Upgrade of the CATS sample changer on FIP-BM30A at the ESRF: towards a commercialized standard.

An upgraded version of the sample changer ;CATS' (Cryogenic Automated Transfer System) that was developed on the FIP-BM30A beamline at the ESRF is presented. At present, CATS is installed at SLS (three systems), BESSY (one system), DLS (two systems) and APS (four systems for the LSCAT beamline). It consists mainly of an automated Dewar with an assortment of specific grippers designed to obtain a fast and reliable mounting/dismounting rate without jeopardizing the flexibility of the system. The upgraded system has the ability to manage any sample standard stored in any kind of puck.

Regulated expression of the proteoglycan SPOCK in the neuromuscular system.

SPOCK is prevalent in developing synaptic fields of the central nervous system (Charbonnier et al., 2000. Mech. Dev. 90, 317-321). The expression of SPOCK during neuromuscular junction (NMJ) formation was compared to agrin and acetylcholine receptor (AChR) distribution. SPOCK is detected within the myogenic masses during the early steps of embryonic development, and distributed in the cytoplasm of myotubes before coclustering with AChRs. In the adult, SPOCK is present in axons and is highly expressed by Schwann cells. SPOCK altered expression pattern after nerve lesioning, or cholinergic transmission blockade, strongly indicate that its cellular distribution at the NMJ depends on innervation.

Myoneurin, a novel member of the BTB/POZ-zinc finger family highly expressed in human muscle.

Initially characterized as Drosophila developmental regulators, the BTB/POZ and zinc finger proteins (BTB/POZ-ZF) constitute a growing family of proteins with gene expression regulatory functions since they have been shown to be involved in both transcriptional activation and repression of various genes in a broad range of species, including mammals. Here we report the cloning of a novel human transcript, coding for a 68-kDa deduced BTB/POZ-ZF protein. This molecule, called myoneurin on the basis of its prevalent expression in the neuromuscular system, contains an amino-terminal BTB/POZ domain and eight tandemly repeated zinc-finger motifs of the C(2)H(2) type. The murine myoneurin, identified in the mouse embryo, is highly homologous to the human protein.

Particle-associated retroviral RNA and tandem RGH/HERV-W copies on human chromosome 7q: possible components of a 'chain-reaction' triggered by infectious agents in multiple sclerosis?

Different groups have observed retrovirus particle (RVP) production in cell cultures from patients with multiple sclerosis (MS). This in vitro production appeared relatively specific for MS versus healthy controls, but was likely to be enhanced or activated by infectious triggers such as Herpesviruses (e.g. HSV, EBV). Independent molecular analysis of retroviral RNA associated with RVP revealed two different genetic families of endogenous retroviral elements (HERV): MSRV/HERV-W and RGH/HERV-H. Interestingly, these sequences were detected by mutually exclusive primers in RT - PCR amplifications. Surprisingly, these two HERV families both contain an ancestral proviral copy inserted in chromosome 7q21-22 region at about 1 kb of distance of each other. Another HERV-W proviral sequence is located within a T-cell alpha-delta receptor (TCR) gene in chromosome 14q11.2 region. Interestingly, these two regions correspond to genetic loci previously identified as potentially associated with 'multigenic' susceptibility to MS and TCR alpha chain genetic determinants have been reported to be statistically associated with MS. A plausible role for infectious agents triggering a co-activation of the chromosome 7q HERV tandem (replicative retrovirus and/or other virus and/or intracellular bacteria) and, eventually, other HERV copies, is discussed. The role of particular HERV polymorphism and the production of pathogenic molecules (gliotoxin and superantigen) possibly associated with retroviral expression are also evoked. An integrative concept of pathogenic 'chain-reaction' in MS involving several step-specific pathogenic 'agents' and 'products' somewhat interacting with particular genetic elements would federate most partial data obtained on MS, including retroviral expression.

New perspectives in multiple sclerosis: retroviral involvement and glial cell death.

Retroviral involvement in the pathogenic cascade in multiple sclerosis (MS) and a cytotoxic activity with narrow specificity towards glial cells have been recently considered as credible working hypotheses to explain some of the complex pathophysiological and neuropathological features of MS. The partial characterization of exogenous retroviral sequences, thought to be associated with MS, has led us to the identification of new human endogenous retroviruses closely related to the extracellular multiple sclerosis associated retrovirus (MSRV). These endogenous retroviruses (HERV-TcR and HERV-7q) have the potential to be transcribed into RNA and proteins. Interestingly, the env domain of HERV-7q could code for a 59.8 kDa secreted glycoprotein (called enverin) with an immunoregulatory region. The presence in various MS biological fluids of a cytotoxic activity able to induce programmed cell death for oligodendrocytes and astrocytes suggests the possibility of a demyelination phenomenon as part of direct glial cell damage. Moreover, both retroviral expression and cytotoxic factor production have been evidenced in MS monocyte/macrophage cultures and MS cerebrospinal fluid. It is now crucial to better characterize the endo/exo retroviruses possibly involved in MS and their pathogenic potential, and to identify the contributing factor(s) to the gliotoxicity found in the MS cerebrospinal fluid or serum, as well as to elucidate the mechanism of induction of the observed programmed glial cell death.

Expression of the proteoglycan SPOCK during mouse embryo development.

SPOCK is a modular proteoglycan, with homology with proteins involved in cell adhesion processes and neurogenesis. We have previously shown that SPOCK transcripts predominate in the adult mouse brain. Here, we report its expression during mouse embryonic development by in situ hybridization, and immunocytochemistry. SPOCK is actively expressed at the onset of neurogenesis during periods of neuron migration and axonal outgrowth. At a later developmental stage, its expression is particularly prevalent within developing synaptic fields. In the peripheral nervous system, SPOCK expression is also developmentally regulated particularly in dorsal root ganglion neurons.

Endogenous retroviruses and multiple sclerosis. II. HERV-7q.

The search for new endogenous retroviral sequences, on the basis of sequence homologies with the pol gene of the recently reported multiple sclerosis associated retrovirus (MSRV), allowed us to identify a full length endogenous retrovirus sequence located on the long arm of human chromosome 7. This retrovirus, HERV-7q, includes in its env region, within a single 1,620 bp open reading frame, a 664 bp domain almost identical to a 3' non-coding region of the rab7 gene. Transcripts encompassing both the env and the 3' LTR regions of HERV-7q have already been identified as expressed sequence tags, suggesting that this env-like gene might code for a 538 amino acid long deduced protein.

[Endogenous retroviral sequences analogous to that of the new retrovirus MSRV associated with multiple sclerosis (part 1)]. / Séquences rétrovirales endogènes analogues à celle du nouveau rétrovirus MSRV associé à la sclérose en plaques (1re partie).

Multiple sclerosis (MS) is still of unknown origin and may involve autoimmune, genetic and viral components in a pathogenic sequence whose relative importance is yet to be determined. A peptide, isolated from the cerebrospinal fluid of MS patients, is similar to a fragment of the pol protein reverse transcriptase (RT) of the newly reported MSRV retrovirus. The 700 amino acid sequence of MSRV-RT is closely related to a novel human retroviral-like sequences. We also identified a gag-like sequence upstream of this human genomic RT-like sequence, which allowed us to identify altogether 4,000 nucleotides, possibly coding for an endogenous retroviruses. Homologous sequences found in other locations in the human genome seem to characterize a new family of retroviral endogenous sequences, which may be of relevance to multiple sclerosis.

An endogenous retrovirus with nucleic acid sequences similar to those of the multiple sclerosis associated retrovirus at the human T-cell receptor alpha, delta gene locus.

Retroviruses are suspected to be involved in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). Here, we describe a complete cartography of a novel human endogenous retroviral sequence with a pol domain which shares a high homology with the pol sequence of the multiple sclerosis associated retrovirus (MSRV). Since this new endogenous retroviral sequence is located in the close vicinity of the locus of the human gene coding for the T-cell receptor (TcR) alpha and delta chains on chromosome 14, it could be of potential interest for the understanding of MS pathogenesis.

Genomic organization of the human SPOCK gene and its chromosomal localization to 5q31.

SPOCK, previously identified as testican, is a modular proteoglycan that carries both chondroitin and heparan sulfate glycosaminoglycan side chains. The overall genomic organization has been established. The SPOCK gene spans at least 70 kb and is composed of 11 exons: the first half of the gene is dramatically expanded, but the second half is more compact. In situ hybridization and YAC mapping independently linked the SPOCK gene to 5q31, a region containing an impressive number of genes encoding growth factors, cytokines, and neurotransmitter and hormone receptors. The gene is located between the IL9 and the EGR1 genes, bordering the smallest commonly deleted region of chromosome 5.

[Cloning of testican/SPOCK in man and mouse. Neuromuscular expression perspectives in pathology]. / Clonage de la testicane/SPOCK chez l'homme et la souris. Expression au niveau neuromusculaire et perspectives en pathologie.

We have recently cloned a novel proteoglycan initially identified in human testis and hence previously called testican. A close examination of the overall protein structure reveals three main regions: four osteonectin/SPARC-like domains encompassing the amino-terminal and central part of the deduced protein, a Kazal-like motif overlapping the third domain, and the CWCV domain in the carboxyl-terminal end region of the protein core. We propose to call it SPOCK, the acronym of SPARC/Osteonectin CWCV and Kazal-like domains proteoglycan, according to its specific multidomain structure. To get further insight into the function, a Northern blot analysis was performed in order to determine the site of expression in various adult tissues; a 5.2 kb transcript appeared only but strongly in mouse brain. The structure of the murine brain proteoglycan was determined through molecular cloning; human and mouse deduced proteins are highly homologous with 95% overall amino acid identity. Murine brain serial sections hybridized with cDNA and immunological probes revealed identical distribution in discrete cerebral regions, such as CA3 hippocampal region and cerebellum. Immunoelectron microscopy showed the antigen selectively localized in the post-synaptic density of scattered pyramidal neurons and Purkinje cells. Structural analysis, a main expression in nervous system and preliminary assignment of the human gene in a critical region for neuropathologies, suggest that SPOCK may be of importance in neural development and neurodegenerative diseases.

Structure and cellular distribution of mouse brain testican. Association with the postsynaptic area of hippocampus pyramidal cells.

The complete deduced primary structure of mouse brain testican has been established from cDNA cloning. The cDNA encodes a polypeptide of 442 amino acids belonging to the proteoglycan family. The mouse brain testican core protein is 95% identical to its human testicular counterpart. In situ hybridization investigations revealed that mouse testican mRNA is mainly present in a subpopulation of pyramidal neurons localized in the CA3 area of the hippocampus. An immunocytochemical approach, with antibodies directed against an overexpressed chimeric antigen, produced in bacterial systems, showed that testican is associated with the postsynaptic region of these pyramidal neurons. Testican includes several putative functional domains related to extracellular or pericellular proteins associated with binding and/or regulatory functions. On the basis of its structural organization and its occurrence in postsynaptic areas, this proteoglycan might contribute to various neuronal mechanisms in the central nervous system.

Amino acid sequence of the Homo sapiens brain 21-23-kDa protein (neuropolypeptide h3), comparison with its counterparts from Rattus norvegicus and Bos taurus species, and expression of its mRNA in different tissues.

The amino acid sequence of neuropolypeptide h3 from Homo sapiens brain has been determined. It revealed that h3 is the exact counterpart of the 21-kDa protein from Bos taurus brain and the 23-kDa protein from Rattus norvegicus brain: The three proteins belong to the same 21-23-kDa protein family. Multiple tissue Northern blots showed that the mRNA encoding the 21-23-kDa protein is expressed in different amounts according to tissues and species; it is particularly abundant in Rattus norvegicus testis.

Proteoglycans in male reproductive tract.

Proteoglycans in male reproductive tract have been mainly characterized in testicular extracellular matrix and somatic cells. Heparan sulfate, chondroitin sulfate and hybrid chondroitin/heparan sulfate proteoglycans coexist within the testes. Their biological roles are not currently established, however, the molecular characterization of some of them is indicative that they might be involved in various regulatory processes during spermatogenesis.

Testican, a multidomain testicular proteoglycan resembling modulators of cell social behaviour.

The molecular characterization of a human testicular proteoglycan, the progenitor of a seminal plasma glycosaminoglycan-bearing peptide, was achieved by cDNA cloning. Its protein core encompasses several domains encountered in various proteins associated with adhesion, migration and cell proliferation. An osteonectin-like domain, a Kazal-like sequence and a 46-amino-acid motif around a Cys-Trp-Cys-Val peptide encountered in cell-surface antigens, cell-adhesion molecules and growth-factor-binding proteins are distributed within the testican protein core. Testican is the progenitor of the unique heparan/chondroitin-sulfate-bearing peptide present in human seminal plasma, a feature which might confer additional potentialities to this hybrid proteoglycan.

Characterization of a human seminal plasma glycosaminoglycan-bearing polypeptide.

A glycosaminoglycan-bearing polypeptide (S.GP), present in human seminal plasma, was purified to homogeneity by a combination of CsCl density-gradient centrifugation, f.p.l.c. ion-exchange chromatography on a Mono Q HR column and Superose 6 gel filtration. The observed polydispersity of S.GP was attributed to the heterogeneity of its glycosaminoglycan content. Enzymic deglycosylation experiments and N-terminal amino-acid sequence determination indicate that it consists of a polypeptide (apparent molecular mass approx. 18 kDa) bearing both chondroitin and heparan sulphate chains. Evidence is given that S.GP contains a glycosaminoglycan-linkage domain of a so far uncharacterized gene product, proteolytically processed in the genital tract.

Expression of the serglycin gene in human leukemic cell lines.

The expression of the human serglycin gene was determined in nine human leukemic cell lines, representing a spectrum of erythrocytic, megakaryocytic, monocytic, granulocytic, and lymphocytic potentialities. By Northern blot analysis, a 1.4 kb transcript was characterized in some of these cell lines, using a cDNA probe coding for human serglycin. Five of these cell lines, HEL, U-937, HL-60, K-562, and KU-812 were treated with phorbol myristic acetate to induce differentiation. Under these conditions the expression of the serglycin gene was modulated compared to the non-induced cells. HL-60, K-562, and KU-812 were also induced with dimethyl sulfoxide and retinoic acid; variations in serglycin transcript level were also observed. The present investigation establishes, at the nucleic acid level, the ability of various cells mimicking different stages in the developmental pathways of the haemopoietic lineage to synthesize proteoglycans belonging to the serglycin family. The results reported here led us to conclude that serglycin expression is closely associated with the haemopoietic cell differentiation pathway. The putative functions of serglycin in the haemopoietic system are briefly discussed.

Localization of human platelet proteoglycan gene to chromosome 10, band q22.1, by in situ hybridization.

A cDNA probe of 527 base pairs coding for the human platelet proteoglycan (PPG) protein core demonstrated that the PPG gene lies on the long arm of chromosome 10, band q22.1. This result and other available data concerning proteoglycans containing serine-glycine repeats indicate that this gene is involved in the expression of a proteoglycan in various blood cell types.

Characterization and N-terminal sequence of human platelet proteoglycan.

Human platelet proteoglycan (P.PG) was prepared from a 4 M-guanidinium chloride platelet extract in the presence of proteinase inhibitors. The purification procedure included CsCl-density-gradient centrifugation, DEAE-Sepharose CL-6B ion-exchange chromatography and f.p.l.c. on a Mono Q HR 5/5 column. P.PG was recovered as a polydisperse molecule, but the protein core appeared to be at least 90% homogeneous. This observation could be due to partial proteolysis of the core protein during extraction. The N-terminal sequence of the human P.PG core protein was determined up to residue 66 and was shown to be highly homologous to the propeptide of an embryonic rat yolk-sac tumour proteoglycan (PG19); the significance of this homology is discussed.

Complete amino acid sequence of a human platelet proteoglycan.

The primary structure of a human platelet proteoglycan (P.PG) core was established by a combination of amino acid sequence analysis and cDNA cloning. The deduced 131 amino acid long protein contains eight Ser-Gly repeats. The significance of homologies observed between P.PG and promyelocytic leukemia cell line proteoglycans is discussed.