High Extracellular Ca2+ Stimulates Ca2+-Activated Cl− Currents in Frog Parathyroid Cells through the Mediation of Arachidonic Acid Cascade

Elevation of extracellular Ca2+ concentration induces intracellular Ca2+ signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca2+ pathways, but the direct mechanism responsible for the rise of intracellular Ca2+ concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca2+ signaling in frog parathyroid cells and show that Ca2+-activated Cl− channels are activated by intracellular Ca2+ increase through an inositol 1,4,5-trisphophate (IP3)-independent pathway. High extracellular Ca2+ induced an outwardly-rectifying conductance in a dose-dependent manner (EC50∼6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca2+-induced and Ca2+ dialysis-induced currents reversed at the equilibrium potential of Cl− and were inhibited by niflumic acid (a specific blocker of Ca2+-activated Cl− channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca2+-induced current, suggesting the change of intracellular Cl− concentration in a few minutes. Extracellular Ca2+-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca2+-induced current. IP3 dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca2+-induced conductance. These results indicate that high extracellular Ca2+ raises intracellular Ca2+ concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl− conductance

Genetic Variations in the Regulator of G-Protein Signaling Genes Are Associated with Survival in Late-Stage Non-Small Cell Lung Cancer

The regulator of G-protein signaling (RGS) pathway plays an important role in signaling transduction, cellular activities, and carcinogenesis. We hypothesized that genetic variations in RGS gene family may be associated with the response of late-stage non-small cell lung cancer (NSCLC) patients to chemotherapy or chemoradiotherapy. We selected 95 tagging single nucleotide polymorphisms (SNPs) in 17 RGS genes and genotyped them in 598 late-stage NSCLC patients. Thirteen SNPs were significantly associated with overall survival. Among them, rs2749786 of RGS12 was most significant. Stratified analysis by chemotherapy or chemoradiation further identified SNPs that were associated with overall survival in subgroups. Rs2816312 of RGS1 and rs6689169 of RGS7 were most significant in chemotherapy group and chemoradiotherapy group, respectively. A significant cumulative effect was observed when these SNPs were combined. Survival tree analyses identified potential interactions between rs944343, rs2816312, and rs1122794 in affecting survival time in patients treated with chemotherapy, while the genotype of rs6429264 affected survival in chemoradiation-treated patients. To our knowledge, this is the first study to reveal the importance of RGS gene family in the survival of late-stage NSCLC patients

Dynamic Imaging of the Effector Immune Response to Listeria Infection In Vivo

Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30–1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ [CD8 superscript +] T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ [CD8 superscript +] T cells in effector sites.National Institutes of Health (U.S.) (Grant P01AI-071195

Studies on the biocompatibility of bacterial cellulose

Bacterial cellulose was functionalized with a chimeric protein containing a cellulose-binding module and the adhesion peptide Arg-Gly-Asp. Small-diameter bacterial cellulose membranes were produced and subcutaneously implanted in sheep for 1–32 weeks. The implants triggered a biological response similar to other high surface-to-volume implants. There were no significant differences in the inflammation degree between the bacterial cellulose coated with the recombinant protein Arg-Gly-Asp–cellulose-binding module and the native bacterial cellulose. The implants were considered to be mildly irritating to the tissue compared to the negative control sample (expanded polytetrafluoroethylene). The analysis of the fluorescence microscopy revealed that, apart from increasing cell adhesion, the presence of Arg-Gly-Asp stimulated an even cell distribution, while the cells on the untreated bacterial cellulose seemed to form aggregates. Furthermore, the cells on the Arg-Gly-Asp–treated bacterial cellulose presented a more elongated morphology. Mechanical tests indicated that the small-diameter bacterial cellulose tubes were more elastic than the human arteries and veins.This work is funded by FEDER Funds through the Operational Programme for Competitiveness Factors COMPETE and National Funds through FCT - Foundation for Science and Technology under the Strategic Project PEst-C/AGR/UI0115/2011, the project PTDC/EBB-EBI/112170/2009 and under the PhD grant reference SFRH/BD/64838/2009. This work was also supported by Fundacao para a Ciencia e Tecnologia (FCT), Ministerio da Educacao e da Ciencia, Portugal, through the research Project PTDC/DES/104036/2008 and by QREN No. 1372 para Criacao de um Nucleo I&DT para Desenvolvimento de Produtos nas Areas de Medicina Regenerativa e de Terapias Celulares - Nucleo Biomat & Cell.Fabia K. Andrade is the recipient of a fellowship from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Brazil). Fabia K Andrade and Nuno Alexandre contributed equally to this work