The regions within the N-terminus critical for human glucagon like peptide-1 receptor (hGLP-1R) cell Surface expression

The hGLP-1R is a target for the treatment of type 2 diabetes and belongs to the class B family of GPCRs. Like other class B GPCRs, the GLP-1R contains an N-terminal signal peptide (SP) and undergoes N-linked glycosylation, which are important for its trafficking and maturation. This study analysed the role of the SP, the hydrophobic region after the SP (HRASP), glycosylation and the conserved residues within the N-terminus in GLP-1R trafficking. HGLP-1R targeted to the cell surface showed no SP, and the SP deleted mutant, but not the mutants defective in SP cleavage, showed cell surface expression, demonstrating the importance of SP cleavage for hGLP-1R cell surface expression. The N-terminal deletions of hGLP-1R revealed that the HRASP, not the SP, is essential for cell surface expression of GLP-1R. Further, inhibition of hGLP-1R glycosylation prevented cell surface expression of the receptor. Mutation of Trp39, Tyr69 and Tyr88, which are required for agonist binding, in the GLP-1R abolished cell surface expression of the receptor independent of the SP cleavage or N-linked glycosylation. In conclusion, the N-terminus of hGLP-1R regulates receptor trafficking and maturation. Therefore this study provides insight into the role of hGLP-1R N-terminus on the receptor cell surface expression

Seizure-mediated iron accumulation and dysregulated iron metabolism after status epilepticus and in temporal lobe epilepsy

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes

Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis

INHIBITION OF mRNA export and dimerization of interferon regulatory factor 3 by Theiler's virus leader protein

Theiler's murine encephalomyelitis virus (TMEV or Theiler's virus) is a neurotropic picornavirus that can persist lifelong in the central nervous system of infected mice, causing a chronic inflammatory demyelinating disease. The leader (L) protein of the virus is an important determinant of viral persistence and has been shown to inhibit transcription of type I interferon (IFN) genes and to cause nucleocytoplasmic redistribution of host proteins. In this study, it was shown that expression of the L protein shuts off synthesis of the reporter proteins green fluorescent protein and firefly luciferase, suggesting that it induces a global shut-off of host protein expression. The L protein did not inhibit transcription or translation of the reporter genes, but blocked cellular mRNA export from the nucleus. This activity correlated with the phosphorylation of nucleoporin 98 (Nup98), an essential component of the nuclear pore complex. In contrast, the data confirmed that the L protein inhibited IFN expression at the transcriptional level, and showed that transcription of other chemokine or cytokine genes was affected by the L protein. This transcriptional inhibition correlated with inhibition of interferon regulatory factor 3 (IRF-3) dimerization. Whether inhibition of IRF-3 dimerization and dysfunction of the nuclear pore complex are related phenomena remains an open question. In vivo, IFN antagonism appears to be an important role of the L protein early in infection, as a virus bearing a mutation in the zinc finger of the L protein replicated as efficiently as the wild-type virus in type I IFN receptor-deficient mice, but had impaired fitness in IFN-competent mice

Abstract P2-16-01: Overall Survival Data from SOLTI-0701: A Multinational, Double-Blind, Placebo-Controlled, Randomized Phase 2b Study Evaluating the Oral Combination of Sorafenib and Capecitabine in Patients with Locally Advanced or Metastatic HER2-Negative Breast Cancer

Abstract Background: Sorafenib (SOR) is an oral multikinase inhibitor with antiangiogenic and antiproliferative activity. We previously reported data from the primary analysis of SOLTI-0701 demonstrating a progressionfree survival (PFS) benefit with the oral regimen of SOR + capecitabine (CAP) vs placebo (PL)+CAP in patients with advanced breast cancer (BC). Here we describe overall survival (OS), a secondary endpoint of SOLTI-0701. Methods: SOLTI-0701 is a multinational (Brazil, France, Spain) phase 2b study in HER2-negative patients with locally advanced or metastatic BC and ≥1 prior chemo regimen for advanced BC. Patients were randomized (1:1) to receive CAP (1000 mg/m2 po, twice daily [BID], 14 of every 21 days) with PL or SOR (400 mg po, BID). The primary endpoint was PFS. The sample size was estimated at 220 patients based on patient accrual projections and the estimated rate of PFS events. OS analysis was planned after 120 events. Results: A total of 229 patients were enrolled (115 SOR+CAP and 114 PL+CAP) from August 2007 to December 2008. Treatment arms were balanced for age (mean 55.2 y), ECOG status 0 (68%) and 1 (30%), stage IV disease (91%), and visceral metastases (75%). In the primary analysis, median PFS was 6.4 mo for the SOR arm vs 4.1 mo for the PL arm (hazard ratio 0.58; 95% CI, 0.41-0.81; 1-sided P=0.0006); the most common Grade 3/4 toxicity was hand-foot skin reaction/syndrome (45% vs 13%, respectively); discontinuation of treatment due to adverse events was infrequent (15% vs 7%, respectively). OS data are expected by the 3rd quarter of this year and will be presented, as well as updated safety data. Conclusions: The oral combination of SOR+CAP significantly improved PFS in patients with advanced BC. The regimen was tolerable with a manageable toxicity profile. An ongoing phase 3 registration trial is evaluating the combination of SOR+CAP for advanced BC. The secondary endpoint of OS will better characterize the potential role of SOR in advanced BC as randomized controlled trials thus far have yet to demonstrate any survival benefit with antiangiogenic agents. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-16-01.</jats:p