Involvement of rBAT in Na(+)-dependent and -independent transport of the neurotransmitter candidate L-DOPA in Xenopus laevis oocytes injected with rabbit small intestinal epithelium poly A(+) RNA

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[(14)C]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na(+)-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[(14)C]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[(14)C]DOPA uptake activity. rBAT was thus responsible for the L-[(14)C]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission.     

Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.     

Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells.     

HLA-DP4, the most frequent HLA II molecule,defines a new supertype of peptide-binding specificity

Among HLA-DP specificities, HLA-DP4 specificity involves at least two molecules, HLA-DPA1*0103/DPB1*0401 (DP401) and HLA-DPA1*0103/DPB1*0402 (DP402), which differ from each other by only three residues. Together, they are present worldwide at an allelic frequency of 20-60% and are the most abundant human HLA II alleles. Strikingly, the peptide-binding specificities of these molecules have never been investigated. Hence, in this study, we report the peptide-binding motifs of both molecules. We first set up a binding assay specific for the immunopurified HLA-DP4 molecules. Using multiple sets of synthetic peptides, we successfully defined the amino acid preferences of the anchor residues. With these assays, we were also able to identify new peptide ligands from allergens and viral and tumor Ags. DP401 and DP402 exhibit very similar patterns of recognition in agreement with molecular modeling of the complexes. Pockets P1 and P6 accommodate the main anchor residues and interestingly contain only two polymorphic residues, beta86 and beta11, respectively. Both positions are almost dimorphic and thus produce a limited number of pocket combinations. Taken together, our results support the existence of three main binding supertypes among HLA-DP molecules and should significantly contribute to the identification of universal epitopes to be used in peptide-based vaccines for cancer, as well as for allergic or infectious diseases.     

A screen for identifying genes interacting with armadillo, the Drosophila homolog of beta-catenin

Applications of quantitative genetics and conservation genetics often require measures of pairwise relationships between individuals, which, in the absence of known pedigree structure, can be estimated only by use of molecular markers. Here we introduce methods for the joint estimation of the two-gene and four-gene coefficients of relationship from data on codominant molecular markers in randomly mating populations. In a comparison with other published estimators of pairwise relatedness, we find these new "regression" estimators to be computationally simpler and to yield similar or lower sampling variances, particularly when many loci are used or when loci are hypervariable. Two examples are given in which the new estimators are applied to natural populations, one that reveals isolation-by-distance in an annual plant and the other that suggests a genetic basis for a coat color polymorphism in bears.     

The French National Registry of patients with Facioscapulohumeral muscular dystrophy

Background

Facioscapulohumeral muscular dystrophy is a rare inherited neuromuscular disease with an estimated prevalence of 1/20,000 and France therefore harbors about 3000 FSHD patients. With research progress and the development of targeted therapies, patients’ identification through registries can facilitate and improve recruitment in clinical trials and studies.

Results

The French National Registry of FSHD patients was designed as a mixed model registry involving both patients and physicians, through self-report and clinical evaluation questionnaires respectively, to collect molecular and clinical data. Because of the limited number of patients, data quality is a major goal of the registry and various automatic data control features have been implemented in the bioinformatics system. In parallel, data are manually validated by molecular and clinical curators. Since its creation in 2013, data from 638 FSHD patients have been collected, representing about 21% of the French FSHD population. The mixed model strategy allowed to collect 59.1% of data from both patients and clinicians; 26 and 14.9% from respectively patients and clinicians only. With the identification of the FSHD1 and FSHD2 forms, specific questionnaires have been designed. Though FSHD2 patients are progressively included, FSHD1 patients still account for the majority (94.9%). The registry is compatible with the FAIR principles as data are Findable, Accessible and Interoperable. We thus used molecular standards and standardized clinical terms used by the FILNEMUS French network of reference centers for the diagnosis and follow-up of patients suffering from a rare neuromuscular disease. The implemented clinical terms mostly map to dictionaries and terminology systems such as SNOMED-CT (75% of terms), CTV3 (61.7%) and NCIt (53.3%). Because of the sensitive nature of data, they are not directly reusable and can only be accessed as aggregated data after evaluation and approval by the registry oversight committee.

Conclusions

The French National Registry of FSHD patients belongs to a national effort to develop databases, which should now interact with other initiatives to build a European and/or an international FSHD virtual registry for the benefits of patients. It is accessible at www.fshd.fr and various useful information, links, and documents, including a video, are available for patients and professionals.

     

Protein unfolding transitions in an intrinsically unstable annexin domain: molecular dynamics simulation and comparison with nuclear magnetic resonance data

Unfolding transitions of an intrinsically unstable annexin domain and the unfolded state structure have been examined using multiple approximately 10-ns molecular dynamics simulations. Three main basins are observed in the configurational space: native-like state, compact partially unfolded or intermediate compact state, and the unfolded state. In the native-like state fluctuations are observed that are nonproductive for unfolding. During these fluctuations, after an initial loss of approximately 20% of the core residue native contacts, the core of the protein transiently completely refolds to the native state. The transition from the native-like basin to the partially unfolded compact state involves approximately 75% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate state adopts for part of the time in one of the trajectories a novel highly compact salt-bridge stabilized structure that can be identified as a conformational trap. The intermediate-to-unfolded state transition is characterized by a large increase in the radius of gyration. After an initial relaxation the unfolded state recovers a native-like topology of the domain. The simulated unfolded state ensemble reproduces in detail experimental nuclear magnetic resonance data and leads to a convincing complete picture of the unfolded domain.     

Endogenously released DOPA is a causal factor for glutamate release and resultant delayed neuronal cell death by transient ischemia in rat striata

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four-vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal L-aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10-30 nM DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration-dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nM, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration-dependently by intrastriatal perfusion of 0.3-1 mM DOPA and stereoselectively by 0.6 mM DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.     

Alteration of the tertiary structure of the major bee venom allergen Api m 1 by multiple mutations is concomitant with low IgE reactivity

We have engineered a recombinant form of the major bee venom allergen (Api m 1) with the final goal of reducing its IgE reactivity. This molecule (Api mut) contains 24 mutations and one deletion of 10 amino acids. The successive introduction of these sequence modifications led to a progressive loss of specific IgE and IgG reactivity and did not reveal any immunodominant epitopes. However, Api mut exhibited a clear loss of reactivity for Api m 1-specific IgE and IgG. Injection of Api mut into mice induced specific antibody production. This humoral response was as high as that induced by the Api m 1 but the cross-reactivity of the antibodies was weak. As inferred by far UV circular dichroism, this mutant was correctly folded. However, near UV circular dichroism and denaturation curves of Api mut showed that it exhibits a dynamic tertiary structure and that it is a highly flexible molecule. Finally, as all the sequence modifications have been introduced outside the human and murine T cell epitope regions, we investigated its T cell properties in mice. We showed that Api mut-specific T lymphocytes induced in vivo were stimulated in vitro by both proteins. These data provide new insights in the design of hypoallergenic molecules.