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Biomedical subjects

J P Hugnot

Publications and source records attributed to J P Hugnot.

At least 19 recordsLinked to original sources
PubMed2003

The human NTERA2 neural cell line generates neurons on growth under neural stem cell conditions and exhibits characteristics of radial glial cells.

NTERA2 cells are a human neural cell line generating neurons after exposure to retinoic acid and, as such, are widely used as a model of neurogenesis. We report that these cells form spheres when grown in serum-free medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). These spheres were found to express markers of radial glial cells such as, Pax6, glutamate transporter (GLAST), tenascin C, brain lipid-binding protein (BLBP), and the 3CB2 antigen. On plating on an adhesive substrate, NTERA2 spheres generate a large percentage of immature neurons (30-50%) together with a minority of cells of the oligodendrocyte lineage. Thus NTERA2 cells share properties with neural stem cells. However, at variance with the latter, we found that they produce their own bFGF implicated in an autocrine or paracrine proliferative loop and that they do not generate astrocytes after differentiation. These results provide an interesting model to study radial glial cells and their role in human neurogenesis.

Animals↗
PubMed2001

Direct cell-cell interactions control apoptosis and oligodendrocyte marker expression of neuroepithelial cells.

During brain development, the neuroepithelium generates neurons and glial cells. Proliferation and differentiation of neuroepithelial cells are controlled by a complex combination of secreted factors and more intrinsic or local mechanisms, such as lateral inhibition and asymmetric division. To obtain further insights into the signals governing neuroepithelial cell fate, we used the immortomouse to derive conditionally immortalised cell lines from mouse E10 neuroepithelium. We isolated a nestin-positive basic fibroblast growth factor (bFGF)-responsive cell line (SVE10-23) which mostly differentiate into astrocytes when cocultured with primary cortical cells. We found that, by simply lowering the cell density, SVE10-23 cells embarked on oligodendrocytic differentiation as indicated by the strong expression of galactocerebroside C and 2'3'-cyclic nucleotide 3'-phosphodiesterase. Apoptosis accompanied the differentiation, and all cells died within 1 week. We present here evidence that direct interactions between cells are the main mechanism regulating this oligodendrocytic differentiation. We demonstrate that SVE10-23 cells contact or proximity inhibit their differentiation, prevent apoptosis, and promote their proliferation. Similarly, others nestin-positive precursor cell lines and nonimmortalised bFGF-grown E10 cells were found to spontaneously differentiate at low density, thus generalising the idea that neural precursor fate is regulated by direct cell-cell interactions. The SVE10-23 cell line provides a valuable tool with which to study further the molecular components implicated in this mode of regulation.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed2001

Regulation of glial differentiation of MHP36 neural multipotent cell line.

MHP36 is a nestin bFGF-dependent cell line isolated from embryonic hippocampus using a thermolabile form of SV40 T antigen. When grafted in ischemic hippocampus MHP36 cells differentiate and alleviate the cognitive deficit associated with the lesion. We report here in vitro features of MHP36 cells. First, we found that T Ag expression was not necessary for MHP36 growth as cells cultured at the nonpermissive temperature carry on proliferating at a normal rate, Second, we observed that part of MHP36 cells spontaneously differentiate into astrocytes when bFGF is removed at39 degrees C. This differentiation was increased 4-fold by leukemia inhibitory factor. Third, we found that the majority of cells spontaneously expressed oligodendrocytic markers (CNPase, A2B5, GalC) when cultured at low density.

Animals↗
PubMed1997

New modulatory alpha subunits for mammalian Shab K+ channels.

Two novel K+ channel alpha subunits, named Kv9.1 and Kv9.2, have been cloned. The Kv9.2 gene is situated in the 8q22 region of the chromosome. mRNAs for these two subunits are highly and selectively expressed in the nervous system. High levels of expressions are found in the olfactory bulb, cerebral cortex, hippocampal formation, habenula, basolateral amygdaloid nuclei, and cerebellum. Interestingly Kv9.1 and Kv9.2 colocalized with Kv2.1 and/or Kv2.2 alpha subunits in several regions of the brain. Neither Kv9.1 nor Kv9.2 have K+ channel activity by themselves, but both modulate the activity of Kv2.1 and Kv2.2 channels by changing kinetics and levels of expression and by shifting the half-inactivation potential to more polarized values. This report also analyzes the changes in electrophysiological properties of Kv2 subunits induced by Kv5.1 and Kv6.1, two other modulatory subunits. Each modulatory subunit has its own specific properties of regulation of the functional Kv2 subunits, and they can lead to extensive inhibitions, to large changes in kinetics, and/or to large shifts in the voltage dependencies of the inactivation process. The increasing number of modulatory subunits for Kv2.1 and Kv2.2 provides an amazingly new capacity of functional diversity.

Amino Acid Sequence↗
PubMed1997

Modes of regulation of shab K+ channel activity by the Kv8.1 subunit.

The Kv8.1 subunit is unable to generate K+ channel activity in Xenopus oocytes or in COSm6 cells. The Kv8.1 subunit expressed at high levels acts as a specific suppressor of the activity of Kv2 and Kv3 channels in Xenopus oocytes (Hugnot, J. P., Salinas, M., Lesage, F., Guillemare, E., Weille, J., Heurteaux, C., Mattéi, M. G., and Lazdunski, M. (1996) EMBO J. 15, 3322-3331). At lower levels, Kv8.1 associates with Kv2.1 and Kv2.2 to form hybrid Kv8.1/Kv2 channels, which have new biophysical properties and more particularly modified properties of the inactivation process as compared with homopolymers of Kv2.1 or Kv2.2 channels. The same effects have been seen by coexpressing the Kv8.1 subunit and the Kv2.2 subunit in COSm6 cells. In these cells, Kv8.1 expressed alone remains in intracellular compartments, but it can reach the plasma membrane when it associates with Kv2.2, and it then also forms new types of Kv8.1/Kv2. 2 channels. Present results indicate that Kv8.1 when expressed at low concentrations acts as a modifier of Kv2.1 and Kv2.2 activity, while when expressed at high concentrations in oocytes it completely abolishes Kv2.1, Kv2.2, or Kv3.4 K+ channel activity. The S6 segment of Kv8.1 is atypical and contains the structural elements that modify inactivation of Kv2 channels.

Amino Acid Sequence↗
PubMed1997

The human inward rectifying K+ channel Kir 2.2 (KCNJ12) gene: gene structure, assignment to chromosome 17p11.1, and identification of a simple tandem repeat polymorphism.

K+ channels are essential for a variety of cellular functions in both excitable and nonexcitable cells, and K+ channel gene alteration has been recently described in cardiac and neurological disorders. To explore further the relations between hereditary human diseases and K+ channels, we isolated from a human cosmid library the gene encoding the inwardly rectifying K+ channel alpha-subunit Kir 2.2 (KCNJ12). PCR analysis performed on this clone indicates that the entire open reading frame is contained in one unique exon. A polymorphic (CA)16 sequence was localized 2.2 kb upstream of the ATG start codon. Fluorescence in situ hybridization on human metaphases assigns the gene to band 17p11.1. The implication of a deletion of the Kir 2.2 gene in the Smith-Magenis syndrome, which is also localized at 17p11, is unlikely since a Kir 2.2-linked microsatellite sequence could be amplified from the DNA of a Smith-Magenis syndrome affected patient bearing a 17p interstitial deletion.

Abnormalities, Multiple↗
PubMed1996

Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels.

Outward rectifier K+ channels have a characteristic structure with six transmembrane segments and one pore region. A new member of this family of transmembrane proteins has been cloned and called Kv8.1. Kv8.1 is essentially present in the brain where it is located mainly in layers II, IV and VI of the cerebral cortex, in hippocampus, in CA1-CA4 pyramidal cell layer as well in granule cells of the dentate gyrus, in the granule cell layer and in the Purkinje cell layer of the cerebellum. The Kv8.1 gene is in the 8q22.3-8q24.1 region of the human genome. Although Kv8.1 has the hallmarks of functional subunits of outward rectifier K+ channels, injection of its cRNA in Xenopus oocytes does not produce K+ currents. However Kv8.1 abolishes the functional expression of members of the Kv2 and Kv3 subfamilies, suggesting that the functional role of Kv8.1 might be to inhibit the function of a particular class of outward rectifier K+ channel types. Immunoprecipitation studies have demonstrated that inhibition occurs by formation of heteropolymeric channels, and results obtained with Kv8.1 chimeras have indicated that association of Kv8.1 with other types of subunits is via its N-terminal domain.

Amino Acid Sequence↗
PubMed1995

Heterologous multimeric assembly is essential for K+ channel activity of neuronal and cardiac G-protein-activated inward rectifiers.

The family of G-protein-activated inward-rectifiers K+ channels presently comprise at least 3 cloned members called GIRK1, GIRK2 and GIRK3. A close structural parent of GIRK channels has recently been described as being an ATP-sensitive K+ channel. This paper shows that Xenopus expression of this new channel that we call GIRK4 does not produce an ATP-inhibitable activity with a pharmacological activation by pinacidil as previously described but instead a G-protein activated inward-rectifier. While oocyte expression of single subunits is infrequent and relatively modest in intensity, expression of GIRK1,2, GIRK1,4 and GIRK2,4 mixtures leads to routine and robust expression of K+ channels indicating that heterologous subunit assembly is necessary for activity. Activity of GIRK1,2, GIRK1,4 and GIRK2,4 channels required the presence of ATP acting as an activator at the cytoplasmic face and is further activated by the beta gamma subunits.

Adenosine Triphosphate↗
PubMed1994

Cloning provides evidence for a family of inward rectifier and G-protein coupled K+ channels in the brain.

MbIRK3, mbGIRK2 and mbGIRK3 K+ channels cDNAs have been cloned from adult mouse brain. These cDNAs encode polypeptides of 445, 414 and 376 amino acids, respectively, which display the hallmarks of inward rectifier K+ channels, i.e. two hydrophobic membrane-spanning domains M1 and M2 and a pore-forming domain H5. MbIRK3 shows around 65% amino acid identity with IRK1 and rbIRK2 and only 50% with ROMK1 and GIRK1. On the other hand, mbGIRK2 and mbGIRK3 are more similar to GIRK1 (60%) than to ROMK1 and IRK1 (50%). Northern blot analysis reveals that these three novel clones are mainly expressed in the brain. Xenopus oocytes injected with mbIRK3 and mbGIRK2 cRNAs display inward rectifier K(+)-selective currents very similar to IRK1 and GIRK1, respectively. As expected from the sequence homology, mbGIRK2 cRNA directs the expression of G-protein coupled inward rectifier K+ channels which has been observed through their functional coupling with co-expressed delta-opioid receptors. These results provide the first evidence that the GIRK family, as the IRK family, is composed of multiple genes with members specifically expressed in the nervous system.

Amino Acid Sequence↗
PubMed1994

Expression cloning in K+ transport defective yeast and distribution of HBP1, a new putative HMG transcriptional regulator.

The rat HBP1 cDNA was cloned by its capacity to suppress the potassium transport-defective phenotype of mutant Saccharomyces cerevisiae cells. HBP1 cDNA encodes a 513 amino acids protein which, unexpectedly, does not share any homology with K+ transporters or K+ channels. However, a search in protein databases reveals that HBP1 contains a putative DNA-binding domain called HMG-box. Northern blot analysis shows that HBP1 is expressed in a variety of tissues and that in adipocyte and myogenic cell lines, its expression is directly related to differentiation. Taken together, the results suggest that the rat HBP1 is a new member of the HMG class of transcriptional regulators involved in cell differentiation pathways.

Adipocytes↗
PubMed1993

Multiple mRNA isoforms encoding the mouse cardiac Kv1-5 delayed rectifier K+ channel.

The mouse Kv1-5 K+ channel cDNA has been cloned from heart. This channel was highly expressed in heart and, to a lesser extent, in other tissues, including brain and thymus. Two alternatively spliced isoforms were found. The longer form encoded a 602-amino acid protein, while in the short form (Kv1-5 delta 5'), the first 200 amino acids lying upstream the transmembrane segment S1 were deleted. RNase protection experiments showed that both Kv1-5 mRNA isoforms are present in the mouse tissues examined, the longer form being predominant. The short mRNA (Kv1-5 delta 5') arose by an unusual splicing event within the exonic sequence. An additional short cDNA clone (Kv1-5 delta 3') that codes for a carboxyl-terminal truncated protein has been isolated. The gene coding sequence contained a single exon and has been mapped on human chromosome 12 (p13) and on mouse chromosome 6 (band F). Expression in Xenopus oocytes revealed that the long (Kv1-5) and the amino-terminal deleted (Kv1-5 delta 5') isoforms elicited similar K+ currents with a drastically decreased efficacy for Kv1-5 delta 5'. The carboxyl-terminal truncated Kv1-5 delta 3' clone was not functional but inhibited the expression of the long isoform.

Alternative Splicing↗
PubMed1993

Expression of the dystrophin gene in cultured fibroblasts.

The dystrophin whose defect is responsible for Duchenne and Becker muscular dystrophies is present in muscle, brain and cerebellum. We describe here the detection of dystrophin in human cultured skin fibroblasts, L809 cells and murine 3T6 cell line. Dystrophin transcripts initiated at the muscle specific first exon can also be amplified by cDNA-PCR from various fibroblastic cells. The expression of the dystrophin gene in fibroblasts could account for some abnormalities observed in patient's fibroblast cultures.

Animals↗
PubMed1992

Distal transcript of the dystrophin gene initiated from an alternative first exon and encoding a 75-kDa protein widely distributed in nonmuscle tissues.

A transcript generated by the distal part of the Duchenne Muscular Dystrophy (DMD) gene was initially detected in cells where the full size 14-kilobase (kb) messenger RNA is not found at a significant level. This transcript, approximately 4.5 kb long, corresponds to the cysteine-rich and carboxyl-terminal domains of dystrophin. It begins with a novel 80- to 100-nucleotide exon containing an ATG start site for a new coding sequence of 17 nucleotides in-frame with the consecutive dystrophin cDNA sequence from exon 63. This result suggests the existence of a third promoter that would be localized about 8 kilobases upstream from exon 63 of the DMD gene. The distal transcript is widely distributed but is absent in adult skeletal and myometrial muscle. It is much more abundant in fetal tissues. With an antibody directed against the dystrophin carboxyl terminus, the protein corresponding to this transcript was detected as a 70- to 75-kDa entity on Western blots. It was found in all tissues analyzed except in skeletal muscle. It was not found in lymphoblastoid cells from a Duchenne patient with a complete deletion of the dystrophin gene. The role and subcellular localization of this protein is not known. It may explain extramuscular symptoms exhibited by some Duchenne patients.

Animals↗
PubMed1992

Positive and negative regulatory DNA elements including a CCArGG box are involved in the cell type-specific expression of the human muscle dystrophin gene.

The muscle-specific promoter of the dystrophin gene is active in skeletal, cardiac, and smooth muscles and is specifically stimulated during differentiation of myoblasts into multinucleated myotubes. An 850-base pair (bp) DNA fragment upstream from the cap site is able to confer a partial muscle specificity to a reporter gene. The region between -850 and -140 bp includes nonspecific negative and positive regulatory sequences. A continuous stretch of 140 bp upstream from the cap site exhibits a striking conservation between rodents and human (93% homology) and still retains muscle preference of expression. It contains two putative binding sites for factors involved in regulation of other muscle-specific genes, a CCArGG box and an E box. This latter element, however, is unable to confer the ability to be transactivated by MyoD1 to the dystrophin promoter. The -140-bp promoter fragment exhibits antagonist effects contributed by one inhibiting sequence (nucleotide -140/-96), active in all cell types, and one activating region, from nucleotide -96 to the cap site, sufficient to confer a muscle preference of expression, in which the CCArGG box seems to play a major role.

Animals↗
PubMed1992

Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein.

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22-58), and complex-specific (amino acids 304-332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60-97 and 298-397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60-135; amino acids 60-205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298-397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.

Amino Acid Sequence↗
PubMed1992

Are cysteine-rich and COOH-terminal domains of dystrophin critical for sarcolemmal localization?

It has been hypothesized that the tight localization of dystrophin at the muscle membrane is carried out by its cysteine-rich and/or carboxyl domains. We report the results of biochemical and immunocytochemical investigations of dystrophin in muscle from a 1-yr-old patient with a large deletion that removes the distal part of the dystrophin gene, thus spanning the exons coding for the cysteine-rich and the carboxy-terminal domains, and extends beyond the glycerol kinase and congenital adrenal hypoplasia genes. Immunological analysis of muscle dystrophin shows that the deletion results in the production of a truncated, but stable, polypeptide correctly localized at the sarcolemma. These data indicate that neither the cysteine-rich domain, nor the carboxyl domain, are necessary for the appearance of normal dystrophin sarcolemmal localization.

Cysteine↗
PubMed1991

Illegitimate (or ectopic) transcription proceeds through the usual promoters.

Illegitimate transcription corresponds to the low level presence of specific transcripts in nonspecific cells. This phenomenon allows to analyse any tissue-specific disease transcript in any easily accessible cell. We demonstrate here that the start sites of transcription are the same in specific and non-specific cells, which indicates that illegitimate transcription is due to a low level activity of the normal promoter. In addition, it is possible to increase about 10 fold the abundance of illegitimate transcripts through the use of cycloheximide. This treatment should, therefore, facilitate detection and qualitative analysis of illegitimate transcripts.

Base Sequence↗