Mutant products of the NF2 tumor suppressor gene are degraded by the ubiquitin-proteasome pathway

Neurofibromatosis type 2 (NF2), a syndrome associated with multiple tumors of the nervous system, mostly schwannomas, is caused by mutations in the NF2 tumor suppressor gene that encodes schwannomin (Sch). Here we examined NF2 pathogenetic mutations that result in misfolding of the FERM domain. We found that these mutant forms of Sch were efficiently degraded by the ubiquitin-proteasome pathway. In transfected cells, Sch Delta F118 was 3-fold more efficiently degraded than the related molecule ezrin bearing the equivalent mutation. In heterozygous Nf2 knock-out mouse fibroblasts, endogenous mutant Sch Delta 81-121, but not wild type Sch, was also degraded by proteasomes. We further show that this degradation pathway is functional in primary Schwann cells. We analyzed Sch Delta 39-121 expressed in a transgenic mouse model of NF2 and found that Sch Delta 39-121, but not the endogenous wild type Sch, was unstable due to proteasome-mediated degradation. Altogether these results suggest that degradation of mutant Sch mediated by the ubiquitin-proteasome pathway is a physiopathological pathway contributing to the loss of Sch function in NF2 patients.     

Cloning,expression and mutagenesis of a subunit contact of rabbit muscle-specific (betabeta) enolase

The cDNA for rabbit muscle-specific (betabeta) enolase was cloned, sequenced and expressed in Escherichia coli. This betabeta-enolase differs at eight positions from that sequenced by Chin (17). Site-directed mutagenesis was used to change residue 414 from glutamate to leucine, thereby abolishing a salt bridge involved in subunit contacts. Recombinant wild-type and mutant enolase were purified from E. coli and compared to enolase isolated from rabbit muscle. Molecular weights were determined by mass spectrometry. All three betabeta-enolases had similar kinetics, and UV and circular dichroism (CD) spectra. The mutant enolase was far more sensitive to inactivation by pressure, by KCl or EDTA, and by sodium perchlorate. We confirmed, by analytical ultracentrifugation, that the sodium perchlorate inactivation was due to dissociation. DeltaG(o) for dissociation of enolase was decreased from 49.7 kJ/mol for the wild-type enzyme to 42.3 kJ/mol for the mutant. In contrast to the wild-type enzyme, perchlorate inactivation of E414L was accompanied by a small loss of secondary structure.     

Daily melatonin supplementation in mice increases atherosclerosis in proximal aorta

Considerable evidence supports the hypothesis that LDL oxidation plays an important role in atherosclerosis. Even though high melatonin doses inhibit LDL oxidation in vitro, the effect of melatonin on atherosclerosis has never been studied. We have demonstrated that the feeding of hypercholesterolemic mice with an atherogenic diet supplemented with melatonin highly increases the surface of atherosclerotic lesions in the proximal aorta. These observations occur without detectable lipidic or glucidic phenotype alteration. Melatonin treatment increased highly the sensitivity of atherogenic lipoprotein to Cu(2+) and gamma-radiolysis generated oxyradical ex vivo oxidation during the fasting period. Moreover, these altered lipoproteins were less recognized by the LDL receptor metabolic pathway of murine fibroblasts while they transferred many more cholesteryl esters to murine macrophages. This study suggests that caution should be taken as regards high melatonin dosage in hypercholesterolemic patients.     

Role of the N-terminus of epidermal growth factor in ErbB-2/ErbB-3 binding studied by phage display

Epidermal growth factor (EGF) binds with high affinity to the EGF receptor, also known as ErbB-1, but upon replacement of the N-terminal linear region by neuregulin (NRG) 1 or transforming growth factor (TGF) alpha sequences it gains in addition high affinity for ErbB-2/ErbB-3 heterodimers. However, these chimeras weakly bind to ErbB-3 alone. To further dissect the ligand binding selectivity of the ErbB network, we have applied the phage display technique to examine the role of the linear N-terminal region in EGF for interaction with ErbB-2/ErbB-3 heterodimers. A library of EGF variants was constructed in which residues 2, 3, and 4 were randomly mutated, followed by selection for binding to intact MDA-MB-453 cells that overexpress ErbB-2 and ErbB-3 but lack ErbB-1. Analysis of the selected phage EGF variants revealed clones with high binding affinity to ErbB-2/ErbB-3 while maintaining high affinity to ErbB-1. In these variants, Trp (or alternatively His) was almost exclusively present at position 2, while specific combinations of hydrophobic, basic, and small residues were found at positions 3 and 4. The mitogenic activity of the phage EGF variants corresponded with their relative binding affinity. Two of the selected EGF variants, EGF/WVS and EGF/WRS, were further characterized as recombinant proteins. In contrast to previously characterized chimeras of EGF with NRG-1 or TGF-alpha, these variants did not only show high binding affinity for ErbB-2/ErbB-3 heterodimers but also for ErbB-3 alone. These data show that the linear N-terminal region of EGF-like growth factors is directly involved in binding to ErbB-3.     

Structure/function relationships within the RNA recognition motif family applied to the hermes gene product

The RNA Recognition Motif (RRM) family of RNA-binding domains comprises distinct structural subclasses which can be equated to various types of cognate RNA(s) in relation to biological functions. By identifying structural templates within the appropriate RRM subclass, we have homology-modelled the three-dimensional structure of the hermes gene-encoded RRM. Our findings lead us to propose potential RNA targets for the corresponding protein and to predict possible functions in RNA metabolism during heart development.     

Gyrodactyloides bychowskii (Monogenea: Gyrodactylidae) from sea-caged Atlantic salmon Salmo salar in Scotland: occurrence and ribosomal RNA sequence analysis

Gyrodactyloides bychowskii has been recorded in Scottish waters for the first time. The parasite was found on the gills of Atlantic salmon reared in seawater. An integrated morphological and molecular examination of the parasite was carried out. Prevalence of the parasite was greatest in February and declined to 0 by June in 1999. During 2000, parasites were located in March and November. An overdispersion of parasites was recorded, with intensity of infection reaching over 200 parasites per gill arch in some fish. Parasitised gill tissue showed hyperplasia and hypertrophy but not always at the site of parasite attachment. The internal transcribed spacer of the ribosomal RNA gene array was amplified by PCR and sequenced. This sequence shared greatest similarity with the internal transcribed spacer of Gyrdicotylus gallieni, followed by Gyrodactylus species. This is the first molecular analysis of this parasite and provides sequence data that may be used in comparison of G. bychowskii from other locations or in phylogenetic analysis of this group of Monogenea.     

Photosynthetic Bradyrhizobia Are Natural Endophytes of the African Wild Rice Oryza breviligulata

We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulata could significantly enhance cultivated rice production.