N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.     

Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.     

Molecular biology of nickel carcinogenesis: Identification of differentially expressed genes in morphologically transformed C3H10T1/2 Cl 8 mouse embryo fibroblast cell lines induced by pecific insoluble nickel compounds

Inhalation of mixtures of insoluble and soluble nickel compounds by humans during nickel refining has been associated with excess lung and nasal sinus cancers. Insoluble nickel subsulfide (Ni3S2) and nickel oxide (NiO) are carcinogenic to rodents by inhalation. We previously showed that insoluble Ni3S2, crystalline nickel monosulfide (NiS), and green (high temperature, HT) and black (low temperature, LT) NiO, induced morphological transformation in cultured C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells. To understand molecular mechanisms of carcinogenesis by insoluble nickel compounds, we used random, arbitrarily primed-polymerase chain reaction (RAP-PCR) mRNA differential display and identified nine cDNA fragments that were differentially expressed between nontransformed and nickel-transformed cell lines in approximately 10.0% of the total mRNA. Expression of the calnexin gene (encoding a type I membrane protein/molecular chaperone), the ect-2 proto-oncogene, and the stress-inducible gene, Wdr1, was upregulated. Expression of six genes--the vitamin D interacting protein/thyroid hormone activating protein 80 (DRIP/TRAP-80) gene, the insulin-like growth factor receptor 1 (IGFR1) gene, the small nuclear activating protein (SNAP C3) gene, and three unknown genes, was down-regulated, in nickel-transformed cell lines. We hypothesize that these resulting aberrations in gene expression could contribute to the induction and/or maintenance of morphological transformation induced by specific insoluble nickel compounds.     

The role of shear stress in atherosclerosis

Atherosclerotic lesions preferentially localize near side branches or curved vessels. During the last few decades, research has been shown that low or low and oscillating shear stress is associated with plaque location. Despite ample evidence, the precise mechanism is unknown. This is mainly because of a lack of appropriate animal models. We describe two novel methods to study the hypothesis that shear stress acts through endothelial gene expression or shear stress acts through localizing of inflammation. Both literature evidence and own findings support a role for both mechanisms in atherosclerosis.     

Safe and successful endoarterial infusion of liposomal amphotericin B in treatment of mucormycosis

Mucormycosis is a rare invasive mycotic infection treated by antifungini or amphotericin B. We describe the case of a patient with septic fever and a necrotic lesion, with phlegmon of medial left thigh. Surgery was performed to drain the abscess content and to remove the necrotic tissue; mucormycosis was diagnosized by histological and culture tests and treated by intravenous amphotericin B. Since the lesion worsened, liposomal amphotericin B was directly infused into the left common iliac artery, with progressive improvement, and treatment was continued until complete recovery. Therefore, the endoarterial infusion of liposomal amphotericin B was a safe and successful treatment of advanced lesions of mucormycosis. In such lesions, intravenous general antibiotic administration probably is not sufficient to reach the whole infected area.     

Kinetics of development and characteristics of antibodies induced in cancer patients against yeast expressed rDNA derived granulocyte macrophage colony stimulating factor (GM-CSF)

We have determined the presence and kinetics of granulocyte macrophage colony stimulating factor (GM-CSF) antibodies induced after repeated administration of a yeast expressed GM-CSF product in prostate cancer patients with minimal recurrent disease using a panel of assays for detection and characterization of antibodies. Results showed that all 15 prostate cancer patients treated with GM-CSF developed GM-CSF reactive antibodies during the course of therapy. Most patients (87%) developed GM-CSF reactive antibodies within 3 months while in other patients (13%), these antibodies were induced after additional cycles of GM-CSF treatment. For most patients, the timing of occurrence of these antibodies was the same regardless of whether the ELISA or surface plasmon resonance (SPR) assays were used for detection. However, in two patients, the recognition of GM-CSF reactive antibodies by SPR assays preceded their detection by ELISA. A significant number of patients (n=9, 60%) developed GM-CSF antibodies which neutralized the biological activity of GM-CSF in vitro in a cell-line based bioassay. These antibodies also recognized GM-CSF protein from different expression systems including the non-glycosylated protein from E. coli indicating that the antibody response is directed towards the amino acid backbone of the protein. A significant effect of GM-CSF antibodies on PSA modulation was not observed in this small cohort of patients despite an alteration in PSA levels in some treated patients. The study design used here did not allow conclusions regarding the relationship between neutralizing antibodies and the PSA levels which were used as a marker for clinical outcome. Implementation of a clinical strategy which permits monitoring for antibody development and for levels of a relevant pre-determined clinical marker at appropriate time-points is necessary for assessing the impact of antibody development on the therapeutic efficacy of the protein.     

Crystallization and preliminary X-ray diffraction studies of a pea lectin-methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside complex

The seed lectin isolated from garden peas (Pisum sativum) has been co-crystallized with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 64.3 A, b = 73.4 A and C = 108.5 A. The asymmetric unit contains one pea lectin dimer (alpha 2 beta 2). The crystals are suitable for high-resolution structure analysis.     

Focus Group Abstracts

Sleep and Biological Rhythms -     

Seed germinability and longevity influences regeneration of <Emphasis Type="Italic">Acacia gerrardii</Emphasis>

Acacia gerrardii is the only native tree species of the Kuwaiti desert ecosystem. However, anthropogenic disturbances and harsh arid climate have contributed towards the disappearance of this keystone species from its habitat. In this study, effects of different seed pretreatments to break dormancy, water entry pathway, and ecology (seasonal timing) of dormancy loss and germination of A. gerrardii were investigated. Effects of mechanical scarification, hot water treatment (30 s, 1, 2, and 5 min), and concentrated acid scarification (10, 20, and 30 min) on germination percentage and rate (time to 50% germination and final germination) were also examined. Pretreatment with mechanical scarification produced the highest germination in the least time and 20 °C, 40% RH with 12 h of light (2370 Lux) were found to provide the best germination environment. Seeds were rapidly aged at 60% RH and 45 or 50 °C to determine longevity, and the results were analyzed using probit analysis. Times taken for viability of A. gerrardii seeds aged at 45 and 50 °C to fall to 50% (p₅₀) were 38.6 and 9.3 days, respectively, and therefore the seeds can be considered to have medium longevity. Experiments to find the water entry pathway in A. gerrardii indicated that the micropyle region was the primary point of water entry into the seed. Seed burial experiments indicated that though seed retention decreased over time, there was no significant decrease in number of viable seeds after 31 weeks. The findings of this study are important to nursery managers, seed banks, and those involved in conservation and restoration activities.