Biomedical subjects

P Thiébaud

Publications and source records attributed to P Thiébaud.

11 recordsLinked to original sources

PubMed2001

Developmental program expression of myosin alkali light chain and skeletal actin genes in the rainbow trout Oncorhynchus mykiss.

We have isolated MLC1(F) (tMLC1(F)), MLC3(F) (tMLC3(F)) and skeletal actin cDNAs from the teleost Oncorhynchus mykiss. Sequence analysis indicates that tMLC1(F) and tMLC3(F) are not produced from differentially spliced mRNAs as reported in avians and rodents but are encoded by different genes. Results from RNase protection analysis showed that the corresponding transcripts are expressed in fast skeletal muscles. Whole-mount in situ hybridisation revealed distinct expression patterns of the myosin alkali light chains and skeletal actin genes during skeletal muscle development in the embryo.

Actins↗

PubMed2001

Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures.

In this study we examined the passive biocompatibility of a three-dimensional microelectrode array (MEA), designed to be coupled to organotypic brain slice cultures for multisite recording of electrophysiological signals. Hippocampal (and corticostriatal) brain slices from 1-week-old (and newborn) rats were grown for 4-8 weeks on the perforated silicon chips with silicon nitride surfaces and 40 microm sized holes and compared with corresponding tissue slices grown on conventional semiporous membranes. In terms of preservation of the basic cellular and connective organization, as visualized by Nissl staining, Timm sulphide silver-staining, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunostaining, the slice cultures grown on chips did not differ from conventionally grown slice cultures. Neither were there any signs of astrogliosis or neurodegeneration around the upper recording part of the 47-microm-high platinum-tip electrodes. Slice cultures grown on a separate set of chips with platinum instead of silicon nitride surfaces also displayed normal MAP2 and GFAP immunostaining. The width of the GFAP-rich zone (glia limitans) at the bottom surface of the slice cultures was the same ( approximately 20 microm) in cultures grown on chips with silicon nitride and platinum surfaces and on conventional insert membranes. The slice cultures grown on chips maintained a normal, subfield differentiated susceptibility to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) and the neurotoxin trimethyltin (TMT), as demonstrated by the cellular uptake of propidium iodide (PI), which was used as a reproducible and quantifiable marker for neuronal degeneration. We conclude that organotypic brain slice cultures can grow on silicon-based three-dimensional microelectrode arrays and develop normally with display of normal subfield differentiated susceptibilities to known excito- and neurotoxins. From this it is anticipated that the set-up, designed for recording of electrophysiological parameters, can be used for long-term studies of defined neuronal networks and provide valuable information on both normal, neurotoxicological and neuropathological conditions.

Animals↗

PubMed2000

Glider and Vision: two new families of miniature inverted-repeat transposable elements in Xenopus laevis genome.

We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the alpha-tropomyosin (alpha-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated alpha-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the Xenopus laevis genome. Every MITEs elements but two described in our study are found either in 5' or in 3' regulatory regions of genes suggesting a potential role in gene regulation.

Animals↗

PubMed1999

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices.

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

Animals↗

PubMed1999

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.

Animals↗

PubMed1999

Two skeletal alpha-tropomyosin transcripts with distinct 3'UTR have different temporal and spatial patterns of expression in the striated muscle lineages of Xenopus laevis.

The Xenopus laevis alpha-tropomyosin (TM) gene, like its vertebrates counterparts, encodes muscle and non-muscle isoforms through two promoters and alternatively spliced exons. In the present study we describe a cDNA clone (XTMalpha7) encoding a skeletal muscle isoform of the gene that differs from the previously described skeletal TM transcript (XTMalpha2) by its 3'UTR sequence. The two skeletal alpha-TM encoding mRNAs are generated through distinct 3'end processing using different polyA signals and distinct patterns of exon splicing. Using RNAse protection and RNA in situ hybridization, we have analysed the developmental and spatial expression of the two transcripts. Both are expressed in the embryo, but XTMalpha7 is by far the most prevalent of the two. In contrast, only XTMalpha2 is expressed in adult striated muscle tissues. In the embryo, the spatial expression of XTMalpha7 is restricted to the somites whereas XTMalpha2 is expressed in both somites and embryonic heart.

3' Untranslated Regions↗

PubMed1999

Differential expression of two skeletal muscle beta-tropomyosin mRNAs during Xenopus laevis development.

A cDNA clone for a Xenopus laevis skeletal muscle beta-tropomyosin (beta-TMad) isoform was isolated from an adult skeletal muscle cDNA library. Sequence analysis revealed that this clone corresponded to a second beta-tropomyosin mRNA distinct from the one that was previously characterized (beta-TMemb). The two skeletal beta-TM mRNAs originate from distinct genes and are differentially expressed during development. Beta-TMemb mRNA is expressed only in the somites of the early embryo while beta-TMad mRNA is expressed in pre-metamorphic tadpoles and adult skeletal muscles. We have isolated the promoter region of the beta-TMemb gene and shown that a DNA construct containing 2.9 kb of promoter region is properly expressed after injection in the embryo.

Animals↗

PubMed1998

A novel tropomyosin isoform encoded by the Xenopus laevis alpha-TM gene is expressed in the brain.

A full-length cDNA clone encoding a novel non-muscle tropomyosin (nmTM) termed X alpha TMB5, as yet unidentified in vertebrates, was isolated from a Xenopus laevis (Xl) brain cDNA library. X alpha TMB5 is derived from the XL alpha-tropomyosin gene (X alpha TM), which was previously found to express striated muscle and nmTM isoforms via an alternative splicing mechanism. The deduced amino-acid sequence reveals that X alpha TMB5 contains 248 amino acids. The protein differs from the skeletal muscle alpha-TM isoform only at its NH2-terminal region. The mRNA encoding X alpha TMB5 is expressed mainly in brain and striated muscle. Genomic clone analysis reveals that, unlike mammals and avian, the X alpha TM gene is devoid of the brain-specific exon 9c. The amphibian alpha-TM gene is a complex transcription unit containing 14 exons, including two alternative promoters, two internal mutually exclusive exons, and two alternatively spliced 3' exons that encode two different COOH-terminal coding regions. Therefore, a total of at least five distinct mRNAs are expressed from the X alpha TM gene in a cell-type-specific manner.

Amino Acid Sequence↗

PubMed1998

Alpha-tropomyosin gene expression in Xenopus laevis: differential promoter usage during development and controlled expression by myogenic factors.

Tropomyosins (TMs) constitute a group of contractile proteins encoded by a multigene family showing distinct cell-type-specific and developmental expression patterns. In mammals and birds, the alpha-TM gene is the most complex and can produce several muscle and non-muscle isoforms. We report here the characterization of the 5' region of the Xenopus laevis alpha-TM gene and its developmental expression. The 5' region of the gene is structurally related to the avian and mammalian cognates and presents two promoters flanking a pair of alternatively spliced exons, 2a/2b, where exon 2a is a smooth-muscle-specific exon. The internal promoter is used to generate a non-muscle low molecular weight TM whilst muscle TM isoforms originate from the distal promoter. RNase protection analysis shows that the two promoters have distinct temporal programs of activation. The internal promoter is activated early in oogenesis and non-muscle transcripts are found throughout oogenesis, embryogenesis and in adult tissues. Only low molecular weight non-muscle TM-encoding mRNAs are expressed in oogenesis. The distal promoter is silent during oogenesis, and the skeletal muscle alpha-TM transcripts accumulate from stage 15 in the embryo and are expressed in adult striated muscle tissues. In situ hybridization indicates that these transcripts are expressed in both the somites and heart of the embryo. Ectopic expression of myogenic factors, but not the MEF2 myocyte-specific enhancer factor 2 factors SL1 and SL2, can induce the expression of the alpha-TM gene suggesting that the gene is a direct target for myogenic but not for MEF2 factors. The amphibian alpha-TM gene constitutes a gene marker for studying the developmental control expression of muscle genes in the different myogenic lineages.

Alternative Splicing↗

PubMed1997

Towards amperometric immunosensor devices.

In contrast to optical immunosensors, the electrochemical detection of an immunanalytical reaction does require a labeling, but allows an easier discrimination of specific and non-specific binding. We present a concept and first results for a multivalent amperometric immunosensor system which is based on silicon technology. The capture molecule streptavidin, covalently immobilized on silica, allows the immobilization of biotinylated antigens at a defined density. A nanostructured gold electrode serving as a stable network of nanowires is expected to be beneficial for the electrochemical detection of bound ferrocene-labeled antibody molecules. The results presented focus on site-specific immobilization of streptavidin on silica and reduction of non-specific binding of proteins.

Bacterial Proteins↗

PubMed1997

Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures.

A three-dimensional platinum (Pt) microelectrode array embedded on a micromachined silicon (Si) substrate (porosity of 13%, via hole diameter of 40 microns) has been developed. Electrodes are 35-micron wide and 20-microns high, spaced 200 microns apart and arranged in an elliptic geometry. Integrated within a microperfusion chamber, the devices were used for stimulation and recording experiments of hippocampal slice cultures over a period of several days.

Animals↗