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D Paulin

Publications and source records attributed to D Paulin.

At least 73 records · Page 4Linked to original sources
PubMed1995

Induction of blood-brain barrier differentiation in a rat brain-derived endothelial cell line.

An immortalized brain capillary endothelial cell line displaying blood-brain barrier characteristics may represent a useful tool for studying blood-brain barrier endothelial cell differentiation and for the in vitro prediction of drug brain penetration. In the present study, we have established a rat cerebral capillary endothelial cell line (CR3) by genomic introduction of the immortalizing SV40 large T gene under the control of the human vimentin promoter. The CR3 cell line displayed endothelial morphological and biochemical characteristics for up to 30 passages. However, the CR3 cell line did not spontaneously express the specific blood-brain barrier markers gamma-glutamyl transpeptidase and mdr P-glycoprotein. However, when the cells were treated with the cell differentiating agent all-trans-retinoic acid, the blood-brain barrier markers were induced. Retinoic acid-treated CR3 cells may thus represent a useful tool for biological and pharmacological research related to the blood-brain barrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Mouse chick chimera: a new model to study the in ovo developmental potentialities of mammalian somites.

Chimeras were prepared by transplanting somites from 9-day post-coïtum mouse embryos or somitic dermomyotomes from 10-day post-coïtum mouse embryos into 2-day-old chick embryos at different axial levels. Mouse somitic cells then differentiated in ovo in dermis, cartilage and skeletal muscle as they normally do in the course of development and were able to migrate into chick host limb. To trace the behavior of somitic myogenic stem cells more closely, somites arising from mice bearing a transgene of the desmin gene linked to a reporter gene coding for Escherichia coli beta-galactosidase (lacZ) were grafted in ovo. Interestingly, the transgene was rapidly expressed in myotomal muscles derived from implants. In the limb muscle mass, positive cells were found several days after implantation. Activation of desmin nls lacZ also occurred in in vitro cultures of somite-derived cells. Our experimental method facilitates investigation of the mechanisms of mammalian development, allowing the normal fate of implanted mouse cells to be studied and providing suitable conditions for identification of descendants of genetically modified cells.

Animals↗
PubMed1995

What can be learned from intermediate filament gene regulation in the mouse embryo.

In recent years, intermediate filaments (IFs) have attracted much interest, largely because their constitutive polypeptide units are specifically expressed in various cell types and thus represent excellent differentiation markers. Data obtained through biochemical studies and molecular cloning have allowed the classification of IFs into five types according to their protein structure. The expression of most IF types is characteristic of a given cell type: cytokeratins (IF types I and II) are produced in epithelia, neurofilaments and alpha-internexin (type IV) in neurons and nestin (type IV) in neuroblast and myoblast. On the other hand the four type III IFs are highly related proteins which are expressed in different cell types. Thus the study of type III IF gene regulation provides an excellent approach towards the analysis of cell-specific transcription. This review focuses on type III IF gene regulation during mouse embryogenesis and describes the latest data obtained through the combination of both in vitro (in cell lines) and in vivo (in transgenic mice) approaches. It appears that, while intragenic sequences play a major role in the regulation of the expression of the genes encoding other types of IFs, a major contribution to the transcriptional regulation of type III IF genes is brought by 5' upstream sequences. However, recent evidence obtained through the use of transgenic mice indicate that upstream sequences must cooperate with intragenic elements to establish the complex and dynamic expression pattern characteristic of type III IF genes. The very high similarity between the coding sequences of type III IF genes raises the question of the significance of the occurrence of four members of this class. We propose a model for the amplification of this small gene family based on the increasing complexity of expression patterns in higher organisms. This could have led first to the requirement for a highly sophisticated control region in an ancestral type III IF gene, followed by two successive gene duplications, thus leading to the appearance of four different regulatory regions directing the cell-specific transcription of nearly identical genes in different cell types.

Animals↗
PubMed1995

Desmin transgene expression in mouse somites requires the presence of the neural tube.

Transgenic mice were used to study the effect of the neural tube on somite myogenesis. These mice express a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene which codes for E. coli beta-galactosidase. In order to determine whether the developmental fate of cells, specifically the prospective myogenic population, in newly developed somites was pre-determined, newly formed somites were isolated from the caudal region of day 9.5 transgenic embryos and transplanted into 8.5 day non-transgenic host embryos. Even though the implanted somites were not oriented in the host embryos, all the specimens examined developed normally at the graft site forming a dermatome, myotome and sclerotome in the correct anatomical positions. The myotome even expressed the desmin transgene. In addition, we isolated the 3 most caudal somites, that is, the most recently developed somites, from day 9.5 transgenic embryos and maintained them on gelatin-coated coverslips in culture for up to 4 days. While these somite explants did not develop myoblasts, it was possible to induce myogenesis by introducing pieces of neural tube into the explant cultures. These results suggest that the developmental fate of cells within the newly formed somite is not predetermined, but is dependent on the microenvironment surrounding the developing somite.

Animals↗
PubMed1994

Mice lacking vimentin develop and reproduce without an obvious phenotype.

To address the biological role of vimentin in the context of the living organism, we have introduced a null mutation of the vimentin gene into the germ line of mice. Surprisingly, animals homozygous for this mutation developed and reproduced without an obvious phenotype. Immunoblotting, immunofluorescence, and immunogold labeling analysis confirmed the absence of vimentin and of the corresponding filament network. Furthermore, no compensatory expression of another intermediate filament could be demonstrated. While these results leave open the question of the possible role of vimentin in unusual situations or pathological conditions, they show that a conspicuous developmental and cell-specific structure that is an integral part of the cytoskeleton can be eliminated without apparent effect on mouse reproduction and development.

Animals↗
PubMed1994

Sequence-specific interaction of alpha-beta-anomeric double-stranded DNA with the p50 subunit of NF kappa B: application to the decoy approach.

The potential use of alpha-beta-anomeric duplex oligonucleotides to inhibit transcription factor activity by the decoy approach is investigated in this report. Indeed, several alpha-beta-anomeric heteroduplexes display a sequence-specific interaction with the p50 subunit of the transcription factor NF kappa B. Used in a decoy approach, these duplexes interact strongly enough with this transcription factor to modulate the expression of a reporter gene, under the control of NF kappa B. However, all the alpha-beta-anomeric heteroduplexes do not interact with the p50 subunit; the sequence of the chirally natural beta-anomeric strand may explain the different recognition properties of the protein. The analysis of the appropriate beta-anomeric sequences is consistent with a preferential interaction of the p50 subunit with one strand of double-stranded DNA.

Base Sequence↗
PubMed1994

Endothelial cells are activated by cytokine treatment to kill an intravascular parasite, Schistosoma mansoni, through the production of nitric oxide.

Like many pathogens that undergo an intravascular stage of development, larvae of the helminth parasite Schistosoma mansoni migrate through the blood vessels, where they are in close contact with endothelial cells. In vitro exposure of murine endothelial cells to various cytokines (interferon gamma, tumor necrosis factor alpha, and interleukin 1 alpha or 1 beta) resulted in their activation to kill schistosomula through an arginine-dependent mechanism involving production of nitric oxide (NO). Cytokine-treated endothelial cells showed increased expression of mRNA for the inducible form of the NO synthase, and both NO production and larval killing were suppressed by treatment with competitive inhibitors. The effector function of cytokine-treated endothelial cells was similar to that of activated inflammatory tissue macrophages, although activation appeared to be differentially regulated in these two cell types. Activated endothelial cells killed older (18-day) forms of the parasite, such as those currently thought to be a primary target of immune elimination in the lungs of mice previously vaccinated with radiation-attenuated cercariae, as well as newly transformed larvae. In C57BL/6 mice, which become resistant to S. mansoni infection as a result of vaccination with irradiated cercariae, endothelial cell morphology characteristic of activation was observed in the lung by 1-2 weeks after challenge infection. Similar endothelial cell changes were absent in P-strain mice, which do not become resistant as a result of vaccination. Together, these observations indicate that endothelial cells, not traditionally considered to be part of the immune system, may play an important role in immunity to S. mansoni and, by means of NO-dependent killing, could serve as effectors of resistance to other intravascular pathogens.

Amino Acid Oxidoreductases↗
PubMed1994

Immortalization of multiple cell types from transgenic mice using a transgene containing the vimentin promoter and a conditional oncogene.

Differentiated clonal cell lines were obtained from transgenic mice carrying a recombinant gene composed of DNA coding for a temperature-sensitive mutant of the simian virus large T antigen under the control of regulatory elements of the human vimentin gene. In response to mitogenic factors the vimentin promoter is activated in the presence of serum in almost all cultured cells independently of their origin. The expression of the T antigen could be controlled both at the level of transcription since the vimentin promoter is growth-regulated and at the level of the protein structure through the temperature stability of the T antigen. Indeed, the switch-off of the oncogene protein is obtained by serum deprivation of the culture and achieved with enhancement of the growth temperature. From transgenic mice several types of clonal differentiated cell lines were established and characterized including melanocytes, macrophages, mesangial, muscle, and endothelial cells. Melanocytes displayed melanin while endothelial cells from brain and heart expressed the related factor VIII and low density lipoprotein absorption capacities. Mesangial cells from kidney exhibited numerous desmosomes. Typical markers of macrophages from bone marrow were observed while skeletal muscle cells fused and contracted.

Animals↗
PubMed1993

Different factors interact with myoblast-specific and myotube-specific enhancer regions of the human desmin gene.

We have previously reported that high level human desmin expression depends on a 280-base pair muscle-specific enhancer which can function not only in myotubes, but can also activate gene expression in myoblasts. We report here that this enhancer contains two different regions, one active in myotubes and the other in myoblasts. In the myotube-specific region, one MyoD1 site and one MEF2 site are necessary for full enhancer activity. Site-directed mutation of the MyoD1 binding site revealed that the intact site is essential for gene expression in myotubes and for transactivation by MyoD1 or myogenin in co-transfected fibroblasts. In the myoblast-specific region, four regions are protected by nuclear factors from the myogenic cell line C2, 7; three regions contain a GC-rich sequence sharing homology with the Krox binding site. Deletion and site-directed mutation experiments demonstrated that at least two Krox-like sequences are required for enhancer activity in myoblasts. In addition, another GC-rich sequence, designated Mb, is also required for full enhancer activity in myoblasts.

Animals↗
PubMed1993

Activation of the human vimentin gene by the Tax human T-cell leukemia virus. I. Mechanisms of regulation by the NF-kappa B transcription factor.

The molecular basis for transactivation of the human vimentin gene by the Tax protein from the human T-cell leukemia virus (HTLV-1) is analyzed in this report. We first demonstrate that the factor NF-kappa B binds to the vimentin promoter. Using gel retardation assays, we found the putative NF-kappa B protein. Specific antibodies and competition experiments between the NF-kappa B-binding site in the interleukin-2R alpha and HIV-1 promoters and the vimentin promoter show that the three sites have identical affinity for the factor. We further show that the mechanisms of activation of NF-kappa B by the Tax protein involve a cellular inducer. Nuclear extract from lymphoid cells expressing Tax can induce in vitro a NF-kappa B binding activity in nonlymphoid cytosolic extract. This inducer, if preexisting in an inactivate state in T-cells which are not expressing Tax, cannot be switched to an active state by addition of partially purified Tax protein. While found in the nucleus of Tax-expressing cells in our experiments, this inducer might be cytoplasmic as well. In a first attempt to identify and characterize the inducer, we present the results of fractionation assays of nuclear extract.

Base Sequence↗
PubMed1993

Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control.

Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII-related antigen. Treatment of IVEC cells with Interleukin-1 beta (IL-1 beta) at 10 U.ml-1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 microM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT-PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium.

Antigens, Polyomavirus Transforming↗
PubMed1993

Identification of a negative element in the human vimentin promoter: modulation by the human T-cell leukemia virus type I Tax protein.

The vimentin gene is a member of the intermediate filament multigene family and encodes a protein expressed, in vivo, in all mesenchymal derivatives and, in vitro, in cell types of various origin. We have previously demonstrated that the expression of this growth-regulated gene could be trans activated by the 40-kDa Tax protein of HTLV-I (human T-cell leukemia virus type I) and that responsiveness to this viral protein was mediated by the presence of an NF-kappa B binding site located between -241 and -210 bp upstream of the mRNA cap site (A. Lilienbaum, M. Duc Dodon, C. Alexandre, L. Gazzolo, and D. Paulin, J. Virol. 64:256-263, 1990). These previous assays, performed with deletion mutants of the vimentin promoter linked to the chloramphenicol acetyltransferase gene, also revealed the presence of an upstream negative region between -529 and -241 bp. Interestingly, the inhibitory activity exerted by this negative region was overcome after cotransfection of a Tax-expressing plasmid. In this study, we further characterize the vimentin negative element and define the effect of the Tax protein on the inhibitory activity of this element. We first demonstrate that a 187-bp domain (-424 to -237 bp) behaves as a negative region when placed upstream either of the NF-kappa B binding site of vimentin or of a heterologous enhancer such as that present in the desmin gene promoter. The negative effect can be further assigned to a 32-bp element which is indeed shown to repress the basal or induced activity of the NF-kappa B binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice.

During the development of the mouse embryo, desmin is one of the first muscle proteins detected in both the heart and the somites. The expression of the desmin gene differs from most other muscle genes, since it is initiated in replicating myoblasts and accumulates as the muscle differentiates. We have characterized a muscle-specific enhancer which directs the expression of desmin in vitro in the myoblasts and myotubes of C2 cells but not in non-myogenic cells. We report here on the generation and characterization of transgenic mice bearing a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coli beta-galactosidase (Des1-nlacZ). The enhancer activity of the desmin promoter is very strong and the reporter gene expression is easily detected in tissue sections. We have demonstrated that the regulatory elements present in the transgene Des1-nlacZ are sufficient to direct muscle-specific and developmentally regulated expression of nlacZ in skeletal muscles. Endogenous desmin expression and transgene activity were found to be correlated during the development of skeletal muscles. The transgene was expressed in the committed mononucleate myoblasts as well as in the myotubes. In addition, we have shown that the desmin-derived sequences direct a highly selective expression of nlacZ in cells that leave the somites and invade the limb bud, indicating that the cells that migrate from the somites are already predetermined for myogenesis. In contrast, smooth and cardiac muscle cells were beta-galactosidase negative both during embryonic and foetal development. Interestingly, the transgene was found to be expressed in the conduction system of the heart, which exhibits many features characteristic of skeletal muscles.

Animals↗
PubMed1993

Organization and expression of intermediate filaments in epithelial cells expressing the HTLV-I Tax protein.

Intermediate filaments (IF) represent major components of the cytoskeletal network. These proteins which are differentially expressed according to the cell type, constitute a dynamic structure which not only contributes to the cell architecture but also defines its state of differentiation. Furthermore, numerous observations have shown that the IF network is altered in cells transformed by tumorigenic viruses. We have previously demonstrated that HTLV-I (human T-cell leukemia virus type I) transformed T cells were characterized by a high level of vimentin transcripts and that the HTLV-I Tax regulatory protein was able to transactivate the vimentin promoter transfected into Jurkat and HeLa cells. To enlarge the scope of this study, we investigated the effects of the Tax protein on the expression and organization of IF of epithelial cells in which the IF network is composed of vimentin and cytokeratin. To this aim, we have developed a model of epithelial cells (HeLa) stably expressing the tax sequences which were introduced by using retrovirus-mediated gene transfer. Half of the Tax expressing HeLa clones were loosely adherent to the culture surface and were displaying remarkable morphological alterations, as ascertained by the presence of round-shaped or spindle-shaped cells. In these cells, expression of this viral protein correlated to a pronounced disruption in the distribution of both the vimentin and the cytokeratin networks, as shown by immunofluorescence and ultrastructural analysis. Indeed, vimentin filaments appeared to be concentrated in discrete spots throughout the cytoplasm, while the cytokeratin filaments appeared to form a dense ring around the nucleus. More importantly, mRNA and protein analysis indicate an enhanced expression of the cytokeratin 7 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelium↗
PubMed1992

Identification and characterization of neutral endopeptidase in endothelial cells from venous or arterial origins.

Neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) is a cell membrane-associated zinc metalloprotease, which cleaves peptides like atrial natriuretic peptide (ANP) on the amino side of hydrophobic amino acids. Although NEP is mainly located in reabsorptive epithelia (kidney proximal tubule), it is also present in non-epithelial cells such as neuronal cells. As the renal NEP cannot account for the entire ANP metabolism, other locations were postulated. The present experiments show its expression in endothelial cells (EC) from arterial (bovine pulmonary, porcine, and human aorta) and venous (human umbilical, rabbit ear marginal) origins. Three different methods were used to demonstrate the presence of the protein and its mRNA. 1) NEP enzymatic activity was estimated using both a synthetic ([D-Ala2,Leu5]enkephalin) and a natural substrate (bradykinin). Using the synthetic substrate, the enzymatic activity in EC was completely blocked by thiorphan, a specific NEP inhibitor with an IC50 value in the nanomolar range. In contrast, captopril, bestatin, [2-guanidinoethylmercapto]succinic acid, inhibitors of angiotensin-converting enzyme, aminopeptidases, and carboxypeptidases, respectively, were 10,000 times less active, revealing an inhibition profile similar to that of the purified enzyme. Bradykinin, a natural substrate of NEP, was in part metabolized by NEP, in the presence of captopril, since 50% of the formation of the major metabolite bradykinin 1-7 was inhibited by thiorphan. 2) Immunoreactive NEP was detected on the plasma membrane of rabbit EC using a monoclonal antibody directed against the homologous renal enzyme. 3) NEP mRNA was detected by Northern blot analysis of rabbit EC as a major transcript of 3.9 kilobases. Reverse transcriptase polymerase chain reaction amplification showed the presence of a specific transcript in all EC tested. Therefore, endothelial NEP may play an important role in the inactivation of ANP, bradykinin, and endothelins by its localization facing the circulating vasoactive peptides.

Animals↗
PubMed1992

Mechanism of entry of a xenotropic MMuLV-derived recombinant retrovirus into porcine cells using the expression of the reporter nlslacZ gene.

A xenotropic Moloney murine leukemia virus-derived recombinant retrovirus (MMuLVSVnlslacZ) has been utilized to study the mechanism of virus entry into endothelial and epithelial porcine cells. In the genome of this recombinant retrovirus, the nlslacZ reporter gene is under the transcriptional control of both LTR and SV40 early promoter. The entry of the retrovirus has been determined from the expression of this transduced reporter gene after its integration into the infected cells. This allows the detection of a very low level of viral infection and hence entry of the virus. Exposure of the virus-cell mixture to acidic pH (less than 6) during the early phase of interaction reduces the level of internalization. Cellular infection in presence of weak bases, ammonium chloride and amantadine and an ionophore monensin at concentrations sufficient to neutralize the endosomal pH does not modify the extent of viral entry into the cells. The results indicate that the entry of the recombinant retrovirus into porcine cells takes place by a pH-independent viral membrane-cell plasma membrane fusion mechanism.

Amantadine↗
PubMed1991

High level desmin expression depends on a muscle-specific enhancer.

Expression of the desmin gene is increased several-fold during myogenesis. To identify the DNA sequences involved in this regulation, a 16-kilobase fragment containing a 4.5-kilobase 5'-flanking region and the complete sequence of the human desmin gene has been isolated. The transcription initiation sites were determined by S1 nuclease mapping and primer extension analysis. A series of constructs have been made by fusing different lengths of human desmin 5'-flanking fragments to the bacterial chloramphenicol acetyl transferase (CAT) gene, these constructs were introduced either into mouse myogenic cells or into nonmyogenic cells. Comparison of the transient expression of CAT between myogenic and nonmyogenic cells demonstrated that the sequence including 228 base pairs upstream of the transcription initiation site is sufficient to confer low level, muscle-specific expression of the desmin gene. A negative region was located between -693 and -228 base pairs relative to the desmin mRNA start site. High level expression of the gene depends on a 280-base pair muscle-specific enhancer located between -693 and -973 base pairs upstream of the transcription initiation site. This enhancer can activate either the desmin promoter or heterologous promoters about 20-70-fold in myogenic cells. The enhancer, which also acts in myoblast, is discussed in the context of early expression of the desmin during skeletal myogenesis in the mammalian embryo.

Animals↗