Functional segregation of the highly conserved basic motifs within the third endoloop of the human secretin receptor

In this study, a mutagenesis-based strategy was employed to assess the roles of two highly conserved motifs (KLR and RLAR) within the third endoloop of the human secretin receptor. Block deletion of KLRT and mutation of Lys323 (K323I) significantly reduced cAMP accumulation, and these mutations did not affect ligand interaction and receptor number expressed on the cell surface. Thus, the KLRT region at the N terminus of the third endoloop, particularly Lys323, is important for G protein coupling. For the RLAR motif, receptors with substitutions at positions 339 and 342 from Arg to Ala (R339, 342A), Glu (R339, 342E), or Ile (R339, 342I) as well as block deletion of the RLAR motif were all found to be defective in both secretin-binding and cAMP production. Interestingly, a single mutation at the corresponding positions of Arg339 or Arg342 responded as the wild-type human secretin receptor in all functional assays, indicating that the presence of one Arg at either position within the RLAR motif is sufficient for a normal receptor function. Immunofluorescent staining of these mutant receptors showed that these Arg residues are responsible for surface presentation and/or receptor stability.link_to_subscribed_fulltex

Classification of functional motifs of the secretin receptor on signal transduction

Recently, secretin has been suggested as a neuropeptide and is be involved in autism and other diseases such as pancreatic adenomas. Thus, its biological functions and relationships with diseases have become a research focus. Therefore, understanding the signal transduction through secretin receptor is a pre-requisite for further investigation and interpretation. Secretin receptor has a relatively large N-terminal domain containing N-glycoslation sites for ligand recognition and conserved cysteine residues for receptor conformations. As one of the G protein coupled receptors, secretin receptor also contains the common molecular architecture of 7 transmembrane domains that intertwined by 3 exoloops and 3 endoloops. These transmemrbane domains were enriched by hydrophobic residues, which help to anchor the receptor into the plasma membrane. Whereas, many conserved basic residues were located in the 2nd and 3rd endoloops and proved to be involved in receptor activation and G protein coupling. Nevertheless, receptor signaling is a complicated process and the interaction and selectivity of G proteins that certainly rely on a combination of functional motifs. For the Carboxyl tail, conflicting research findings were published in the aspect of secretin receptor internalization and desensitization. In this article, we summarize the research studies and implications on function motifs of the secretin receptor and hence provide a general picture on its function

Functional identification of an intronic promoter of the human glucose-dependent insulinotropic polypeptide gene

Glucose-dependent insulinotropic polypeptide (GIP), a physiological incretin and enterogastrone, plays a vital role in regulating glucose-dependent insulin release from the pancreas and gastric acid secretion from the stomach. By using a transgenic mouse approach, we previously reported that the distal 1.2. kb promoter region of the human GIP (hGIP) gene (-2545/-346, relative to the ATG) was able to target the transgene expression in the stomach but not in the small intestine where the majority of GIP-producing cells are located. In the present study, in order to identify the cis-acting element(s) that is/are required for intestinal expression, a 1.6. kb (-1580/-) DNA fragment within the first intron of the hGIP gene was isolated and characterized in three GIP-expressing cell lines including HuTu80 (duodenal cells), PANC-1 (pancreatic ductal cells) and Hs746T (stomach cells). By 5' and 3' deletion analysis, a proximal promoter element was confined within the nucleotides -102/-1. This promoter element, functions in an orientation-dependent manner, was able to drive 15.1 and 18.3 fold increases in promoter activities in HuTu80 and PANC-1 cells, respectively. Site-directed mutation analysis indicated that the region -54/-23 was essential for promoter function while the region -22/-1 might possess opposite effects in HuTu80 and PANC-1 cells. In competitive and antibody supershift assays, interactions of the progesterone receptor (PR) and some unknown protein factors from HuTu80 and PANC-1 with the motif(s) at -54/-23 were evident. Consistent with this finding, we demonstrated the transcriptional regulation of the hGIP promoter by progesterone via the PR-B isoform and that progesterone treatment in both HuTu80 and PANC-1 cells resulted in an increase in hGIP transcript level. In addition, a sequence motif (ACATGT) residing -48/-43 was found to be responsible for the binding of potential TFII regulator(s). Taken together, our results suggest that the proximal intronic sequences contain essential cis-acting elements for the cell-specific expression of the hGIP gene. © 2010 Elsevier B.V.link_to_subscribed_fulltex

Involvement of NF-κB subunit p65 and retinoic acid receptors, RARα and RXRα, in transcriptional regulation of the human GnRH II gene

Gonadotropin-releasing hormone (GnRH) I and II are hypothalamic decapeptides with pivotal roles in the development of reproductive competence and regulation of reproductive events. In this study, transcriptional regulation of the human GnRH II gene was investigated. By scanning mutation analysis coupled with transient promoter assays, the motif at -641/-636 (CATGCC, designated GII-Sil) was identified as a repressor element. Mutation of this motif led to full restoration of promoter activity in TE671 medulloblastoma and JEG-3 placenta choriocarcinoma cells. Supershift and chromatin immunoprecipitation assays showed in vitro and in vivo binding of NF-κB subunit p65 and the retinoic acid receptors, RARα and RXRα, to the promoter sequences. Over-expression of these protein factors indicated that p65 is a potent repressor, and the RARα/RXRα heterodimer is involved in the differential regulation of the GnRH II gene in neuronal and placental cells. This was confirmed by quantitative real-time PCR. Treatment of cells with the RARα/RXRα ligands, all-trans retinoic acid and 9-cis-retinoic acid, reduced and increased GnRH II gene expression in TE671 and JEG-3 cells, respectively. Taken together, these data demonstrate the differential roles of NF-κB p65 and RARα/RXRα, interacting with the same sequence in the promoter of the human GnRH II gene to influence gene expression in a cell-specific manner. © 2007 The Authors.link_to_OA_fulltex

Potential mechanisms of prospective antimigraine drugs: A focus on vascular (side) effects

AbstractCurrently available drugs for the acute treatment of migraine, i.e. ergot alkaloids and triptans, are cranial vasoconstrictors. Although cranial vasoconstriction is likely to mediate—at least a part of—their therapeutic effects, this property also causes vascular side-effects. Indeed, the ergot alkaloids and the triptans have been reported to induce myocardial ischemia and stroke, albeit in extremely rare cases, and are contraindicated in patients with known cardiovascular risk factors. In view of these limitations, novel antimigraine drugs devoid of vascular (side) effects are being explored. Currently, calcitonin gene-related peptide (CGRP) receptor antagonists, which do not have direct vasoconstrictor effects, are under clinical development. Other classes of drugs, such as 5-HT1F receptor agonists, glutamate receptor antagonists, nitric oxide synthase inhibitors, VPAC/PAC receptor antagonists and gap junction modulators, have also been proposed as potential targets for acute antimigraine drugs. Although these prospective drugs do not directly induce vasoconstriction, they may well induce indirect vascular effects by inhibiting or otherwise modulating the responses to endogenous vasoactive substances. These indirect vascular effects might contribute to the therapeutic efficacy of the previously mentioned compounds, but may alternatively also lead to vascular side-effects. As described in the current review, some of the prospective antimigraine drugs with a proposed non-vascular mechanism of action may still have direct or indirect vascular effects

Diffusion tensor imaging detects ventilation-induced brain injury in preterm lambs

PURPOSE: Injurious mechanical ventilation causes white matter (WM) injury in preterm infants through inflammatory and haemodynamic pathways. The relative contribution of each of these pathways is not known. We hypothesised that in vivo magnetic resonance imaging (MRI) can detect WM brain injury resulting from mechanical ventilation 24 h after preterm delivery. Further we hypothesised that the combination of inflammatory and haemodynamic pathways, induced by umbilical cord occlusion (UCO) increases brain injury at 24 h. METHODS: Fetuses at 124±2 days gestation were exposed, instrumented and either ventilated for 15 min using a high tidal-volume (VT) injurious strategy with the umbilical cord intact (INJ; inflammatory pathway only), or occluded (INJ+UCO; inflammatory and haemodynamic pathway). The ventilation groups were compared to lambs that underwent surgery but were not ventilated (Sham), and lambs that did not undergo surgery (unoperated control; Cont). Fetuses were placed back in utero after the 15 min intervention and ewes recovered. Twenty-four hours later, lambs were delivered, placed on a protective ventilation strategy, and underwent MRI of the brain using structural, diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) techniques. RESULTS: Absolute MRS concentrations of creatine and choline were significantly decreased in INJ+UCO compared to Cont lambs (P = 0.03, P = 0.009, respectively); no significant differences were detected between the INJ or Sham groups and the Cont group. Axial diffusivities in the internal capsule and frontal WM were lower in INJ and INJ+UCO compared to Cont lambs (P = 0.05, P = 0.04, respectively). Lambs in the INJ and INJ+UCO groups had lower mean diffusivities in the frontal WM compared to Cont group (P = 0.04). DTI colour mapping revealed lower diffusivity in specific WM regions in the Sham, INJ, and INJ+UCO groups compared to the Cont group, but the differences did not reach significance. INJ+UCO lambs more likely to exhibit lower WM diffusivity than INJ lambs. CONCLUSIONS: Twenty-four hours after injurious ventilation, DTI and MRS showed increased brain injury in the injuriously ventilated lambs compared to controls. DTI colour mapping threshold approach provides evidence that the haemodynamic and inflammatory pathways have additive effects on the progression of brain injury compared to the inflammatory pathway alone

Metalothionein - imunohistochemický biomarker rakoviny: Meta-analýza

Metallothionein (MT) has been extensively investigated as a molecular marker of various types of cancer. In spite of the fact that numerous reviews have been published in this field, no meta-analytical approach has been performed. Therefore, results of to-date immunohistochemistry-based studies were summarized using meta-analysis in this review. Web of science, PubMed, Embase and CENTRAL databases were searched (up to April 30, 2013) and the eligibility of individual studies and heterogeneity among the studies was assessed. Random and fixed effects model meta-analysis was employed depending on the heterogeneity, and publication bias was evaluated using funnel plots and Eggers tests. A total of 77 studies were included with 8,015 tissue samples (4,631 cases and 3,384 controls). A significantly positive association between MT staining and tumors (vs. healthy tissues) was observed in head and neck (odds ratio, OR 9.95; 95% CI 5.82– 17.03) and ovarian tumors (OR 7.83; 1.09–56.29), and a negative association was ascertained in liver tumors (OR 0.10; 0.03–0.30). No significant associations were identified in breast, colorectal, prostate, thyroid, stomach, bladder, kidney, gallbladder, and uterine cancers and in melanoma. . However, a high degree of inconsistence was observed in several tumor types, including colorectal, kidney and prostate cancer. Despite the ambiguity in some tumor types, conclusive results are provided in the tumors of head and neck, ovary and liver and in relation to the tumor grade and patient survival.Metalothionein (MT) byl rozsáhle zkoumán jako molekulární marker různých typů rakoviny. Navzdory tomu, že v této oblasti byly zveřejněny četné hodnocení , byl proveden meta-analytický postup. Proto výsledky na aktuální studie imunohistochemické bázi byly shrnuty pomocí meta-analýzy v této recenzi. Web of Science, PubMed, Embase a centrálních databází byly vyhledávány (až do 30.dubna 2013) a způsobilosti jednotlivých studií a heterogenity mezi studiích byla hodnocena. Náhodné a fixní efekty modelu meta-analýza byla použita v závislosti na různorodosti, a publikace zaujatost byla hodnocena pomocí trychtýř pozemky a testy Eggers.Celkem 77 studií bylo zahrnuto s 8015 vzorky tkání (4631 případů a 3384 kontrol).Významně pozitivní asociace mezi MT barvení a nádorů (vs. zdravých tkání) byl pozorován v oblasti hlavy a krku (poměr šancí, OR 9,95; 95% CI 5.82- 17,03) a ovariální nádory (OR 7,83, 1,09 - 56,29), a negativní asociace byla zjištěna v jaterních nádorů (OR 0,10; 0,03 - 0,30). Žádné významné asociace byly identifikovány v prsu, tlustého střeva, prostaty, štítné žlázy, žaludku, močového měchýře, ledvin, žlučníku, a rakoviny dělohy a melanomu. , Nicméně, vysoký stupeň nesouladu byl pozorován u několika typů nádorů, včetně tlustého střeva, ledvin a prostaty. Navzdory nejednoznačnosti některých typů nádorů, průkazné výsledky jsou uvedeny v nádorů hlavy a krku, vaječníků a jater a ve vztahu ke stupni nádoru a přežití pacienta