1/2- and PI3K/AKT-induced NRF2-mediated HO1 signaling pathway.