Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the SACS gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown.

The clavesin family, neuron-specific lipid- and clathrin-binding Sec14 proteins regulating lysosomal morphology.

Clathrin-coated vesicles (CCVs) originating from the trans-Golgi network (TGN) provide a major transport pathway from the secretory system to endosomes/lysosomes. Herein we describe paralogous Sec14 domain-bearing proteins, clavesin 1/CRALBPL and clavesin 2, identified through a proteomic analysis of CCVs. Clavesins are enriched on CCVs and form a complex with clathrin heavy chain (CHC) and adaptor protein-1, major coat components of TGN-derived CCVs.

Direct protein detection with a nano-interdigitated array gate MOSFET.

A new protein sensor is demonstrated by replacing the gate of a metal oxide semiconductor field effect transistor (MOSFET) with a nano-interdigitated array (nIDA). The sensor is able to detect the binding reaction of a typical antibody Ixodes ricinus immunosuppressor (anti-Iris) protein at a concentration lower than 1 ng/ml. The sensor exhibits a high selectivity and reproducible specific detection.

Synthesis and palladium-catalysed isomerisation of fused polycyclic tetrahydrofurans: efficient and stereoselective one-pot domino construction of functionalised bridged bicyclo[n.2.1] ring systems.

A new one-pot domino reaction for a general entry to functionalised bridged bicyclo[n.2.1] ring systems from alpha, alpha'-diactivated cyclic ketones and trans-1,4-dihalides is described.

Farm diversity, classification schemes and multifunctionality.

Diversity is not only intrinsic to agriculture; it can be considered also as one of its main assets as it provides a wide range of responses that can help to face uncertain futures. The ongoing encounter between changing spatial and temporal frameworks and a set of diverse farming strategies is leading to the emergence of an ongoing flow of development models that could materialize in a wide range of farming practices, contrasting enterprise models, changing relations between rural households and agricultural holdings, and differentiated patterns that link farming and farming families to the wider context in which they are embedded. The many-sided diversity encountered in agriculture is not only the outcome of the agency and polyvalence of the actors involved; their agency and polyvalence are in turn inspired and strengthened by the material and symbolic diversity, which contributes to a further unfolding of diversity.

Isolation of clathrin-coated vesicles by differential and density gradient centrifugation.

Clathrin-coated vesicles (CCVs) are an important class of transport organelles that mediate the endocytosis of proteins and lipids at the plasma membrane and the transport of proteins from the trans-Golgi network to the endosomal/lysosomal system. The authors describe a protocol for isolating CCVs from adult rat brain using differential centrifugation, Ficoll and D(2)O-sucrose density gradient centrifugation, and velocity sedimentation in linear sucrose gradients. The application of this basic method to the isolation of CCVs from developing rat brains and to the generation of relatively crude CCVs from cultured cells is also described.

Extraction of impacted mandibular third molars: postoperative complications and their risk factors.

Objective: The purpose of this prospective study was to evaluate the incidence of various complications, including alveolitis, infection and paresthesia of the inferior alveolar nerve, in association with removal of impacted mandibular third molars. The relation between these 3 complications and several clinical variables (age, sex, degree of impaction, surgical difficulty and use of oral contraceptives) was also examined.

Materials And Methods: Data were collected prospectively for all patients who underwent extraction of an impacted third molar in a single private dental practice over a 12-month period.

Elimination of redundant protein identifications in high throughput proteomics.

Tandem mass spectrometry followed by data base search is the preferred method for protein identification in high throughput proteomics. However, standard analysis methods give rise to highly redundant lists of proteins with many proteins identified by the same sets of peptides. In essence, this is a list of all proteins that might be present in the sample.

The DnaJ-domain protein RME-8 functions in endosomal trafficking.

Through a proteomic analysis of clathrin-coated vesicles from rat liver we identified the mammalian homolog of receptor-mediated endocytosis 8 (RME-8), a DnaJ domain-containing protein originally identified in a screen for endocytic defects in Caenorhabditis elegans. Mammalian RME-8 has a broad tissue distribution, and affinity selection assays reveal the ubiquitous chaperone Hsc70, which regulates protein conformation at diverse membrane sites as the major binding partner for its DnaJ domain. RME-8 is tightly associated with microsomal membranes and co-localizes with markers of the endosomal system.

Non-stoichiometric relationship between clathrin heavy and light chains revealed by quantitative comparative proteomics of clathrin-coated vesicles from brain and liver.

We used tandem mass spectrometry with peptide counts to identify and to determine the relative levels of expression of abundant protein components of highly enriched clathrin-coated vesicles (CCVs) from rat liver. The stoichiometry of stable protein complexes including clathrin heavy chain and clathrin light chain dimers and adaptor protein (AP) heterotetramers was assessed. We detected a deficit of clathrin light chain compared with clathrin heavy chain in non-brain tissues, suggesting a level of regulation of clathrin cage formation specific to brain.

Tuning the orientation of an antigen by adsorption onto nanostriped templates.

We investigate the adsorption of a globular protein (P.69 pertactin, also known as antigen 69k) on protein-repellent hydrophilic substrates bearing regularly spaced hydrophobic nanostripes, for stripe widths comprised between 20 and 160 nm. Protein adsorption is shown to be remarkably well-controlled by the templating substrates, with a near-to-perfect reproduction of stripes by the protein monolayer down to 20 nm width, except for a 5-10 nm broadening.

Molecular mechanisms in clathrin-mediated membrane budding revealed through subcellular proteomics.

Subcellular proteomics is a powerful new approach that combines subcellular fractionation and MS (mass spectrometry) to identify the protein complement of cellular compartments. The approach has been applied to isolated organelles and major suborganellar structures and each study has identified known proteins not previously understood to associate with the compartment and novel proteins that had been described only as predicted open-reading frames from genome sequencing data. We have utilized subcellular proteomics to analyse the protein components of CCVs (clathrin-coated vesicles) isolated from adult brain.

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis.

Identification of a family of endocytic proteins that define a new alpha-adaptin ear-binding motif.

Endocytosis by clathrin-coated vesicles (CCVs) is an important mechanism mediating protein internalization. Here, we show that two proteins identified through a proteomics analysis of CCVs are new components of the endocytic machinery. The proteins, named NECAP (adaptin-ear-binding coat-associated protein) 1 and 2, are paralogues that display no sequence similarity or common domains with any known protein.

Expression of myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase in muscle cell lines: involvement of PtdIns(3)P in insulin-stimulated glucose transport.

X-linked myotubular myopathy is a muscle disorder caused by mutations on the myotubular myopathy-1 (MTM-1) gene, coding for myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A myotubularin.

Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics.

Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs).

The PtdIns3P phosphatase myotubularin is a cytoplasmic protein that also localizes to Rac1-inducible plasma membrane ruffles.

Myotubularin, the phosphatase mutated in X-linked myotubular myopathy, was shown to dephosphorylate phosphatidylinositol 3-monophosphate (PtdIns3P) and was also reported to interact with nuclear transcriptional regulators from the trithorax family. We have characterized a panel of specific antibodies and investigated the subcellular localization of myotubularin. Myotubularin is not detected in the nucleus, and localizes mostly as a dense cytoplasmic network.

Functional redundancy in the myotubularin family.

Myotubularin-related genes define a novel highly conserved family of eukaryotic proteins of at least 11 human members. The hMTM1 gene that codes for myotubularin is mutated in X-linked myotubular myopathy, a severe congenital disease. Recently, we and others have characterized myotubularin as a potent and specific phosphatidylinositol 3-phosphate 3-phosphatase.

A multi-centre study of Osseotite implants supporting mandibular restorations: a 3-year report.

This multi-centre study evaluated the performance of the Osseotite implant in the mandibular arch. Osseotite implants (n = 688) were placed in 172 patients; 43.5% were placed in the anterior mandible and 66.

The myotubularin family: from genetic disease to phosphoinositide metabolism.

The myotubularin-related genes define a large family of eukaryotic proteins, most of them initially characterized by the presence of a ten-amino acid consensus sequence related to the active sites of tyrosine phosphatases, dual-specificity protein phosphatases and the lipid phosphatase PTEN. Myotubularin (hMTM1), the founder member, is mutated in myotubular myopathy, and a close homolog (hMTMR2) was recently found mutated in a recessive form of Charcot-Marie-Tooth neuropathy. Although myotubularin was thought to be a dual-specificity protein phosphatase, recent results indicate that it is primarily a lipid phosphatase, acting on phosphatidylinositol 3-monophosphate, and might be involved in the regulation of phosphatidylinositol 3-kinase (PI 3-kinase) pathway and membrane trafficking.

The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy.

Disorganization of the neurofilament network is a prominent feature of several neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy and axonal Charcot-Marie-Tooth disease. Giant axonal neuropathy (GAN, MIM 256850), a severe, autosomal recessive sensorimotor neuropathy affecting both the peripheral nerves and the central nervous system, is characterized by neurofilament accumulation, leading to segmental distension of the axons. GAN corresponds to a generalized disorganization of the cytoskeletal intermediate filaments (IFs), to which neurofilaments belong, as abnormal aggregation of multiple tissue-specific IFs has been reported: vimentin in endothelial cells, Schwann cells and cultured skin fibroblasts, and glial fibrillary acidic protein (GFAP) in astrocytes.

Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway.

Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres. The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus. It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot-Marie-Tooth disease.

MTM1 mutations in X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM; MIM# 310400) is a severe congenital muscle disorder caused by mutations in the MTM1 gene. This gene encodes a dual-specificity phosphatase named myotubularin, defining a large gene family highly conserved through evolution (which includes the putative anti-phosphatase Sbf1/hMTMR5). We report 29 mutations in novel cases, including 16 mutations not described before.

[Physician-patient relations in family medicine].

PROBLEM ADDRESSED: In addition to clinical instruction, residents need "people" skills that will enable them to deal with all sorts of patients in difficult clinical situations. We planned a series of 12 seminars to teach these skills to first-year residents. OBJECTIVES OF PROGRAM: To ask relevant questions typical of the patient-centred approach; with empathy and respect, to encourage patients to express their emotions; to become more aware of one's own emotions and reactions in one's work as a physician; to negotiate with patients, taking into account both the patient's agenda and one's own.

Genomic organization of the MTM1 gene implicated in X-linked myotubular myopathy.

X-linked recessive myotubular myopathy (XLMTM) is a very severe congenital muscular disease characterised by an impaired maturation of muscle fibres, and caused by defects in the MTM1 gene. This gene defines a new family of putative tyrosine phosphatases conserved through evolution. We have determined intronic flanking sequences for all the 15 exons to facilitate the detection of mutations in patients and genetic counselling.