Biomedical subjects

E Schaeffer

Publications and source records attributed to E Schaeffer.

At least 37 records · Page 2Linked to original sources

PubMed1996

Chicken ovalbumin upstream promoter transcription factor, a transcriptional activator of HIV-1 gene expression in human brain cells.

Viral infection of the central nervous system by the human immunodeficiency virus type 1 leads to a wide range of neuropathological disorders. However, the molecular mechanisms governing transcription of the human immunodeficiency virus type 1 genome in brain remain unclear. We have recently established that in brain cells, proteins belonging to the steroid/thyroid/retinoic acid receptor family bind to the -352 to -320 region of the long terminal repeat (LTR). Here, by supershift experiments, we have identified chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of this nuclear receptor family, as one of the major proteins interacting with this LTR site. Cotransfection studies revealed that COUP-TF is able to dramatically activate LTR-directed gene transcription in human oligodendroglioma but not in astrocytoma cells. This activation occurs through two mechanisms, depending on the LTR sequence. Moreover, in neuronal cells COUP-TF and dopamine, a catecholamine neurotransmitter, enhance LTR-directed transcription by acting on the proximal LTR region. These results reveal the importance of COUP-TF and the dopamine signaling pathway as activators of human immunodeficiency virus type 1 gene expression in brain.

Brain↗

PubMed1996

Regulation of human immunodeficiency virus type 1 gene transcription by nuclear receptors in human brain cells.

Infection of cells of the central nervous system by the human immunodeficiency virus type-1 (HIV-1) leads to HIV-1-associated neuropathology. Recent studies have demonstrated the importance of long terminal repeat (LTR) binding sites in determining the pathogenicity of HIV. Here we have investigated the presence and the functional role of transcription factors that have the potential to interact, directly or indirectly, with the nuclear receptor-responsive element in the LTR of HIV-1, in different human cell lines of the brain. Cotransfection experiments showed that in oligodendroglioma TC-620 cells, the retinoic acid receptor and the retinoid X receptor activate LTR-driven transcription in the absence of ligand. Addition of all-trans- or 9-cis-retinoic acid reverses this effect. In contrast, in astrocytoma, neuronal, and microglial cells, no significant effect of the retinoid acid pathway was detected. This retinoid response is mediated by distinct molecular interactions in the lymphotropic LAI and the neurotropic JR-CSF HIV-1 strains. Moreover, retinoid receptors were found to antagonize the chicken ovalbumin upstream promoter transcription factor- as well as the c-JUN-mediated LTR transactivation. Our findings demonstrate the importance of the retinoic acid signaling pathway and of cross-coupling interactions in the repression of HIV-1 LTR gene expression.

Brain↗

PubMed1996

Distinct positive and negative regulatory elements control neuronal and hepatic transcription of the human transferrin gene.

Transferrin (Tf), the iron-transport protein, plays an essential role in the central nervous system development, plasticity, and aging. As a first step toward elucidating the role of each transcription factor involved in the regulation of Tf gene expression, we have recently shown that similar promoter elements direct cell-type specific transcription in oligodendrocytes, epithelial choroid plexus cells, and in the neuronal cell line B103. Here we have analyzed the regulatory elements that control the level of expression of the Tf gene in neuronal cells. Transient expression experiments in B103 cells revealed that the -164/+1 promoter region is stimulated by a position-dependent -1140/-1000 upstream region. DNase I footprinting, gel retardation assays, and antibody reactivity data allowed us to characterize the nuclear factors interacting with this region. The upstream region I-binding protein (URI-BP) belongs to the steroid/retinoid receptor family, while URII-BP is a member of the nuclear factor I (NF-I) family. Interestingly, no enhancer nor silencer activity is detected in B103 cells. This contrasts with our findings in hepatoma cells, where the activity of the -125/+1 promoter can be repressed by a -1000/-819 upstream negative-acting region and stimulated by the -3600/-3300 enhancer. We demonstrate that the negative-acting region presents the characteristics of a silencer that interacts with a nuclear protein present in liver and absent in B103 cells. Similarly, B103 cells lack a nuclear protein able to bind to an essential site of the enhancer. This shows that in B103 cells, the inactivity of the silencer and the enhancer regions results from the absence of at least one essential nuclear protein.

Base Sequence↗

PubMed1996

Different brain areas are involved in memory expression at different times from training.

Rats were trained in a step-down inhibitory avoidance task and tested for retention 1, 31, or 60 days later. Three to 7 days prior to testing, they were bilaterally implanted with cannulae in the CA1 region of the dorsal hippocampus and in the amygdaloid nucleus (H + A), in the entorhinal cortex (EC), and in the posterior parietal cortex (PPC). Ten minutes prior to testing, the animals received, through the cannulae, 0.5-microliter microinfusions of vehicle (20% dimethylsulfoxide in saline) or of 0.5 microgram of CNQX dissolved in the vehicle. A second test session was carried out 90 min after the first. CNQX blocked retention test performance when given into H + A 1 day after training but not later; when given into EC 1 or 31 days after training, but not later; and when given into PPC 1, 31, or 60 days after training. In all cases performance returned to normal levels in the second test session. The data suggest that H and A are involved in memory expression for only a few days after acquisition; that EC is involved in memory expression for up to 31, but less than 60, days after acquisition; and that PPC is involved in memory expression for up to at least 2 months after acquisition.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1995

Transcription of the human transferrin gene in neuronal cells.

We have recently shown that a combination of three transcription factors governs the expression of the human transferrin gene in different brain cell types, oligodendrocytes, choroid plexus cells and neuronal cells. It was essential to elucidate the role of each factor in the regulation of transferrin gene transcription. Site-directed mutagenesis and co-transfection experiments in neuronal cells revealed that chicken ovalbumin upstream promoter transcription factor (COUP-TF), which binds to the promoter region I, acts as a repressor. Overexpression of the CCAAT/enhancer binding protein (C/EBP-alpha), which binds to the promoter region II, transactivates the -164/+1 promoter, even enables the -125/+1 region to promote transcription, and synergistically activates transcription in the presence of CREB. The C/EBP-alpha-mediated activation is antagonized by COUP-TF. The positive action of the cAMP response element-binding protein called CRI-BP is revealed by mutations of the central region I site which repress transcription. Moreover addition of dibutyryl cyclic AMP or overexpression of the catalytic subunit of protein kinase A increase transcription from the wild-type and not from the CRI mutant promoter, which shows that CRI-BP is responsible for mediating cAMP stimulation of Tf gene transcription.

Base Sequence↗

PubMed1995

Interactions of the transcription factor AP-1 with the long terminal repeat of different human immunodeficiency virus type 1 strains in Jurkat, glial, and neuronal cells.

Human immunodeficiency virus type 1 (HIV-1) infection of the neuronal and astroglial cells of the central nervous system has been proposed to contribute to HIV-1-associated dementia. Recently it was shown that differences in the nucleotide sequence of the long terminal repeat (LTR) of different HIV-1 strains govern the tissue-specific pattern of viral expression. The LTR from central nervous system-derived HIV-1 strains JR-FL and JR-CSF directs expression in the neurons of transgenic mice, in contrast with the lymphotropic LAI strain. By in vitro footprinting, gel retardation, and methylation interference experiments, we have studied the interactions of host cell proteins from human neuronal, glial, HeLa, and Jurkat T cells with the LTRs from the neurotropic JR-FL and JR-CSF strains, compared with the LAI strain. Proteins belonging to the nuclear receptor family bind with different affinities to variant -352 to -324 sites. Gel supershift assays with Jun and Fos antibodies showed that the AP-1 transcription factor present in the various cell types was unable to recognize the -352 to -324 and -306 to -285 AP-1 putative binding sites. Interestingly, Jun and Fos components of AP-1 interact with the variant TGGCTCA sequence located in the -247 to -222 region of both neurotropic strains. These interactions were cell type specific, since they were detected only with extracts from glial and HeLa cells and not from neuronal or Jurkat cells. Cotransfection experiments further revealed that the -247 to -222 sequence is able to mediate AP-1-induced transcriptional activation in glial and not neuronal cells.

Animals↗

PubMed1994

Brain-specific expression of the human transferrin gene. Similar elements govern transcription in oligodendrocytes and in a neuronal cell line.

We have identified the regulatory sequences that govern the expression of the human transferrin gene in cultured brain cells and compared them with the data obtained with the neuronal cell line B103. Oligodendrocytes and epithelial choroid plexus cells from rat brain were cultured and used for transient expression experiments. Deletion analysis of 1.8 kilobase pairs of the 5' regulatory sequences revealed a -1530/-1140 positive-acting region in oligodendrocytes. The -164/+1 promoter region was sufficient to confer cell type-specific transcription in oligodendrocytes, epithelial choroid plexus cells, and B103 cells. DNase I footprinting experiments revealed three protected sequences, the proximal regions I and II, and the central region I. Gel retardation and antibody reactivity data allowed us to identify most of the nuclear factors present in oligodendrocytes interacting with the promoter sequences. Chicken ovalbumin upstream promoter transcription factor, a CAAT/enhancer-binding protein, and a cAMP response element-binding protein called CRI-BP interact with the proximal regions I and II and central region I sites, respectively. These data confirm the results obtained with the neuronal cell line and emphasize the importance of the three promoter elements for the transferrin gene-specific expression in the central nervous system compared with only two elements required for liver- and testis-specific expression.

Animals↗

PubMed1994

Synapsin IIa accelerates functional development of neuromuscular synapses.

We have investigated the possible involvement of the synaptic vesicle protein synapsin IIa in synapse development. Synapsin IIa was introduced into Xenopus embryonic spinal neurons by early blastomere injection, and nerve-muscle cultures were prepared. Synaptic currents were measured by comparing synapses in which the presynaptic neuron either contained [syn IIa (+)] or lacked (control) exogenous synapsin IIa. Syn IIa (+) synapses had a 3.6-fold increase in the frequency and a 2.1-fold increase in the amplitude of spontaneous synaptic currents, compared to controls, after 2 days in culture. Synapsin IIa also increased the amplitude of evoked synaptic currents by 2.3-fold in 2-day cultures. The evoked synaptic current amplitudes of syn IIa (+) synapses had a lower coefficient of variation indicating a more stable evoked response. These enhanced synaptic activities were independent of the presence or absence of the protein in the postsynaptic muscle cell. The findings indicate a role for synapsin IIa in synapse maturation.

Acetylcholine↗

PubMed1994

Synapsin IIa bundles actin filaments.

Synapsins are neuron-specific phosphoproteins associated with small synaptic vesicles in the presynaptic nerve terminal. Synapsin I, which has been demonstrated to bundle F-actin in vitro, has been postulated to regulate neurotransmitter release by cross-linking synaptic vesicles to the actin cytoskeleton. To investigate the possible interaction of synapsin II with actin filaments, we expressed synapsin II in Spodoptera frugiperda and High Five insect cells using a recombinant baculovirus. Purified recombinant synapsin IIa was incubated with F-actin, and bundle formation was evaluated by light scattering and electron microscopy. Synapsin IIa was found to bundle actin filaments. Dose-response curves indicated that synapsin IIa was more potent than synapsin I in bundling actin filaments. These data suggest that synapsin IIa may cross-link synaptic vesicles and actin filaments in the nerve terminal.

Actins↗

PubMed1993

A different combination of transcription factors modulates the expression of the human transferrin promoter in liver and Sertoli cells.

We have previously identified the functional regions involved in the regulation of human transferrin (Tf) gene expression in the liver and in Sertoli cells of the testis. Here, we show that a different cellular distribution of transcription factors, interacting with the same proximal promoter regions (PRI and PRII), modulates cell type-specific transcription. In the liver, hepatocyte nuclear factor 4 (HNF-4) and the chicken ovalbumin upstream promoter transcription factor (COUP-TF) act at the PRI site, while CCAAT/enhancer-binding proteins (C/EBPs) act at the PRII site. In the testis, distinct combinations of Sertoli proteins SP-A and SP-D and COUP-TF bind to the PRI site, while SP-alpha and SP-beta bind to the PRII site. Cotransfection experiments in Hep3B cells revealed that mostly HNF-4, C/EBP-alpha, C/EBP-delta, and, to a lesser extent, COUP-TF stimulated transcription driven by the -125/+39 region. In Sertoli cells, HNF-4 and COUP-TF appeared to repress, while the C/EBP factors were able to stimulate transcription driven by the -100/+39 region. However, the specific activating combination remains to be defined among the Sertoli proteins. In the non-Tf-expressing HeLa cells, the Tf promoter could be activated by C/EBP-delta. Our data revealed functional antagonism between HNF-4 and COUP-TF, binding to PRI, as well as cross-coupling interactions between HNF-4 and C/EBP, binding to adjacent sites. Thus, cell type-specific DNA-protein interactions, together with protein-protein interactions, may explain the transcriptional regulation of the Tf gene in different cell types.

Base Sequence↗

PubMed1992

Cancer after bone marrow transplantation. IBMTR and EBMT/EULEP Study Group on Late Effects.

Cancer may be serious late effect of marrow transplantation. Radiation, chemotherapy, immunosuppression and the original disease for which transplantation was performed may predispose to the development of cancer. 116 of 9732 patients reported to the IBMTR (International Bone Marrow Transplant Registry) have developed a new malignancy. Late effects were evaluated by the EBMT-EULEP (European Bone Marrow Transplant-European Late Effect Project) Late Effect Study Group in 147 patients surviving 6 years and 79 patients surviving more than 10 years. New malignancies developed in 11 of these patients. Lymphomas and leukemia comprised 73 cases reported to the IBMTR and one case reported to the EBMT-EULEP study. Tumors of the skin, oropharynx, vulva vagina and cervix prevailed in 41 patients with solid tumors. The distribution of malignancies is similar to that observed in organ transplant patients not given radiation or chemotherapy and suggests immunosuppression as a major contributory factor. In dogs the incidence of malignancies was studied after either chemotherapy or total body radiation in various regimens and marrow transplantation. Both chemotherapy and radiation shortened tumor-free survival in comparison to untreated dogs. Higher doses, larger fractions and shorter treatment schedules enhanced earlier tumor development. Soft tissue sarcomas and thyroid carcinoma were most frequent in treated, mammary carcinoma in untreated dogs. In treated dogs deaths from cancer were observed starting at the age of 5 years as compared to untreated dogs at the age of 9 years. The data from animal experiments indicate that the incidence of solid tumors in marrow transplant patients may still rise in the coming decades.

Animals↗

PubMed1991

Sertoli cell-specific expression of the human transferrin gene. Comparison with the liver-specific expression.

We present a comparative study of the cis- and trans-acting elements governing the expression of the human transferrin (Tf) gene in two tissues, liver and testis, where Tf is expressed at various levels. We have previously identified the elements of the promoter, negative, and enhancer regions involved in the liver-specific expression of the gene. By transfection experiments of primary cultured rat Sertoli cells compared with hepatoma cells, DNase I footprinting, and gel retardation studies, we have analyzed 3.6 kilobase pairs of the Tf regulatory region. The far upstream enhancer functional in Hep3B cells is inactive in Sertoli cells; in the two cell types, different nuclear factors appear to bind to a DNA domain crucial for enhancer activity. Similar negative- and positive-acting elements are present in the distal promoter in both tissues. However different combinations of proximal promoter elements control tissue-specific expression. Liver-specific transcription is governed by the interaction of the Tf-LF1 protein and a C/EBP-related factor with the -125 to -45 region. In Sertoli cells, a -34 to -18 TATA box-binding factor is sufficient to initiate basal-level transcription. Efficient expression is achieved by the association of two factors binding either to the (-82, -1) or to the (-153, -52) region. The addition of a third adjacent element decreases the promoter activity, suggesting that the balance of three factors binding to the proximal sites regulates testis-specific expression.

Animals↗

PubMed1991

The enhancer of the human transferrin gene is organized in two structural and functional domains.

We previously identified a 300-base pair long enhancer, located 3.6 kilobases upstream of the cap site of the human transferrin gene. A 5' deletion up to position 86 of the enhancer resulted in complete loss of the enhancer activity. Here we show by competition footprint analysis, gel retardation assays, and transient expression studies in hepatoma and HeLa cells that the enhancer is composed of two distinct structural and functional domains, A (nucleotides 1-86) and B (nucleotides 87-291). Each domain is a proto-enhancer of a different type. Domain A is a proto-enhancer that, when multimerized, is able by itself to stimulate transcription from the heterologous SV40 promoter, both in Hep3B and HeLa cells. It contains the octanucleotide TGTTTGCT sequence and is the binding site of two liver-specific nuclear factors and of a different HeLa nuclear factor. Domain B contains four binding sites interacting with several liver nuclear proteins. In order to bind, any of these proteins requires the presence of all the others. This domain is able to block the activity of a downstream negative element, but it has no enhancer activity by itself. In the presence of the transferrin promoter, full enhancer activity requires the association of the two domains A and B.

Base Sequence↗

PubMed1989

Isolation and characterization of two new drosophila protein kinase C genes, including one specifically expressed in photoreceptor cells.

We have isolated and characterized two new protein kinase C (PKC) genes from D. melanogaster. One, dPKC98F, maps to chromosome region 98F and displays over 60% amino acid sequence identity with members of a recently described "PKC-related" subfamily in mammals. The other, dPKC53E(ey), maps to region 53E4-7 on the second chromosome and lies within 50 kb of PKC gene previously characterized (dPKC). While dPKC98F transcripts are expressed throughout development, expression of the two genes mapping at cytogenetic location 53E is primarily in adults. dPKC98F and the previously reported 53E gene are transcribed predominantly in brain tissue. In contrast, dPKC53E(ey) is transcribed only in photoreceptor cells. We will discuss the significance of this tissue-specific localization with regard to phototransduction.

Amino Acid Sequence↗

PubMed1989

Cell type-specific expression of the human transferrin gene. Role of promoter, negative, and enhancer elements.

Transferrin (Tf), the iron transport protein of vertebrate serum, is mainly synthesized in the liver. cis-Acting DNA elements required for liver-specific expression of the human Tf gene were identified by transient and stable expression assays in human hepatoma (HepG2 and Hep3B) and epithelial carcinoma (HeLa) cell lines. Deletion analysis of the 5' DNA sequences of the gene have defined four functionally different regions: (a) A cell type-specific promoter located between positions -125 and -45 which interacts with two nuclear factors and is sufficient for liver-specific expression. (b) A distal promoter region from -620 to -125 base pairs containing positive and negative cis-acting elements which regulate the promoter activity. (c) A negative-acting region between -1.0 and -0.6 kilobase pairs which down-regulates transcription from the Tf promoter. (d) An enhancer located between -4.0 and -3.3 kilobase pairs which is more active in hepatoma than in HeLa cells. Thus, Tf gene expression is modulated by a combination of multiple positive and negative cis-acting elements. The expression results are discussed with respect to our previous description of the trans-acting factors interacting with the proximal and distal promoter regions.

Base Sequence↗

PubMed1988

Interactions of DNA-binding proteins with the 5' region of the human transferrin gene.

We have established by transient expression experiments that the 620 base pairs upstream of the cap site of the human transferrin gene contain the information necessary for efficient expression of the gene in hepatoma cells HepG2 or Hep3B but not in HeLa cells. DNase I footprint analysis reveals that at least five distinct factors present in human or rat liver nuclear extracts interact with different sites of this region. One of these factors, binding to nucleotides -193 to -162, is closely related to or identical with the eukaryotic factor CCAAT-binding transcription factor/nuclear factor I; another one, binding to nucleotides -103 to -83 seems to be related to the CCAAT-binding protein. The binding sites of two other factors, not recognized by HeLa nuclear proteins, each contain an identical 10-nucleotide-long sequence (5' TCTTTGACCT 3') in reverse orientation, separated by 400 base pairs. Results of gel retardation assays, cross-competition experiments, and heat inactivation strongly suggest that the proteins binding to these sites are different. One of these sequences and the binding site of the CCAAT-binding protein related factor are located in the region between nucleotides -119 and -45. We have shown by transient expression experiments with 3' deleted vectors that this region is functionally essential for human transferrin gene expression.

Base Sequence↗

PubMed1987

A second type of protein methylation reaction in bacterial chemotaxis.

CheZ is the product of one of six genes required for sensory processing in Escherichia coli and Salmonella typhimurium chemotaxis. This 24-kDa cytoplasmic protein is modified by a posttranslational methylation reaction. The modified residue has been identified by analysis of radioactively labeled protein from two-dimensional electrophoretograms and Edman degradation of CheZ protein isolated by immunoaffinity chromatography using anti-CheZ monoclonal antibodies. The methylated group is an N-monomethylmethionine residue at the amino terminus of CheZ. L16, a ribosomal protein that is required for peptidyltransferase activity during protein synthesis, is also methylated at its amino-terminal methionine (Chen, R., Brosius, J., and Wittmann-Liebold, B. (1977) J. Mol. Biol. 111, 173-181). Homologous sequences at the amino termini of L16 and CheZ raise the possibility that a single S-adenosylmethionine-dependent methyltransferase modifies both proteins.

Bacterial Proteins↗

PubMed1987

Complete structure of the human transferrin gene. Comparison with analogous chicken gene and human pseudogene.

The complete structure of the human transferrin gene is presented. This gene has a total size of about 33.5 kb and is organized in 17 exons separated by 16 introns. The chicken ovotransferrin gene has a size of 10.5 kb and is also organized in 17 exons and 16 introns. The analysis of the structure of the two genes confirm, at the gene level, that transferrins originated by a gene duplication phenomenon. Finally, the existence of a new member of the transferrin family, a human transferrin non-processed pseudogene is demonstrated.

Amino Acid Sequence↗