Search PubMed⌕ Search

Biomedical subjects

J Samarut

Publications and source records attributed to J Samarut.

At least 55 records · Page 3Linked to original sources
PubMed1996

Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities.

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185-189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

Alkaline Phosphatase↗
PubMed1995

Virofection: a one-step procedure for using replication-defective retrovirus vectors.

Virofection is a simple new way to use replication-defective vectors. It consists of the cotransfection of two plasmids: one plasmid bearing the genome of the replication-defective retrovirus vector and a second "helper" plasmid expressing the gag, pol, and env retrovirus sequences. It provides stable integration into the chromosome and long-term expression of only vector-borne sequences. We present here several helper plasmids derived from avian leukosis viruses, which we have used for the virofection of the lacZ reporter gene in chicken cell cultures. Expression can be stabilized at a very high rate in both chicken fibroblasts and blastoderm cells, without giving rise to replication-competent virus.

Animals↗
PubMed1995

A 43-kDa protein related to c-Erb A alpha 1 is located in the mitochondrial matrix of rat liver.

In order to characterize Sterling's triiodothyronine (T3) mitochondrial receptor using photoaffinity labeling, we observed two specific T3-binding proteins in the inner membrane (28 kDa) and in the matrix (43 kDa) of rat liver mitochondria. Western blots and immunoprecipitation using antibodies raised against the T3-binding domain of the T3 nuclear receptor c-Erb A alpha 1 indicated that at least the 43-kDa protein was c-Erb A alpha 1-related. In addition, gel mobility shift assays demonstrated the occurrence of a c-Erb A alpha 1-related mitochondrial protein that specifically binds to a natural or a palindromic thyroid-responsive element. Moreover, this protein specifically binds to a direct repeat 2 sequence located in the D-loop of the mitochondrial genome. Furthermore, electron microscopy studies allowed the direct observation of a c-Erb A-related protein in mitochondria. Lastly, the relative amounts of the 43-kDa protein related to c-Erb A alpha 1 were in good correlation with the known mitochondrial mass in three typical tissues. Interestingly, expression of a truncated form of the c-Erb A alpha 1 nuclear receptor in CV1 cells was associated with a mitochondrial localization and a stimulation of mitochondrial activity. These results supply evidence of the localization of a member of the nuclear receptor superfamily in the mitochondrial matrix involved in the regulation of mitochondrial activity that could act as a mitochondrial T3-dependent transcription factor.

Affinity Labels↗
PubMed1995

In vitro differentiation of embryonic stem cells into glial cells and functional neurons.

Mouse embryonic stem cells were induced to differentiate in culture with retinoic acid. Putative precursors of neurons and glial cells (nestin-positive cells) were clearly identified as early as three days after the onset of differentiation. At day 6, neuron-like cells could be clearly identified, either as isolated cells or as cellular networks. Some of these cells were positive for astrocyte- or oligodendrocyte-specific antigens (GFAP or O4 antigens, respectively). Other cells were positive for neuron-specific antigens (cytoskeleton proteins MAP2, MAP5 and NF200, as well as synaptophysin). Some neuronal-like cells were also positive for acetylcholinesterase activity or glutamic acid decarboxylase expression, indicating that ES cells could differentiate into GABAergic and possibly cholinergic neurons. Electrophysiological analyses performed in voltage clamp conditions showed that cell membranes contained voltage-dependent channels. Overshooting action potentials could be triggered by current injection. Taken together, these data provide evidence that embryonic stem cells can differentiate first into neuron-glia progenitors, and later into glial cells and functional neurons, in vitro. This technique provides an unique system to study early steps of neuronal differentiation in vitro.

Animals↗
PubMed1994

CNRS defended.

Explore the source record for details and available documents.

France↗
PubMed1994

Virofection: a new procedure to achieve stable expression of genes transferred into early embryos.

A new procedure, virofection, designed to stabilize the expression of transfected DNA has been developed. It exploits the capacity of retroviruses to integrate their genome into the chromosomes of host cells. The co-transfection of two plasmids, one carrying the genome of a defective retrovirus vector, the other one encoding all the retroviral proteins, results in a transient production of infectious virus particles. These particles can infect the neighboring cells and this leads to the stable integration of the vector genome. This procedure is time-saving and appears to be quite efficient. When applied to chicken embryonic fibroblasts cultured in vitro, it resulted in the stable expression of the lacZ gene in more than 30% of the cells, and did not induce chronic viremia. Stable lacZ expression was also achieved in chicken embryos in ovo. Virofection appears to be a promising and generally applicable method for implementing stable, safe and efficient gene transfer in vitro and in vivo.

Animals↗
PubMed1994

c-erbA alpha/T3R and RARs control commitment of hematopoietic self-renewing progenitor cells to apoptosis or differentiation and are antagonized by the v-erbA oncogene.

In AEV-transformed erythroleukemic cells the v-erbA gene product is likely to antagonize the function of triiodothyronine (T3) and retinoic acid (RA) receptors and thereby to block cell differentiation. We have thus investigated the effects of T3 and RA on normal early erythrocytic progenitor cells. Here we show: (1) that either RA or T3 play an essential role during the early commitment to erythrocytic differentiation, (2) that both T3 and RA induce death by apoptosis and a strong inhibition of self-renewal in progenitor cells grown in the absence of differentiation-inducing agents and (3) that the v-erbA oncogene renders erythrocytic progenitor cells insensitive to apoptosis and to self-renewal inhibition induced by RA or T3. The behaviour of a non-transforming mutant of v-erbA suggests that this v-erbA-induced protection is related to its transforming potential.

Animals↗
PubMed1994

v-erbA stimulates quail myoblast differentiation in a T3 independent, cell-specific manner.

The v-erbA oncoprotein represents a mutated version of a thyroid hormone receptor, responsible for the induction of a differentiation arrest in chicken erythroid cells. We have studied the influence of v-erbA on proliferation and differentiation of avian myoblasts. Secondary quail myoblast cultures were infected either with an avian retrovirus carrying the v-erbA oncogene in association with the neomycin resistance gene, or with a control deleted v-erbA/neoR alpha retrovirus. We report here that v-erbA expression led to an increase in myoblast proliferation and to a surprising stimulation of quail myoblast terminal differentiation. In addition, these effects occurred in the presence or absence of T3, and v-erbA did not suppress T3 influence on myoblasts. Transient transfection assays demonstrated that, in contrast to its action in HeLa cells, v-erbA was unable to repress the transcriptional activation of a TRE-CAT reporter gene by liganded c-erbA alpha receptors in quail myoblasts. We also observed that the AP-1/c-erbA/v-erbA interactions are not functional in quail myoblasts. These data suggest that, in these cells, v-erbA action does not interfere with T3 induced mechanisms. They also demonstrate a cell specificity for the v-erbA pathway. Lastly, expression of c-erbA/v-erbA chimeric proteins and of the S61G v-erbA mutant indicates that the DNA binding domain of v-erbA, and more specifically serine 61, is directly involved in the enhancement of myoblast differentiation by the oncoprotein.

Animals↗
PubMed1994

c-ErbA, but not v-ErbA, competes with a putative erythroid repressor for binding to the carbonic anhydrase II promoter.

The carbonic anhydrase II (CAII) gene is the only known gene identified as direct target for v-ErbA-mediated repression in avian erythroleukemic cells transformed by Avian Erythroblastosis Virus (AEV). This gene is transcriptionally activated by thyroid hormone (T3) in normal erythrocytic cells. In this work we have analysed the molecular basis of the transcriptional control of the CAII gene by c-ErbA and v-ErbA. We show that several domains in the promoter control hormonal regulation of transcription. One domain proximal to the TATA box mediates T3 response but contains no identified binding site for c-ErbA. An other domain termed PAL2 is approximately 600 bp upstream the transcription initiation site and contains a c-ErbA binding site. We show that when it is associated to a heterologous promoter this site mediates transcriptional repression in erythrocytic cells but not in HeLa cells. Moreover, this site binds a nuclear erythrocyte-specific factor that we called NFX, which is different from c-ErbA. heterodimers between c-ErbA and the 9-cis retinoic acid receptor (RXR) compete with NFX for binding to PAL2. In contrast, v-ErbA alone or in association with RXR is a very poor competitor and is unable to chase NFX out of the PAL2 site. We propose that NFX is a transcription repressor whose activity is inhibited by c-ErbA but not v-ErbA. This mechanism might contribute to the overall regulation of the carbonic anhydrase II promoter. These data illustrate another possible mechanism through which v-ErbA might antagonize the function of c-ErbA in controlling gene expression.

Animals↗
PubMed1994

A nontoxic and versatile protein salting-out method for isolation of DNA.

A pivotal technique in basic and applied molecular biology is the isolation of DNA. However, the present DNA extraction methods are either toxic, expensive, time-consuming and laborious or restricted to certain applications. Here we describe a nontoxic and versatile protein salting-out method for convenient and rapid extraction of large as well as small DNA molecules from vertebrate cells and plasmid DNA from bacteria. Easy and relatively imprecise manipulations of a large number of samples result in high yields of pure mammalian and plasmid DNA that are suitable for transformation of bacteria, restriction enzyme analyses, Southern blotting, end labeling of DNA, PCR and sequencing.

Animals↗
PubMed1994

Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts.

The expression of the retinoblastoma susceptibility (RB-1) gene was investigated in highly proliferating mouse embryonic stem (ES) cells and in slowly proliferating mouse embryonic fibroblasts. The RB protein was expressed at the same level in these two cell types. Mainly hyperphosphorylated RB was detected in exponentially-growing ES cells. Embryonic fibroblasts and embryonic stem cells were synchronized by colcemid block followed by mitotic shake-off. In embryonic fibroblasts, DNA replication started 10-15 h after exit from mitosis and RB was transiently dephosphorylated during the G1 phase as previously described. In ES cells, DNA replication started 2 h after release from the colcemid block but virtually no hypophosphorylated RB was observed after the release. Instead, there was a dramatic decrease in the total RB protein level between exit from mitosis and entry into S phase. These observations were made by using two different monoclonal antibodies, both in immunoblotting and immunoprecipitation experiments. Absence of hypophosphorylated RB and cell cycle-dependent change in total RB protein level may be relevant to the high proliferation rate and to the tumorigenic nature of mouse embryonic stem cells.

Animals↗
PubMed1993

v-jun cooperates with v-erbB to transform the thrombocytic/megakaryocytic lineage.

The transforming properties of v-jun, the viral counterpart of the transcription factor AP1, were investigated in avian hematopoietic cells. Two retroviruses, called JB and JBN, expressing both v-jun and v-erbB, were constructed using an avian erythroblastosis-based vector. We show that the cooperative action of both oncogenes allowed the virus to efficiently transform bone marrow cells. No such transformation was obtained with either oncogene alone. JB-transformed bone marrow cells expressed GATA-1, TAL-1, and histone H5, suggesting that they belong to the erythrocytic/thrombocytic lineage. (Thrombocytes are the avian homologues of mammal megakaryocytes.) Moreover, after induction with phorbol 12-myristate 13-acetate JB-transformed bone marrow cells began to differentiate and synthesized high levels of platelet glycoproteins, indicating that they were of thrombocytic origin. These results were confirmed by c-ets1 analysis since this transcription factor, specifically found in cells with megakaryocytic but not erythrocytic features, was clearly detected in these cells.

Animals↗
PubMed1993

Sequence analysis reveals that the BTG1 anti-proliferative gene is conserved throughout evolution in its coding and 3' non-coding regions.

The human BTG1 gene (expressing an anti-proliferative function) is an evolutionarily conserved gene homologous to the murine PC3/TIS21 genes. Here, we report the cloning and sequencing of the murine BTG1 coding region and chicken BTG1 cDNA. The putative human and mouse BTG1 proteins are 100% identical; the chicken BTG1 cDNA contains an open reading frame of 170 amino acids with a 91% identity to its human and murine counterparts. The 3'-untranslated region of BTG1 is also highly conserved (82% homology between human and chicken), suggesting that it plays a key role in the regulation of BTG1 expression. These data confirm that BTG1 is phylogenetically highly conserved and that BTG1 and PC3/TIS21 may constitute the first members of a new family of functionally related genes.

Amino Acid Sequence↗
PubMed1993

A hepatitis B virus pre-S-retinoic acid receptor beta chimera transforms erythrocytic progenitor cells in vitro.

In this report, we investigated the transforming properties of retinoic acid receptor beta (RAR beta). The v-erbA protein, which is the viral oncogenic homologue of the thyroid hormone receptor, was replaced by either the complete RAR beta (beta R) or a hepatitis B virus pre-S-RAR beta (H beta R) hybrid product in an avian erythroblastosis virus-based vector. In chicken hematopoietic cells, the H beta R protein was able to transform erythroid progenitor cells, whereas no such transformation was observed with the wild-type beta R protein. Moreover, the fully transformed phenotype was observed even in the absence of v-erbB, and H beta R-transformed erythroid cells grew independently of growth factors and transforming growth factor alpha. The analysis of erythrocytic-specific proteins revealed that the transformed cells were blocked at the colony-forming unit-erythroid stage and that the expression of the carbonic anhydrase II gene, a gene normally regulated by thyroid hormones, was repressed by the H beta R protein. Finally, hepatocarcinomas rapidly developed in some chickens infected in ovo with viruses encoding either the normal or the hybrid H beta R, suggesting that an inappropriate expression of the RAR beta gene may represent an important event in oncogenesis.

Amino Acid Sequence↗
PubMed1993

v-erbA cooperates with bFGF in neuroretina cell transformation.

We have studied the transforming ability of the oncogenes erbA and/or erbB in chicken neuroretina (CNR) cells and the effect of basic fibroblast growth factor (bFGF) on the transformed phenotype. The erbA oncogene alone was only transforming in the presence of bFGF. In contrast, cells expressing erbB as well as erbA + erbB were transformed in a bFGF-independent manner and were unresponsive to the growth factor. We studied whether other oncogenes could also block the cooperation between erbA and bFGF. Cytoplasmic or membrane-bound oncogenes (src, ras, or mill raf) increased the transforming potential of erbA but rendered the cells unresponsive to bFGF. Conversely, the nuclear oncogenes tested (fos and myb-ets + myc) also cooperated with erbA in CNR cell transformation but the cells remained responsive to the growth factor. A likely explanation is that CNR cells carrying the cytoplasmic but not the nuclear oncogenes have already activated the bFGF signal transduction pathway.

Animals↗