The gene product of the gp78/AMFR ubiquitin E3 ligase cDNA is selectively recognized by the 3F3A antibody within a subdomain of the endoplasmic reticulum

The receptor for the autocrine motility factor/phosphoglucose isomerase cytokine (gp78 or AMFR) has been extensively characterized using the 3F3A monoclonal antibody. Cloning of AMFR identified a seven-transmembrane domain G-protein-coupled receptor ubiquitin E3 ligase whose identity as AMFR was based on prior expression cloning with the 3F3A mAb that generated a truncated sequence. We show here that the gp78/AMFR gene product is indeed recognized by the 3F3A mAb. The FLAG-taggedAMFR immunoprecipitated with an anti-FLAG antibody was recognized by the 3F3A mAb in Western blot analysis and cells transfected with AMFR exhibit increased labeling with the 3F3A mAb. The 3F3A mAb does not however recognize higher molecular weight isoforms of AMFR. 3F3A labeling colocalizes with tagged AMFR in a peripheral ER network but does not recognize FLAG- or GFP-tagged AMFR localized to a perinuclear ER domain that likely corresponds to misfolded forms of the protein retained in the ER. These data indicate that 3F3A antibody binding is highly specific for a subpopulation of AMFR localized to an ER subdomain. Coexpression of AMFR-GFP and a lumenal ER-targeted RFP presented extensive colocalization in living cells andAMFR-GFP is concentrated in a basal ER network morphologically similar to that labeled by the 3F3A mAb in fixed cells. The3F3A anti-AMFR mAb therefore selectively recognizes a subpopulation of expressed AMFR localized to a subdomain of the ER.     

Adherence of airway neutrophils and inflammatory response are increased in CF airway epithelial cell-neutrophil interactions

Persistent presence of PMN in airways is the hallmark of CF. Our aim was to assess PMN adherence, percentage of apoptotic airway PMN (aPMN), and IL-6 and IL-8 production when aPMN are in contact with airway epithelial cells. Before coculture, freshly isolated CF aPMN have greater spontaneous and TNF-alpha-induced apoptosis compared with blood PMN from the same CF patients and from aPMN of non-CF patients. We then examined cocultures of PMN isolated from CF and non-CF airways with bronchial epithelial cells bearing mutated cftr compared with cftr-corrected bronchial epithelial cells. After 18-h coculture, the number of CF aPMN adhered on cftr-deficient bronchial epithelial cells was 2.3-fold higher compared with the coculture of non-CF aPMN adhered on cftr-corrected bronchial epithelial cells. The percentage of CF apoptotic aPMN (9.5 +/- 0.2%) adhered on cftr-deficient bronchial epithelial cells was similar to the percentage of non-CF apoptotic aPMN adhered on cftr-corrected bronchial epithelial cells (10.3 +/- 0.7%). IL-6 and IL-8 levels were enhanced 6.5- and 2.9-fold, respectively, in coculture of CF aPMN adhered on cftr-deficient bronchial epithelial cells compared with coculture of non-CF aPMN adhered on cftr-corrected bronchial epithelial cells. Moreover, blocking surface adhesion molecules ICAM-1, VCAM-1, and E-selectin on cftr-deficient bronchial epithelial cells with specific MAbs inhibited the adherence of CF aPMN by 64, 51, and 50%, respectively. Our data suggest that in CF patients a high number of nonapoptotic PMN adhered on airway epithelium associated with elevated IL-6 and IL-8 levels may contribute to sustained and exaggerated inflammatory response in CF airways.     

Activation of the Ghrelin Receptor is Described by a Privileged Collective Motion: A Model for Constitutive and Agonist-induced Activation of a Sub-class A G-Protein Coupled Receptor (GPCR)

Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser _[octa]-Phe-NH ₂ and Gly-Ser-Ser _[octa]-Phe-Leu-NH ₂ allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.     

Calcium-dependent regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells

Dysregulation of nuclear factor kappa B (NF-(kappa)B) and increased Ca(2+) signals have been reported in airway epithelial cells of patients with cystic fibrosis (CF). The hypothesis that Ca(2+) signaling may regulate NF-(kappa)B activation was tested in a CF bronchial epithelial cell line (IB3-1, CFTR genotype DeltaF508/W1282X) and compared to the CFTR-corrected epithelial cell line S9 using fluorescence microscopy to visualized in situ NF-(kappa)B activation at the single cell level. Upon stimulation with IL-1beta,we observed a slow but prolonged [Ca(2+)](i) increase (up to 10 min) in IB3-1 cells compared to S9 cells. The IL-1beta-induced [Ca(2+)](i) response was accompanied by an activation of NF-(kappa)B in IB3-1 but not in S9 cells. Pretreatment of IB3-1 cells with the ER Ca(2+) pump inhibitor thapsigargin inhibited the IL-1beta-induced [Ca(2+)](i) response. Treatment with either the calcium chelator BAPTA or an inhibitor of I(kappa)Balpha phosphorylation (digitoxin) led to a drastic [Ca(2+)](i) decrease accompanied by an inhibition of NF-(kappa)B activation of IL-1beta-stimulated IB3-1 cells in comparison to untreated cells. In IB3-1 cells cultured at low temperature (26 degrees C) for 16 h, the IL-1beta-induced [Ca(2+)](i) response was inhibited and no significant NF-(kappa)B activation was observed. To our knowledge, this is the first report of visualization of the Ca(2+)-mediated activation of NF-(kappa)B in individual living airway epithelial cells. Our results support the concept that [Ca(2+)](i) is a key regulator of NF-(kappa)B activation in CF airway epithelial cells.     

Genetic differentiation among migrant and resident populations of the threatened Asian houbara bustard

The Asian houbara bustard Chlamydotis macqueenii is a partial migrant of conservation concern found in deserts of central Asia and the Middle East. In the southern part of the species range, resident populations have been greatly fragmented and reduced by sustained human pressure. In the north, birds migrate from breeding grounds between West Kazakhstan and Mongolia to wintering areas in the Middle East and south central Asia. Extensive satellite tracking has shown substantial partitioning in migration routes and wintering grounds, suggesting a longitudinal barrier to present-day gene flow among migrants. In this context, we explored genetic population structure using 17 microsatellite loci and sampling 108 individuals across the range. We identified limited but significant overall differentiation (F(CT) = 0.045), which was overwhelmingly due to the differentiation of resident Arabian populations, particularly the one from Yemen, relative to the central Asian populations. Population structure within the central Asian group was not detectable with the exception of subtle differentiation of West Kazakh birds on the western flyway, relative to eastern populations. We interpret these patterns as evidence of recent common ancestry in Asia, coupled with a longitudinal barrier to present-day gene flow along the migratory divide, which has yet to translate into genetic divergence. These results provide key parameters for a coherent conservation strategy aimed at preserving genetic diversity and migration routes.     

The neuronal EGF-related gene Nell2 interacts with Macf1 and supports survival of retinal ganglion cells after optic nerve injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).