Regulation of K(ATP) channel by 17β-estradiol in pancreatic β-cells

ATP-sensitive potassium channels (K_ATP) regulate electrical activity and insulin secretion in pancreatic β-cells. When glucose concentration increases, the [ATP]/[ADP] ratio rises closing K_ATP channels, and the membrane potential depolarizes, triggering insulin secretion. This pivotal role of K_ATP channels is used not only by glucose but also by neurotransmitters, hormones and other physiological agents to modulate electrical and secretory β-cell response.In recent years, it has been demonstrated that estrogens and estrogen receptors are involved in glucose homeostasis, and that they can modulate the electrical activity and insulin secretion of pancreatic β-cells. The hormone 17β-estradiol (E2), at physiological levels, is implicated in maintaining normal insulin sensitivity for β-cell function. Long term exposure to E2 increases insulin content, insulin gene expression and insulin release via the estrogen receptor α (ERα), while rapid responses to E2 can regulate K_ATP channels increasing cGMP levels through the estrogen receptor β (ERβ) and type A guanylate cyclase receptor (GC-A). This review summarizes the main actions of 17β-estradiol on K_ATP channels and the subsequent insulin release in pancreatic β-cells.     

Calpain regulates N-terminal interaction of GSK-3β with 14-3-3ζ, p53 and PKB but not with axin

Calpain produces a truncation of GSK3β that removes the N-terminal inhibitory domain. Here we analyze the effect of that truncation on protein-protein interaction. We pulled down GST-tagged proteins in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Commercial GSK-3β was first incubated with calpain for 2.5 min in vitro, and then with GST-tagged proteins in the presence of calpeptin, a synthetic calpain inhibitor. Western blot analyses were performed to determine if there is an interaction between these GST-tagged proteins and truncated GSK-3β. Using axin GST-tagged, we pulled down the protein in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Western blot analyses showed full length GSK-3β in the pellet as well GSK-3β cleaved by calpain. Thus axin was able to bind GSK-3β without the N-terminal end. When the same experiment was carried out with GST-tagged 14-3-3ζ, p53 and PKB, full length GSK-3β was observed in the pellet, but GSK-3β truncated by calpain was not pulled down demonstrating that GSK-3β N-terminal end is necessary to interact with these three proteins. Our data demonstrate that N-terminal end is necessary for 14-3-3ζ, p53 and PKB interaction. However, the interaction of GSK3β with axin is not altered by calpain. These data support a physiological role for GSK3β truncation mediated by calpain.     

Synthesis and radiolabeling of chelator-RNA aptamer bioconjugates with copper-64 for targeted molecular imaging

Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g., copper-64, (64)Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of (64)Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10-3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g., pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with (64)Cu.     

Role of reactive oxygen species in the early stages of liver regeneration in streptozotocin-induced diabetic rats

Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.     

Onward but not always upward: individualistic elevational shifts of tree species in subtropical montane forests

Ongoing global climate change is driving widespread shifts in species distributions. Trends show frequent upwards shifts of treelines, but information on changes in montane forest below the treeline and in the tropics and subtropics is limited, despite the importance of these areas for biodiversity and ecosystem function. Patterns of species shifts in tropical and subtropical regions are likely to be more complex and individualistic than global averages suggest due to high species diversity and strong influence of competition, alongside direct climatic limitations on distributions. To address the question of how subtropical montane tree species are likely to move as climate changes, we used an extensive national forest inventory to estimate distribution shifts of 75 tree species in Taiwan by comparing the optimum elevation and range edges of adults and juveniles within species. Overall there was a significant difference in optimum elevation of adults and juveniles. Life stage mismatches suggested upward shifts in 35% of species but downward shifts of over half (56%), while 8% appeared stable. Upward elevation shifts were disproportionately common in high elevation species, whilst mid to low elevation species suggested greater variation in shift direction. Whilst previous research on mountain forest range shifts has been dominated by work addressing changes in treeline position, we show that although high elevation species shift up, below the treeline species may shift individualistically, heralding widespread changes in forest communities over coming decades. The wide variation of responses indicated is likely driven by individual species responses to interacting environmental factors such as competition, topography and anthropogenic influences across the broad range of forest types investigated. As global environmental changes continue, more detailed understanding of tree range shifts across a wide spectrum of forests will allow us to prepare for the implications of such changes for biodiversity, ecosystem function and dependent human populations.     

Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers

Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.     

Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes

The dramatic cell shape changes during cytokinesis require the interplay between microtubules and the actomyosin contractile ring, and addition of membrane to the plasma membrane. Numerous membrane-trafficking components localize to the central spindle during cytokinesis, but it is still unclear how this machinery is targeted there and how membrane trafficking is coordinated with cleavage furrow ingression. Here we use an arf6 null mutant to show that the endosomal GTPase ARF6 is required for cytokinesis in Drosophila spermatocytes. ARF6 is enriched on recycling endosomes at the central spindle, but it is required neither for central spindle nor actomyosin contractile ring assembly, nor for targeting of recycling endosomes to the central spindle. However, in arf6 mutants the cleavage furrow regresses because of a failure in rapid membrane addition to the plasma membrane. We propose that ARF6 promotes rapid recycling of endosomal membrane stores during cytokinesis, which is critical for rapid cleavage furrow ingression.     

Functional traits and climate drive interspecific differences in disturbance-induced tree mortality

With climate change, natural disturbances such as storm or fire are reshuffled, inducing pervasive shifts in forest dynamics. To predict how it will impact forest structure and composition, it is crucial to understand how tree species differ in their sensitivity to disturbances. In this study, we investigated how functional traits and species mean climate affect their sensitivity to disturbances while controlling for tree size and stand structure. With data on 130,594 trees located on 7617 plots that were disturbed by storm, fire, snow, biotic or other disturbances from the French, Spanish, and Finnish National Forest Inventory, we modeled annual mortality probability for 40 European tree species as a function of tree size, dominance status, disturbance type, and intensity. We tested the correlation of our estimated species probability of disturbance mortality with their traits and their mean climate niches. We found that different trait combinations controlled species sensitivity to disturbances. Storm-sensitive species had a high height-dbh ratio, low wood density and high maximum growth, while fire-sensitive species had low bark thickness and high P50. Species from warmer and drier climates, where fires are more frequent, were more resistant to fire. The ranking in disturbance sensitivity between species was overall consistent across disturbance types. Productive conifer species were the most disturbance sensitive, while Mediterranean oaks were the least disturbance sensitive. Our study identified key relations between species functional traits and disturbance sensitivity, that allows more reliable predictions of how changing climate and disturbance regimes will impact future forest structure and species composition at large spatial scales.