Biomedical subjects

S Champion

Publications and source records attributed to S Champion.

At least 37 records · Page 2Linked to original sources

PubMed1996

UVB irradiation upregulation of the Drosophila 1731 retrotransposon LTR requires the same short sequence of U3 region in a human epithelial cell line as in Drosophila cells.

Phylogenetic analysis of the retrotransposon and retrovirus suggests an evolutionary relationship between them and indicates that transactivation of the long terminal repeat (LTR)-containing retroelements could be ubiquitous. Using constructs expressing a reporter gene under the control of the entire or deleted LTR of 1731, which is a retrotransposable element of Drosophila melanogaster, we were able to show that the UVB-irradiation activation of the 1731-LTR requires the same short sequence of U3 region in a human epithelial cell line as in Schneider's Drosophila cell line (S2). This sequence is similar to the binding sequence of the members of the nuclear factor-kappa B (NF-kappa B)/rel family. In addition, human colonic carcinoma cells (HT29), in response to UVB-irradiation, produce some extracellular factor(s) that activates the 1731-LTR in nonirradiated cells.

Animals↗

PubMed1996

Activation of the transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat by autologous and heterologous cell-to-cell contact.

Recent studies have demonstrated that the activation of HIV-1 provirus and of the Long Terminal Repeat of HIV-1 (HIV-1-LTR) may occur upon cell-to-cell contact between peripheral blood lymphocytes (PBLs) and infected or transfected (HT29) human colonic carcinoma cells. Using transient or stable transfections, we ascertained that the HIV-1 LTR was up-regulated by cell-to-cell contact in various cell lines. The degree of cell-to-cell contact responsiveness was cell type dependent. In contrast, in transient transfection, the HIV-1-LTR was strongly induced by Tat expression in all cell types tested. This indicates that there are differences in the induction mechanism for these two stimuli, even though Tat protein has been previously reported to induce cell adhesion. Except for the PBLs/transfected cells interactions, the results also demonstrate that the cell-to-cell contact-induced HIV-1-LTR activation was highest after contact between autologous as compared to heterologous cells. Previous experiments have shown that, in HT29 cells, cell-to-cell contact activation of the HIV-1-LTR involved the NF-kappa B tandem binding site and activates DNA binding of the nuclear factor NF-kappa B. In this work, we show that in a stably transfected cell line, the principal enhancer element was also involved in the HIV-1-LTR cell-to-cell contact activation. On the other hand, in the HT29 cell line, the NF-kappa B binding appeared to involve the RGD motif of the cell-binding domain, which indicates that a post-integrin receptor event could be implicated.

Cell Communication↗

PubMed1995

The Gag polypeptides of the Drosophila 1731 retrotransposon are associated to virus-like particles and to nuclei.

1731 is a Drosophila melanogaster retrotransposon whose nucleotide sequence shows a proviral architecture with two long terminal repeats (LTRs) framing two internal Open Reading Frames (ORFs). The pol ORF2 of this mobile genetic element was demonstrated to code for an active Reverse Transcriptase (RT) and the ORF1 is expected to code for the structural Gag proteins of the virus-like particles (VLP). Using specific anti-Gag antibodies, we have characterized the 1731 Gag polypeptides expressed either in vitro or in Kc Drosophila melanogaster cultured cells. Together with the 1731 RT, the largest, likely post-translationaly-modified Gag polypeptides are gathered into cytoplasmic virus-like particles. Moreover and consistent with the nuclear localization signal present in the Gag sequence, we observed that a short 1731 Gag polypeptide is associated to the cell nuclei.

Amino Acid Sequence↗

PubMed1995

X irradiation-induced transcription from the HIV type 1 long terminal repeat.

It has been previously shown in vitro and in vivo that the human immunodeficiency virus type 1 can be dramatically enhanced by certain heterologous viral, chemical, and physical (ultraviolet irradiation) agents. A common denominator shared by these agents is their ability to cause stress responses in cells. To analyze if a similar effect could occur by X irradiations, we tested the in vitro effect of X rays on HIV LTR-directed gene expression. The results demonstrate that the HIV-1 LTR is activated by X irradiation in a dose- and time-dependent manner, in all cell types tested, including epitheloid, fibroblast, and lymphoid cell lines. This study raises the possibility that exposure of AIDS patients to ionizing radiation (e.g., during treatment of epidemic Kaposi's sarcoma) could play a role in the activation of HIV-1 in vivo.

Cell Line↗

PubMed1994

Physical contact with lymphocytes is required for reactivation of dormant HIV-1 in colonic epithelial cells: involvement of the HIV-1 LTR.

HIV-1 transmission from mucosal epithelial cells to lymphocytes is a potential mechanism of HIV-1 contamination during sexual intercourse. The human colon epithelial cell line HT-29, that is infectable by various HIV-1 strains, is a useful model for studying the molecular mechanisms involved in this process. In the present study, we show that HT-29 cells, when exposed to either HIV-1(LAI) or HIV-1(NDK) at a low multiplicity of infection, became infected but did not produce infectious virions. Using two-compartment cell culture chambers separated by a porous membrane, we showed that PBL were able to rescue infectious HIV-1 from latently infected HT-29 cells following a physical interaction between the two cell populations. In contrast, HT-29 cells, infected with the same viruses at a high multiplicity of infection, were able to produce mature viral particles that were infectious to PBL in absence of cellular contacts. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that cell-to-cell contact induced an activation of the HIV-1 promoter. These observations provide a putative molecular mechanism for transmission of HIV-1 from mucosal epithelial cells to lymphocytes.

Cell Communication↗

PubMed1993

Translation and fates of the gag protein of 1731, a Drosophila melanogaster retrotransposon.

An entire copy of 1731, a Drosophila melanogaster retrotransposon, was tagged by fusing in frame its putative gag gene with the reporter LacZ sequence. The high transfection efficiency of Drosophila virilis cells added to the absence of 1731 in their genome allowed, by combining histochemical staining and immunological detections, the demonstration of the translation of the 1731 gag gene. The gag protein is gathered in virus-like particles. Its occurrence in nuclei is consistent with a nuclear localization signal. The expression of the sense construction was inhibited by cotransfections with its antisense homologue.

Amino Acid Sequence↗

PubMed1993

Structural and functional analysis of the human vasoactive intestinal peptide receptor glycosylation. Alteration of receptor function by wheat germ agglutinin.

The vasoactive intestinal peptide (VIP) receptor from the human melanoma cell line IGR39 has been shown to be a 60-kDa glycoprotein. Using serial lectin affinity chromatography, as well as specific glycosidases, we demonstrate that VIP receptor-linked carbohydrates are predominantly tri- or tetraantennary sialylated N-linked oligosaccharides, 27% of which are fucosylated, and some may have terminal galactose residues. Treatment of 125I-VIP receptor complexes with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase revealed the presence of at least three N-linked carbohydrate chains/receptor polypeptide. To investigate the functional role of the carbohydrate moiety, 125I-VIP binding to IGR39 cell membranes was tested in the presence of soluble lectins. Among the lectins tested, only wheat germ agglutinin (WGA) was found to markedly inhibit VIP binding in a dose-dependent manner. Binding data indicated that the presence of the lectin led to a 3-fold increase in Kd value, from 0.15 to 0.44 nM, without any change in the number of available binding sites. The potent inhibitor of WGA binding, N,N',N"-triacetylchitotriose, completely reversed the effect of the lectin. On the other hand, VIP binding inhibition persisted even after neuraminidase treatment, suggesting that sialic acids were not directly involved. Furthermore, WGA inhibition was not abolished although most, if not all, VIP receptor oligosaccharides were converted to high mannose type structures by treating IGR39 cells with deoxymannojirimycin. Finally, whereas the pharmacological profile of VIP receptor was virtually identical, the presence of WGA greatly reduced the VIP-stimulated cAMP in IGR39 cells, indicating that the lectin alters the ability of the receptor to interact with the adenylate cyclase system.

Binding, Competitive↗

PubMed1992

Characterization of the reverse transcriptase of 1731, a Drosophila melanogaster retrotransposon.

The nucleotide sequence of 1731, a retrotransposon cloned from the genome of Drosophila melanogaster, reveals a structural similarity with the proviral form of the retroviruses including a pol-like gene containing a putative reverse-transcriptase(RT)-coding sequence. Diverse parts of that sequence were subcloned and expressed in Escherichia coli. It has been demonstrated that the expression of the RT-like sequence, when translated, gives rise to peptides displaying enzyme activity characteristic of a true RT enzyme. In addition, rabbit antisera directed against such recombinant proteins allowed us to detect an immunoreactive protein of around 110 kDa, which was only present in D. melanogaster cell lines, but not in cells derived from Drosophila virilis or Drosophila hydei, whose genomes do not bear the 1731 element. This protein is expected to correspond to a non-processed pol-gene translated product and cosediments with virus-like particles exhibiting RT activity.

Amino Acid Sequence↗

PubMed1988

Thymotaxin: a thymic epithelial peptide chemotactic for T-cell precursors.

The embryonic thymus is seeded by invading hemopoietic precursor cells that differentiate intrathymically into T lymphocytes. We have recently reported that avian thymic epithelial cells secrete chemotactic peptides, which provoke oriented migration of hemopoietic precursor cells in vitro. The established rat thymic epithelial cell line IT-45 R1 produced a polypeptide that resolves as a single band in the region of 11 kDa on NaDodSO4/polyacrylamide gels. This molecule, which we have named thymotaxin, induced a chemotactic response in a subpopulation of hemopoietic cells from juvenile rat bone marrow. Responding cells were generated by short-term coculture of rat bone marrow hemopoietic cells with mouse bone marrow stroma in a steroid-free medium. Cells selected in a chemotactic chamber have a lymphoid or blast cell morphology. The phenotype of the responding cells is Thy-1+, CD4- [corrected] and CD8-. In contrast, CD8 T-lymphocyte differentiation antigen was expressed after coculture with embryonic thymic monolayers, suggesting that the responding cells correspond to the precursors colonizing the thymus.

Animals↗

PubMed1986

The embryonic thymus produces chemotactic peptides involved in the homing of hemopoietic precursors.

During ontogeny, T cell precursors must colonize the thymus to acquire immunocompetency. Using migration assays, a chemotactic activity was detected in conditioned media from avian embryonic thymic epithelial cells. The responding cells were shown to acquire T lymphocyte markers after homing into the thymus. Absorption experiments demonstrated surface receptors for the chemotactic substance on these hemopoietic precursors, which were not found on thymus-derived lymphocytes. Two peaks of chemotactic activity in the 1 kd-4 kd molecular weight range were detected after fractionation of thymic epithelial cell-conditioned medium. One of these activities was retained after heating to 95 degrees C but was destroyed after proteolytic treatment. Thus chemotactic peptides may be responsible for the thymic recruitment of the first hemopoietic precursors and may also be involved in the renewal of these precursors throughout adult life.

Animals↗

PubMed1986

Relationships between the GTP content and the TSH-stimulated adenylate cyclase activity of cultured thyroid cells.

The adenylate cyclase activity of a crude membrane fraction derived from cells cultured for 4 days in the presence of TSH (0.1 mU/ml), when acutely stimulated with 25 mU/ml, is 5-8 times higher than that derived from control cells. It has been suggested that changes in the intracellular content of GTP resulting from TSH chronic treatment were the cause of the modified responsiveness of the cyclase. To investigate this hypothesis, a method for GTP determination was developed. The steady-state concentration of GTP in 4-day TSH-treated cells is 2-3 times higher than in 4-day control cells. The increase in GTP content is concentration dependent between 5 and 500 microU/ml TSH in the culture medium. It presents a maximum on day 4 of culture, but remains elevated up to day 5. Nevertheless the GTP content is not the only factor controlling the cyclase activity, indeed the addition of 0.1 mM GTP to membranes from control cells does not increase the response up to the level reached by membranes from TSH-treated cells. Treatment of the cells with virazole, a drug inhibiting the biosynthesis of guanyl nucleotides, greatly decreases the GTP level, but is unable to suppress the positive effect of the TSH chronic treatment on adenylate cyclase activity. These results show that the increase in GTP level resulting from culture of the cells in the presence of minute amounts of TSH is not exclusively linked to adenylate cyclase responsiveness to TSH.

Adenosine Triphosphate↗

PubMed1985

Tetradecanoyl phorbol-13-acetate counteracts the responsiveness of cultured thyroid cells to thyrotropin.

We have studied the effects of TPA on the metabolism of porcine thyroid cells cultured for 1-4 days in the absence (control cells) and in the presence of 0.1 mU/ml TSH (TSH cells). The phospholipid turnover, evaluated after a 2 hr incorporation of 32P-phosphate into phospholipids, is markedly modified by the presence of TPA (1.5 microM, 2 hr) in the incubation medium of control and TSH treated cells. The total incorporation is 3-4 times higher than untreated cells, the labelling of phosphatidylinositol (PI) is slightly decreased or unchanged whereas that of phosphatidylcholine (PC) is strongly increased. The increased labelling of PI, promoted by an acute TSH treatment is counteracted by TPA. This TPA effect is not observed when prelabelled cells are challenged for 5 min with the drug. A similar effect is observed when 10 nM TPA is added in the culture medium for 20 hr. The addition of TPA does not affect significantly the protein iodine content in 3 or 4 days control cells incubated for 45 min or 2 hr with 125I-iodine, but dramatically decreases the very high iodination rate of TSH cells. We have tested the TPA effect on the cyclic AMP accumulation for the last 5 min of a 2 hr incubation. TPA inhibits by about 50-80% the stimulation evoked by TSH and only by 10% that evoked by forskolin (0.1 mM). These results suggest a possible link between the PC turnover and the adenylate cyclase responsiveness to TSH and the iodination rate.

Adenylyl Cyclases↗

PubMed1985

Comparative effects of cholera and Bordetella pertussis toxins on cyclic AMP and GTP levels and on lipolysis in rat adipocytes incubated in vitro.

The respective effects of cholera and Bordetella pertussis toxins were studied in time and concentration dependent experiments, following glycerol and fatty acid release, GTP and cAMP levels. Cholera toxin, after a lag time of 30 min, stimulated linearly GTP and cAMP accumulation and lipolysis (maximal effect: 2-fold increase at 5 micrograms/ml). Pertussis toxin presented a biphasic effect both in time and concentration dependent studies. Up to a maximum reached after 2 h with 1.4 units LPF/ml the stimulation affected GTP (3 fold) and cAMP (7 fold) levels, glycerol and fatty acid release (15 fold). Beyond this, an inhibition occurred, yielding a decrease towards basal values of GTP and cAMP content whereas the glycerol and fatty acid release was stopped. These results, which are the first reporting the fluctuation of the GTP content of intact cells challenged with bacterial toxins, show a close relationship between GTP and cyclic AMP levels and lipolytic activity.

Adipose Tissue↗

PubMed1985

Chronic and acute effects of forskolin on isolated thyroid cell metabolism.

The chronic treatment (2 days or more) of cultured thyroid cells with 1-10 microM forskolin (forskolin-treated cells) sensitizes the response of adenylate cyclase to further acute stimulation by 100 microM forskolin or 10 mU/ml thyrotropin (TSH). This positive regulation, similar to that produced by 0.1 mU/ml TSH (TSH-treated cells), is obtained between 2 and 3 days of culture. The acute response to TSH or forskolin of cells treated for 4 days with forskolin increases with the concentration of forskolin present during the chronic treatment. This result is different from that obtained after a chronic treatment with TSH which induces refractoriness beyond 0.1 mU/ml. These cells are then desensitized to TSH but not to forskolin. When both agonists are mixed together, their acute effect is additive on control, TSH- and forskolin-treated cells. The chronic treatment of cultured thyroid cells with 1-10 microM forskolin produces, just like 0.1 mU/ml TSH, a chronic phospholipid effect characterized by enhanced incorporation of 32Pi into phosphatidylinositol (PI) and phosphatidic acid. The acute challenge of these cells with 100 microM forskolin evokes a reverse phospholipid effect, i.e. a decreased incorporation of 32Pi into PI. The acute stimulation of TSH-treated cells with TSH produces a reverse phospholipid effect whereas the acute stimulation of forskolin-treated cells with TSH gives a normal phospholipid effect as it does on control cells. These results show that the observed effects of TSH on cAMP accumulation and phospholipid turnover are not independent and are regulated in an inverse reciprocal pattern.

Animals↗

PubMed1984

Interactions between thyreostimulin and PAF-acether on thyroid cell metabolism.

1-O-Alkyl, 2-acetyl sn-glycerylphosphorylcholine (platelet activating factor (PAF)-acether) originally described as a platelet activating factor, is effective on various parameters in different cells. It seemed interesting to us to test it on porcine thyroid cells cultured for 1 to 5 days in the absence (control cells) or in the presence of 0.1 mU/ml thyreostimulin (TSH cells). At concentrations ranging from 0.5 to 2.5 microM, PAF-acether inhibited significantly the accumulation of cyclic AMP resulting from a 5 min incubation of the cells with TSH (40 mU/ml) or forskolin (0.1 mM). PAF-acether alone did not affect basal cyclic AMP accumulation. The maximal inhibition was obtained on a 3 day culture and amounted to 40-50%. The inhibition was transient and vanished after a 30 min incubation. The effects of PAF-acether (0.5 microM) on phospholipid metabolism depended closely upon the physiological state of the cells and upon the age of the culture. When PAF-acether was incubated for 2 h with [32P]phosphate, it mimicked the effects of TSH, i.e. it increased phosphatidylinositol (PI) labelling on 1 day control cells (expected effect) and decreased it with 1 day TSH cells (reverse effects). The PAF-acether effect was rapid in onset. After cell prelabelling for 2 h in the presence of TSH, PAF-acether added for 15 min completely counteracted the hormone effects on PI and phosphatidylcholine (PC) but increased the phosphatidic acid (PA) labelling. The effect of PAF- acether on PI labelling was partially antagonized by forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1982

Muscarinic cholinergic receptors on cultured thyroid cells. I. Biological effect of carbachol and characterization of the receptors.

A muscarinic cholinergic effect on thyrotropin-stimulated cyclic AMP accumulation in cultured porcine thyroid cells is characterized. The muscarinic agonists carbachol, acetylcholine, oxotremorine, and pilocarpine decreased the acute cyclic AMP response to stimulation with thyrotropin (20 mU/ml). A 50% decrease was obtained in the presence of 0.1 mM carbachol. Maximal effects were observed when cells were cultured in the presence of thyrotropin (100 microU/ml) or prostaglandin E2 (1 microM), whereas cells cultured in basal medium or in the presence of dibutyryl cyclic AMP showed a low response to carbachol. Evidence is reported suggesting that carbachol acutely decreases cyclic AMP synthesis. The properties of the muscarinic receptors present in thyroid cells were defined by using the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate to cell homogenates. Scatchard plots revealed a single population of binding sites with a KD of 0.3 nM. The numbers of binding sites per cell after 4 days in culture were 880 in control cells, 4335 in thyrotropin (100 microU/ml)-treated cells, 5600 in prostaglandin (1 microM)-treated cells, and 1420 in dibutyryl cyclic AMP-treated cells. Therefore the sensitivity to carbachol appeared to be related to the number of antagonist binding sites and to be independent of the sensitivity of the cells to acute thyrotropin stimulation.

Animals↗

PubMed1982

Muscarinic cholinergic receptors on cultured thyroid cells. II. Carbachol-induced desensitization.

Muscarinic cholinergic receptors were previously characterized on cultured porcine thyroid cells. The receptor number was increased by chronic thyrotropin or prostaglandin E2 treatments [Champion, S., and J. Mauchamp. Mol. Pharmacol. 21:66-72 (1982)]. The long-term effect of carbachol was studied. After chronic treatment with carbachol, cells were completely desensitized to acute carbachol stimulation. This process was blocked by muscarinic antagonists. A complete desensitization was obtained after 6 hr of treatment with 100 microM agonist. Under these conditions the quinuclidinyl benzilate binding capacity of cell homogenates was reduced by 50%. Withdrawal of carbachol allowed the complete restoration of the sensitivity of cells within 6 hr with only a partial recovery of the binding capacity (25%). The complete complement of receptors was obtained after 24 hr of recovery. Desensitization and agonist-induced decrease in receptor number were not affected by cycloheximide, whereas the recovery of both effect and binding sites required active protein synthesis.

Animals↗

PubMed1981

Variability of electromyographic patterns for level-surface walking through a range of self-selected speeds.

The surface electromyographic patterns of eight muscles and the foot-contact patterns exhibited by 25 normal persons between the ages of 20 and 40, were measured and studied. These persons were walking indoors on a level surface without shoes at several different self-selected walking speeds. The electromyographic patterns demonstrated a considerable amount of interindividual variability at each speed. There were definite trends in the number of individuals exhibiting each pattern type as walking speed increased. The foot-contact patterns showed a consistent sequencing of foot events at all speeds with the double-limb support stages and stance phase comprising a smaller percentage of the gait cycle as speed increased.

Adult↗