The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

Modelling the Effects of Water Stress and Temperature on Seed Germination of Radish and Cantaloupe

Cantaloupe (Cucumis melo L.) and radish (Raphanus sativus L.) are considered as important vegetables with potential for national and international markets due to their sugars, vitamins and minerals. This study arranged, therefore, to simultaneously investigate the effect of temperature (T) and water potential (ψ) on seed germination (SG) of these plants using two hydrothermal time (HTT) models and to determine cardinal Ts and base water potential (ψ_b(50)) for both species. The results indicated that SG of both species was more affected by ψ than T (p ≤ 0.001). At Ts below an optimum temperature (T_o) the ψ_b(50) was constant (− 0.582 and − 0.760 MPa for radish and cantaloupe, respectively) and then increased linearly by 0.0481 and 0.0446 MPa °C⁻¹ as T increased above T_o (as thermoinhibition) until 0 MPa at the ceiling temperature (T_c), respectively. As the first report, however, we observed that the T at which ψ_b(50) begins to change was the same here (that is, T_d = T_o), when determined by either model for both species. This result suggests that the assumption in Rowse and Finch-Savage’s model (T_d is often less and or very close to T_o) may be invalid in some cases. For both species, the base temperature (T_b) and T_o were not affected by ψ and were constant while there was an exception only for T_c for which the value declined with decreasing ψs (more negative). In general, the estimated T_b, T_o and T_c were 9.64, 21.3 and 33.0 °C for radish and 11.8, 28.3 and 45.7 °C for cantaloupe in the control condition (ψ = 0 MPa), respectively. The HTT models used here and their parameters, each with strengths and weaknesses, can be used as a predictive tool in both cantaloupe and radish SG simulation models. However, at first, we need to select an appropriate HTT model based on SG behavior of plant species and then use the best model for quantifying the response of SG across Ts and ψs.

     

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR·RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.     

Short-Term,Intermittent Fasting Induces Long-Lasting Gut Health and TOR-Independent Lifespan Extension

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Quantification of soybean seed germination response to seed deterioration under PEG-induced water stress using hydrotime concept

This experiment was arranged to investigate the ability of hydrotime model (θ_H) for estimating soybean seed germination (cv. ‘JK’) under different accelerated aging periods (AAP, 0, 24, 48, and 72 h) at each of the following water potentials (ψ, 0, ??0.12, ??0.24, and ??0.36 MPa). Results indicated that both germination percentage (GP) and germination rate (GR) significantly influenced by ψ, AAP, and their interactions (P?<?0.01). GP and GR decreased by 62.6 and 47.3% with longer AAP from 0 to 72 h and by 90.7 and 81.5% with lower ψ from zero to ??0.36 MPa as compared to the control, respectively. Therefore, the effect of ψ on GP and GR was more than AAP. The θ_H value was constant (~?6.71 MPa h^?1) till 50.6 h AAP and then linearly declined with the rate of 0.1545 MPa h^?1 per hour increase in AAP until 72 h (~?50% lower than its initial value). The ψ_b(50) value was ? 0.343 MPa in the control and then increased (became more positive) by ~?70% until 72 h AAP (? 0.104 MPa). In general, GP and GR of soybean declined with the increasing ψ_b(50) which can be due to cell membrane damage and reduce the activity of enzymes and organelles during AAP. Based on our findings, the θ_H model could describe well these relationships and their parameters can nicely be used for simulating soybean seed germination under this condition.     

A new two-color Fab labeling method for autoantigen protein microarrays

Antigen microarrays hold great promise for profiling the humoral immune response in the settings of autoimmunity, allergy and cancer. This approach involves immobilizing antigens on a slide surface and then exposing the array to biological fluids containing immunoglobulins. Although these arrays have proven extremely useful as research tools, they suffer from several sources of variability. To address these issues, we have developed a new two-color Fab labeling method that allows two samples to be applied simultaneously to the same array. This straightforward labeling approach improves reproducibility and reliably detects changes in autoantibody concentrations. Using this technique we profiled serum from a mouse model of systemic lupus erythematosus (SLE) and detected both expected and previously unrecognized reactivities. The improved labeling and detection method described here overcomes several problems that have hindered antigen microarrays and should facilitate translation to the clinical setting.     

Serum level and tumor tissue expression of Ribonucleotide-diphosphate Reductase subunit M2 B: a potential biomarker for colorectal cancer

Molecular Biology Reports - This study explored the applicability of serum level and tissue expression of Ribonucleotide-diphosphate Reductase subunit M2 B (RRM2B) as reliable biomarkers for...