Biomedical subjects

P Pognonec

Publications and source records attributed to P Pognonec.

At least 19 recordsLinked to original sources

PubMed2001

MICE, a program to track and monitor animals in animal facilities.

BACKGROUND: A growing number of laboratories are using the mouse as a model system in developmental biology as well as in molecular biology. Surprisingly, most of these laboratories do not have reliable computerized systems to track these animals, and the few commercial solutions available are expensive. We thus developed MICE (Mouse Information and Classification Entity), a program aimed at facilitating the monitoring of animals in animal facilities. RESULTS: This program consists of a virtual facility in which scientists can perform all the tasks done in the real world (i.e., receiving animals, breeding them, preparing cage labels, etc.). Recording of each animal (birth date, cage number, ID number, tail analysis number, parents, genetic status, genetic background, etc.) enables reliable tracking. According to any parameter of interest, animals can then be identified, grouped, sorted, moved, and so forth. Crossings are automatically processed by the program. For example, new genetic backgrounds, generation number, and anticipated due dates are determined. The program also reminds the user when new births are expected and entering newborn animals only requires a few clicks. The genealogy of each animal can be determined in two different ways, one being the visualization of a genealogical tree from which information of ancestors can be retrieved. CONCLUSION: This standalone program, that will be distributed free of charge to academic laboratories requesting a license, represents a new and valuable tool for all animal facility users, and permits simple and reliable tracking and retrieving of animals.

Animals↗

PubMed2001

Bcl-XL expression correlates with primary macrophage differentiation, activation of functional competence, and survival and results from synergistic transcriptional activation by Ets2 and PU.1.

Depriving primary bone marrow-derived macrophages of colony-stimulating factor-1 (CSF-1) induces programmed cell death by apoptosis. We show that cell death is accompanied by decreases in the expression of anti-apoptotic Bcl-x(L) protein and the Ets2 and PU.1 proteins of the Ets transcription factor family. Macrophages require both priming and triggering signals independent of CSF-1 to kill neoplastic cells or microorganisms, and this activation of macrophage competence is accompanied by increased expression of bcl-x(L), ets2, and PU.1. Furthermore, we show that only Ets2 and PU.1, but not Ets1, function in a synergistic manner to transactivate the bcl-x promoter. The synergy observed between PU.1 and Ets2 is dependent on the transactivation domains of both proteins. Although other transcription factors like Fos, c-Jun, Myc, STAT3, and STAT5a are implicated in the activation of macrophage competence or in CSF-1 signaling, no synergy was observed between Ets2 and these transcription factors on the bcl-x promoter. We demonstrate that the exogenous expression of both Ets2 and PU.1 in macrophages increases the number of viable cells upon CSF-1 depletion and that Ets2 and PU.1 can functionally replace Bcl-x(L) in inhibiting Bax-induced apoptosis. Together, these results demonstrate that PU.1 and Ets2 dramatically increase bcl-x activation, which is necessary for the cytocidal function and survival of macrophages.

Animals↗

PubMed2001

Transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Ets, Rel/NFkappaB, STAT and AP1 transcription factor families.

Transcription factors play an essential role in determining the fate of a cell by affecting the expression of target genes involved in proliferation, in differentiation and in programmed cell death. Under certain conditions, some of these factors are capable of deregulating expression of genes involved in the cell cycle and/or in programmed cell death resulting in uncontrolled proliferation of the cell. The focus of this review is on the transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein. Since 1999, several papers have implicated members of the Ets, Rel/NFkappaB, STAT and AP-1 families as transcription factors regulating bcl-x expression. A specific emphasis of these different transcription factor families on bcl-x regulation in hematopoietic cells is discussed.

Animals↗

PubMed2001

[Molecular (de)regulation and cancer: new therapeutic strategies].

The considerable progress of molecular biology within the past twenty years has permitted a more and more detailed characterization of the molecular mechanisms regulating cell proliferation. The corollary to these discoveries has been the identification of different deregulations yielding to cell transformation and cancer. The goal of this review is to present new therapeutic tools that stemmed from the now well understood logic underlying cell transformation. These tools, based on the intimate understanding of signalization pathways, aim at restoring the molecular controls which had been abrogated during the process of cell transformation. We present a survey of these new proposed therapeutic strategies. These new approaches will probably allow the clinician, in the near future, to combine traditional therapies with more targeted ones, and thus to limit side effects often associated with classical cancer therapies, while improving the overall effect of the treatment.

Animals↗

PubMed2000

Virtual nitrogen tank to monitor frozen cell stocks.

We developed a program to facilitate the monitoring of biological samples (cell lines, sera, etc.) that are stored in liquid nitrogen containers. The program consists of a "virtual" container in which scientists can store their samples and a program that records the location of each sample, cell characteristics, storage dates, names of the manipulators and much more. Additional comments and a photograph can be associated with each vial, allowing for reliable tracking of samples. Vials can then be identified according to any parameter of interest to the scientist, including associated comments. Once identified, the program visually presents the location of these vials, which simplifies retrieving them from the real container. The program records the thawing of vials, along with the date and the name of the operator. Any academic laboratory requesting this standalone program will be granted a free license for its use.

Blood↗

PubMed1999

The Ets2 transcription factor inhibits apoptosis induced by colony-stimulating factor 1 deprivation of macrophages through a Bcl-xL-dependent mechanism.

Bcl-xL, a member of the Bcl-2 family, inhibits apoptosis, and its expression is regulated at the transcriptional level, yet nothing is known about the transcription factors specifically activating this promoter. The bcl-x promoter contains potential Ets binding sites, and we show that the transcription factor, Ets2, first identified by its sequence identity to v-ets of the E26 retrovirus, can transactivate the bcl-x promoter. Transient expression of Ets2 results in the upregulation of Bcl-xL but not of Bcl-xS, an alternatively spliced gene product which induces apoptosis. Ets2 is ubiquitously expressed at low levels in a variety of cell types and tissues but is specifically induced to abundant levels during macrophage differentiation. Since Bcl-xL is also upregulated during macrophage differentiation, we asked whether the bcl-x could be a direct downstream target gene of Ets2 in macrophages. BAC1.2F5 macrophages, which are dependent on macrophage colony-stimulating factor 1 (CSF-1) for their growth and survival, were used in these studies. We show that CSF-1 stimulation of BAC1.2F5 macrophages results in the upregulation of expression of ets2 and bcl-xL with similar kinetics of induction. In the absence of CSF-1, these macrophages undergo cell death by apoptosis, whereas constitutive expression of Ets2 rescues these cells from cell death, and bcl-xL is upregulated. These results strongly suggest a novel role of Ets2 in affecting apoptosis through its regulation of Bcl-xL transcription.

Alternative Splicing↗

PubMed1998

A reliable way of obtaining stable inducible clones.

Inducible gene expression systems provide a powerful tool for the analysis of gene product functions. The 'Tetracycline (Tc) expression system' has been widely and successfully used in many instances. However, this system remains somewhat tedious to use due to: (i) the establishment of a primary cell line constitutively and stably expressing the Tc-regulated transactivator and (ii) the obtention of a secondary line expressing the gene of interest in a Tc-dependent manner. In order to facilitate these two critical steps, we devised an efficient and molecular biology-free strategy allowing the successful selection of clones expressing any cDNA under tight regulation.

Cell Line↗

PubMed1998

Synergistic effects of colony-stimulating factor 1 and leukemia inhibitory factor in inducing early myeloid cell differentiation.

Cells of the M1D+ murine myeloid leukemic cell line differentiate into macrophages in response to either leukemia inhibitory factor (LIF) or interleukin 6. Previously, it was shown that LIF treatment of M1D+ cells leads to an increased expression of colony-stimulating factor (CSF) receptor mRNA encoded by c-fms. CSF-1, a macrophage growth factor, induces the survival, growth, and differentiation of mononuclear phagocytes but has not been implicated in the regulation of early myeloid cell differentiation. Here we show that low-dose LIF treatment of M1D+ cells results in CSF-1 secretion and CSF-1 receptor up-regulation. CSF-1, when applied alone, induces some M1D+ adherence and the up-regulation of lysozyme M, a macrophage-specific marker. Finally, we show that when applied together, LIF and CSF-1 act synergistically to induce macrophage morphology, phagocytosis, and the expression of the macrophage-specific markers CD11b/Mac-1 alpha chain, lysozyme M, FcgammaRII, and JE/MCP.1. These results indicate that instead of being part of exclusive pathways, as thought until this work, LIF and CSF-1 can function synergistically to further stimulate the early stages of myeloid differentiation.

Animals↗

PubMed1997

Cross-family interaction between the bHLHZip USF and bZip Fra1 proteins results in down-regulation of AP1 activity.

Heterodimerization among the basic-leucine zipper (bZIP) proteins or among the basic-helix-loop-helix-leucine zipper (bHLHZip) proteins confers a multitude of combinational activities to these transcription factors. To further examine the function of the bHLHZip protein, USF, we screened for cellular proteins which could directly interact with USF using the yeast two-hybrid system. A bZip protein, Fra1, was found to efficiently interact with USF. USF specifically interacts with Fra1 but not with other closely related family members, c-Fos, Fra2, FosB, or with c-Jun. Both the bHLHZip and the N-terminal regions of Fra1 are required for efficient interaction with USF. In vivo association between USF and Fra1 has been demonstrated by co-immunoprecipitation. Expression of exogenous USF led to a decrease in AP1-dependent transcription in F9 cells. Co-expression of exogenous Fra1 restored the AP1 activity in a dose-dependent manner. These data show that USF and Fra1 physically and functionally interact demonstrating that cross-talk occurs between factors of distantly related transcription families.

Animals↗

PubMed1996

Complete sequencing of the murine USF gene and comparison of its genomic organization to that of mFIP/USF2.

USF is a transcription factor able to stimulate promoter activity upon binding to an upstream sequence identical to that recognized by the protooncogene Myc. However, despite extensive biochemical characterization, nothing is known concerning its physiological function. A USF-related protein able to interact with Fos and known as FIP/USF2 has been reported. Its genomic structure in mouse has been recently characterized. We present here the cloning and characterization of the murine USF gene. It consists of 10 exons, the first of which is noncoding, and the gene spans 8 kb of DNA. We show that the murine USF protein is almost identical to its human counterpart, but that an intron not conserved between human and murine USF genes curiously has been conserved between human USF and murine FIP/USF2. Otherwise, the splicing pattern of murine USF and FIP/USF2 is exactly conserved. We also demonstrate that the murine USF promoter is located more than 2.5 kb upstream of the first coding ATG, in a region displaying divergent promoter activity. Finally, we show that an Mx1-related sequence is present less than 3 kb downstream of the murine USF gene, in a tail-to-tail position. Taken together, these data indicate that the murine USF gene is very similar to the murine FIP/USF2 gene and is potentially bracketed by two other transcription units on the other DNA strand.

Amino Acid Sequence↗

PubMed1996

The basic region/helix-loop-helix/leucine repeat transcription factor USF interferes with Ras transformation.

Upstream stimulatory factor (USF) is a transcription factor of the basic region/helix-loop-helix/leucine repeat family. It shares the same DNA-binding sequence as the myc oncogene. Based on the three-dimensional structures, its DNA-binding domain is structurally related to that of Max, the partner of Myc. In addition, USF can form heterodimers with a related factor, Fos-interacting protein/upstream stimulatory factor 2 (FIP/USF2), which has been shown to directly interact with Fos. In view of the provocative relationship of USF with other factors involved in cell proliferation, we investigated whether USF could also play a role in cellular growth control. In this study, we report that USF is not an oncogene, but interferes with Ras-driven transformation. This inhibitory effect is independent of USF transactivating domains, but requires its DNA-binding activity. However, the minimal USF DNA-binding domain does not display this inhibitory effect, and even slightly enhances Ras transformation. On the basis of these data, we propose that USF may play an important role in the control of cell growth and proliferation, through both binding to promoter sequences and specific protein/protein interactions.

3T3 Cells↗

PubMed1996

The mutation Gly142-->Glu in human lipoprotein lipase produces a missorted protein that is diverted to lysosomes.

While the molecular characterization of lipoprotein lipase (LPL) activation is progressing, the intracellular processing, transport, and secretion signals of LPL are still poorly known. The aim of this paper is to study are involvement of glycine 142 in LPL secretion and to elucidate the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutagenesis in order to produce the G142e hLPL in which the glycine 142 was replaced by a glutamic acid. The wild type human LPL (WT hLPL) and the mutant G142E hLPL were expressed by transient transfection in COS1 cells. Using Western blot assays we identified a single band that had the same molecular weight for both proteins. However, Western blots of culture media did not reveal any specific band for the mutant protein, and ELISA experiments showed that the extracellular mass of the mutant LPL was only 25% of the WT protein, indicating defective secretion of the altered enzyme. Heparin increased LPL secretion in the case of the WT hLPL but did not have any stimulatory effect when acting on G142E hLPL-transfected cells. However, heparin-Sepharose chromatography revealed that both proteins presented the same heparin affinity. Metabolic labeling and radioimmunoprecipitation studies showed that both the WT and the mutant hLPL intracellular levels decreased upon chase time. Furthermore, leupeptin had a greater effect on the intracellular level of the mutant enzyme, thus indicating its higher intracellular degradation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeling in G142E hLPL-expressing cells. This result was confirmed using immunoelectron microscopy, which in addition showed gold labeling in Golgi stacks. This finding together with experiments performed with endoglycosidase H digestion of immunoprecipitated radiolabeled LPL, indicated that the mutant enzyme entered the Golgi compartment. The results reported in this paper show that the G142E hLPL is not efficiently secreted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal missorting might be a mechanism of cell quality control of secreted LPL.

Animals↗

PubMed1996

Constitutive c-ets2 expression in M1D+ myeloblast leukemic cells induces their differentiation to macrophages.

The expression of c-ets2 is rapidly induced in a variety of myelomonocytic cell lines as they differentiate into macrophages. We find that constitutive expression of c-ets2 in the M1D+ myeloblast leukemic cell line (M1ets2) is sufficient to push these cells to a more differentiated state. The expression of several differentiation-specific genes is upregulated in M1ets2 cells, including those encoding macrophage-specific lysozyme M and tumor necrosis factor alpha, which are involved in bacteriolytic and inflammatory processes, respectively. Transcription factors c-jun and junB, previously shown to induce partial macrophage differentiation when overexpressed in myelomonocytic leukemia cell lines, are also upregulated in M1ets2 cells. The upregulation of junB is the result of a direct interaction of Ets2 with ets binding sites of the junB promoter, since transient or constitutive Ets2 expression in M1D+ cells activates junB transcription via ets binding sites. In addition, transfection of a dominant negative mutant of Ets2, devoid of its transcriptional activation domain, greatly reduces transcriptional activities of the junB promoter in M1ets2 cells. Finally, unlike their parental M1D+ counterparts, M1ets2 cells secrete the macrophage colony-stimulating factor, CSF-1, and are able to phagocytize. Taken together, these results show that when the immature myeloid M1D+ cell line constitutively expresses c-ets2, these cells acquire different functions of mature macrophages.

Animals↗

PubMed1995

cDNA cloning and characterization of the transcriptional activities of the hamster peroxisome proliferator-activated receptor haPPAR gamma.

We have isolated a cDNA corresponding to the hamster peroxisome proliferator-activated receptor haPPAR gamma, a member of the steroid nuclear hormone receptor superfamily of transcription factors. haPPAR gamma mRNA is highly expressed in adipose tissue, and is expressed in lung, heart, kidney, liver and spleen to a lower extent. Thus, haPPAR gamma may function in activating the transcription of target genes in a variety of tissues, including those not particularly subjected to peroxisomal beta-oxidation. haPPAR gamma binds efficiently in the presence of retinoid X receptor alpha (RXR alpha) to a peroxisome proliferator response element (PPRE) first identified in the acyl-CoA oxidase (ACO) promoter, the rate-limiting enzyme of peroxisomal beta-oxidation. The gene (ACO) encoding this enzyme has been previously shown to be under the transcriptional control of mouse PPAR (mPPAR). Although binding of haPPAR gamma/RXR alpha on the PPRE of the ACO promoter in vitro is similar to that observed for mPPAR/RXR alpha, we show that the transcriptional activities of mPPAR and haPPAR gamma are regulated differently in vivo in response to peroxisome proliferators and heterodimerization with RXR.

Amino Acid Sequence↗

PubMed1995

Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase induces its accumulation in the rough endoplasmic reticulum and alters this cellular compartment.

Lipoprotein lipase (LPL) is the enzyme responsible for the hydrolysis of plasma triglycerides from apolipoprotein C-II-containing lipoproteins at the capillary endothelium and it is synthesized in parenchymal cells of several tissues. Intracellular LPL processing is a major aspect of LPL regulation. The present study aims to determine the intracellular accumulation site of the LPL that is not glycosylated at Asn43. Human LPL (hLPL) cDNA was mutated by site-directed mutagenesis. An Ala residue was substituted for Asn at position 43 of the protein generating N43A hLPL. Wild type hLPL and the mutant hLPL were expressed in COS1 cells. Using immunofluorescence and immunoelectron microscopy we found that wild type hLPL in addition to being secreted into the medium was present in the rough endoplasmic reticulum (ER), Golgi compartments, and vesicles. Neither LPL activity nor protein was found in medium of cells expressing the mutant hLPL and all detectable protein was present exclusively in the ER identified witha specific antibody against the protein disulfide isomerase (PDI), an ER marker. In addition, the intracellular distribution of the ER of the cells that expressed the mutant protein was grossly altered. Treatment of COS1 cells with tunicamycin for 24 h had the same effect on wild type hLPL processing and edoplasmic reticulum distribution. Next, we investigated the influence of the accumulation of mutant hLPL on the intracellular transport of three other proteins that are N-glycosylated before reaching the plasma membrane: the related Bo,+ amino acid transporter (rBAT), the insulin-regulated glucose transporter (GLUT4), and the placental alkaline phosphatase (PLAP) protein. Coexpression of the mutant hLPL (but not wild type) caused the accumulation of rBAT and GLUT4 in the ER while PLAP reached the plasma membrane. Our findings demonstrate that glycosylation of Asn43 of human lipoprotein lipase in the endoplasmic reticulum is essential for its efflux from this compartment and that the retention of the non-glycosylated LPL induces morphological changes in the ER that could also affect its ability to modify the transport of other proteins.

Asparagine↗

PubMed1994

Structure and function of the b/HLH/Z domain of USF.

The basic/helix-loop-helix/leucine zipper (b/HLH/Z) transcription factor upstream stimulatory factor (USF) and its isolated DNA binding domain undergo a random coil to alpha-helix folding transition on recognizing their cognate DNA. The USF b/HLH cocrystal structure resembles the structure of the b/HLH/Z domain of the homologous protein Max and reveals (i) that the truncated, b/HLH DNA binding domain homodimerizes, forming a parallel, left-handed four-helix bundle, and (ii) that the basic region becomes alpha-helical on binding to the major groove of the DNA sequence CACGTG. Hydrodynamic measurements show that the b/HLH/Z DNA binding domain of USF exists as a bivalent homotetramer. This tetramer forms at the USF physiological intranuclear concentration, and depends on the integrity of the leucine zipper motif. The ability to bind simultaneously to two independent sites suggests a role in DNA looping for the b/HLH/Z and Myc-related families of eukaryotic transcription factors.

Amino Acid Sequence↗

PubMed1992

The helix-loop-helix/leucine repeat transcription factor USF can be functionally regulated in a redox-dependent manner.

We showed previously that the DNA-binding capacity of the helix-loop-helix/leucine repeat transcription factor USF43 is lowered dramatically under nonreducing conditions. This report defines the molecular basis of this effect by showing (i) that the only two USF43 cysteine residues, both present within the helix-loop-helix protein-protein interface domain, are required for this regulation, (ii) that the sulfhydryl groups of these cysteine residues are the actual targets of this regulation, (iii) that oxidation of these groups results in both intra- and intermolecular nonrandom covalent links, (iv) that this redox modulation of USF43 DNA-binding potential can translate in vitro into a specific modulation of its ability to activate transcription from a USF-responsive promoter. The implications of these modulations of USF43 function in response to redox changes are discussed with regard to the apparent paradox of USF strong activation potential and its ubiquitous distribution in all cell types tested.

Amino Acid Sequence↗

PubMed1992

Identification of USF as the ubiquitous murine factor that binds to and stimulates transcription from the immunoglobulin lambda 2-chain promoter.

To study the specificity and identity of NF-lambda 2, a ubiquitous murine nuclear factor that interacts specifically with the promoter of the lambda 2-chain gene and stimulates its transcription, competition experiments were carried out using DNA fragments from various immunoglobulin regulatory elements. The results showed that a fragment containing the H-chain enhancer competed efficiently for the binding of NF-lambda 2. Dissection of the H-chain enhancer revealed that the microE3 motif contributed the competing ability. Additionally, a regulatory region found in the adenovirus major late promoter, which interacts with the human general transcription factor USF, competed very efficiently for binding to NF-lambda 2. This region contains a sequence, CACGTGAC, which is identical to a region within the NF-lambda 2 motif. The pattern of complexes formation using oligonucleotide probes corresponding to the NF-lambda 2 and USF motifs were identical, and they both differed from that displayed by the E3 probes. Antisera against different domains of USF also react specifically with NF-lambda 2 showing that this factor is antigenically related, if not identical, to USF. Furthermore, the activity of the lambda 2 promoter in an in vitro transcription assay was significantly reduced when the nuclear extract used was USF-depleted. Addition of exogenous USF to this extract restored the transcription activity. Therefore, we conclude that NF-lambda 2 is the murine homologue of USF.

Animals↗