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T Heidmann

Publications and source records attributed to T Heidmann.

At least 55 records · Page 3Linked to original sources
PubMed1991

Retrotransposition of a mouse IAP sequence tagged with an indicator gene.

We have marked a cloned mouse IAP sequence with a neomycin-containing indicator gene whose expression is conditioned by passage of the transposon through an RNA intermediate. Transposition of the marked IAP introduced into tumor cells could be detected by simple selection of the cells in G418, at a frequency of 10(-6) per cell per generation. Southern blot analysis and nucleotide sequencing after PCR amplification demonstrated "retrotransposition" of the marked element, with splicing out of an intron contained in the indicator gene, and retroviral-like reverse transcription and integration of the transposed IAPs, with 6 bp duplications of the identified target sites. Transposition was found to be mutagenic for the element, as might be expected if the identified marked and endogenous IAP transcripts were coencapsidated into IAP particles as dimers.

Animals↗
PubMed1991

An indicator gene for detection of germline retrotransposition in transgenic Drosophila demonstrates RNA-mediated transposition of the LINE I element.

We have marked a cloned Drosophila transposable element--the I element--with an engineered neomycin-containing indicator gene, whose expression is conditioned by passage of the transposon through an RNA intermediate. Mobility of the marked element introduced into Drosophila as a transgene could be detected in vivo, upon in toto selection of developing embryos on G418-containing medium. For resistant individuals, Southern blot analysis and nucleotide sequencing after PCR amplification disclosed transposition of the marked element into new loci, with target site duplications and splicing out of the intron in the indicator gene. It demonstrates that the I element, which is closely related to the widespread mammalian LINEs, transposes through an RNA intermediate, as up to now only conjectured from sequence singularities of this class of 'non-viral retrotransposons'. The developed indicator gene provides a potent new genetic tool for detection and quantitative analysis of retrotransposon mobility and its regulation as it occurs in vivo.

Animals↗
PubMed1991

Defective retroviruses can disperse in the human genome by intracellular transposition.

Using an assay for retrotransposition detection (T. Heidmann, O. Heidmann, and J. F. Nicolas, Proc. Natl. Acad. Sci. USA 85:2219-2223, 1988), we demonstrated that a defective retrovirus deleted for the gag, pol, and env open reading frames can disperse in the genome of human HeLa cells by intracellular transposition, at a frequency close to 10(-6) events per cell per generation. Transposition requires cooperation in trans for the gag and pol gene products and may be associated with the release of low amounts of noninfectious retroviruslike particles which are the hallmarks but not the intermediates of this transposition process. Similar events could account for the dispersion at high copy number of some of the human endogenous sequences related to retroviruses and for the occurrence of noninfectious retroviruslike particles in human placenta and several tumor cell lines (reviewed by E. Larsson, N. Kato, and M. Cohen, Curr. Top. Microbiol, Immunol, 148:115-132, 1989).

Defective Viruses↗
PubMed1988

An indicator gene to demonstrate intracellular transposition of defective retroviruses.

An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo-MLV(neo)RT]. NeoRT contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)RT provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo-MLV(neo)RT provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 10(7) cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected.

DNA Transposable Elements↗
PubMed1987

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the beta and delta chains.

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled beta chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the beta chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain [Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y., & Changeux, J.-P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2719-2723]. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.

Amino Acid Sequence↗
PubMed1986

Characterization of the transient agonist-triggered state of the acetylcholine receptor rapidly labeled by the noncompetitive blocker [3H]chlorpromazine: additional evidence for the open channel conformation.

The kinetics of covalent labeling of the alpha, beta, gamma, and delta chains of the acetylcholine receptor (AcChR) from Torpedo marmorata by the noncompetitive blocker [3H]chlorpromazine ([3H]CPZ) are investigated by using rapid mixing photolabeling techniques. In an initial study [Heidmann, T., & Changeux, J. P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1897-1901], it was shown that the rate of [3H]CPZ labeling increases 100-1000-fold upon simultaneous addition of nicotinic agonists to the AcChR and that prior addition of these agonists abolishes the effect. The data were interpreted in terms of the rapid labeling of the transient active state of the AcChR where the ion channel is in its open configuration. This interpretation was recently challenged [Cox, R. N., Kaldany, R. R. J., Di Paola, M., & Karlin, A. (1985) J. Biol. Chem. 260, 7186-7193] on the ground of studies with a different noncompetitive blocker, [3H]quinacrine azide, and the suggestion was made that this compound labels the rapidly desensitized closed channel conformation of the AcChR. In this paper it is shown that the rate of rapid labeling of the AcChR by [3H]CPZ decreases to negligible values upon exposure of the AcChR to nicotinic agonists, in the 100-500-ms time range. The absolute values of the rate constants of this decrease (10-15 s-1 for saturating concentrations of acetylcholine and carbamoylcholine) and their variation with agonist concentration (apparent dissociation constants of 40 microM and 0.4 mM for acetylcholine and carbamoylcholine, respectively) are those expected for the rapid desensitization of the AcChR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine.

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of agonist. Incorporation of radioactivity into all subunits occurred and was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The delta subunit was purified and digested with trypsin, and the resulting fragments were fractionated by reversed-phase HPLC. The labeled peptide could not be purified to homogeneity because of its marked hydrophobic character, but a combination of differential CNBr subcleavage and cosequencing of partially purified fragments enabled us to identify Ser-262 as being labeled by [3H]chlorpromazine. The labeling of this particular residue was prevented by phencyclidine and thus took place at the level of, or in proximity to, the high-affinity site for noncompetitive blockers. Ser-262 is located in a hydrophobic and potentially transmembrane segment termed MII.

Affinity Labels↗
PubMed1985

Allosteric effects of diprobutine on acetylcholine receptors.

The nicotinic effects of a novel antiparkinsonian compound, diprobutine were investigated on the acetylcholine receptor (AChR) from Torpedo marmorata electric organ and on rat brain membranes by a variety of techniques including stopped flow measurements. On the nicotinic AChR from Torpedo, diprobutine behaved as a typical noncompetitive blocker: it inhibited the agonist-regulated 22Na+ efflux from excitable microsacs; it shifted in the ms-s time-range the conformation of the AChR towards a high affinity state for agonists; it competed with [3H]PCP bound to its high affinity 'allosteric' site. On rat brain membrane, it displaced [3H]PCP bound to its high affinity site. The pharmacological properties of diprobutine are discussed in the context of its biochemical effects.

Allosteric Site↗
PubMed1984

Time-resolved photolabeling by the noncompetitive blocker chlorpromazine of the acetylcholine receptor in its transiently open and closed ion channel conformations.

A rapid-mixing photolabeling apparatus is developed to resolve the kinetics of association of the noncompetitive channel blocker [3H]chlorpromazine (CPZ) with the membrane-bound acetylcholine (AcCho) receptor from Torpedo marmorata and to photolabel its subunits in the 100-milli-seconds to seconds time range. Rapid mixing of AcCho and [3H]CPZ with the receptor followed by brief (less than 20 msec) UV irradiation results in the selective labeling of the four chains of the AcCho receptor, according to a rapid bimolecular association process close to diffusion-controlled. Rapid association is not observed with the competitive antagonists d-tubocurarine or flaxedil or the snake venom alpha-toxins. Its initial rate increases with agonist concentration, with maxima of 0.6 for carbamoylcholine and 0.2 for phenyltrimethylammonium taking 1 for AcCho, with apparent dissociation constants of 30 microM, 400 microM, and 300 microM for AcCho, carbamoylcholine, and phenyltrimethylammonium, respectively, and with sigmoid shape (Hill coefficients of 1.1-1.3). Under conditions in which the receptor "desensitizes" and the ionic channel closes (preincubation with AcCho), rapid [3H]CPZ association decreases in parallel. It is concluded that the agonist-dependent rapid association of [3H]CPZ takes place at the level of a site common to all five subunits, which lies within the ion channel and becomes accessible when the channel opens.

Acetylcholine↗
PubMed1984

[Selective stabilization of neuronal representations by resonance between spontaneous prerepresentations of the cerebral network and percepts evoked by interaction with the outside world].

Changeux et al. (Changeux, Heidmann and Patte, in "The Biology of Learning" Dahlem Conference, 1984, pp. 115-133, Springer Verlag) have recently discussed a model of "learning by selection" in which the storage of patterns of activity--or prerepresentations--within a network of neurons, results from the coincidence or "resonance" between a spontaneous activity of the neurons and external signals applied to the network--for instance sensory stimuli. In this Note, a mathematical formulation of the model is presented, based on that proposed by Little and Shaw (Little, Math. Biosci., 19, 1974, pp. 101-120; Little and Shaw, Math. Biosci., 39, 1978, pp. 281-290) for the statistical analysis of neuronal activity within a network, and on a rule for modulation of synaptic efficacies derived from that proposed by Hebb (Hebb, The Organisation of Behaviour, 1949, Wiley). The effect of an external signal sigma on the probability P(beta) of occurrence of a given prerepresentation beta under stationary conditions has been analytically derived [cf. equation (16) in text]. Taking into account that the system spontaneously fluctuates between various prerepresentations, it is shown that P(beta) is increased by the external signal sigma when (1) beta is close to sigma--namely the external signal significantly modifies the probabilities of those prerepresentations that resemble sigma--, and (2) when the external signal sigma sets the neurons precisely in the state that they would have more probably reached at the moment when the external signal was applied. Namely there should exist a "resonance" between sigma and the prerepresentation of the network when sigma is applied.

Animals↗
PubMed1983

Rapid kinetics of agonist binding and permeability response analyzed in parallel on acetylcholine receptor rich membranes from Torpedo marmorata.

Excitable acetylcholine receptor rich membrane fragments from Torpedo marmorata have been used to measure, in parallel, (1) the permeability response to the fluorescent cholinergic agonist Dns-C6-Cho (in the 0.1 microM to millimolar concentration range) characterized by both the initial rate of Li+ transport and the rate of channel closure using the rapid-mixing quench-flow technique and (2) the kinetics of interaction of Dns-C6-Cho with the acetylcholine receptor sites using the rapid-mixing stopped-flow technique. Analysis of the kinetics of Dns-C6-Cho binding in the millisecond to minute time scale leads to the identification of at least three conformational states of the acetylcholine receptor: a "low-affinity" one (approximately 50 microM) that can be interconverted in the fraction of a second to a transient state of "intermediate affinity" (approximately 1 microM), followed by the final stabilization, in the second to minute time range, of a state of "high affinity" (approximately 3 nM). Comparison of Dns-C6-Cho binding data with the permeability response to the same agonist demonstrates that the binding to the low-affinity conformation(s) of the acetylcholine receptor sites coincides with the triggering of the permeability increase--or "activation"--and the transitions to the intermediate- and high-affinity states with the two-step process of channel closing--or "desensitization". The data are interpreted in terms of a minimum four-state "allosteric" model for the acetylcholine receptor.

Animals↗
PubMed1982

[Molecular model of the regulation of chemical synapse efficiency at the postsynaptic level].

The regulation of efficacy of a chemical synapse at the postsynaptic level is discussed in terms of the reversible allosteric transitions of the receptor for the neurotransmitter. The proposed model accounts for the regulation of a synapse by its own state of activity and by that of neighbouring synapses on the same neuron. The model predicts persisting changes of efficacy in the time-scale of seconds-minutes compatible with short-term memory processes following, in particular, the scheme of classical conditioning.

Animals↗
PubMed1982

[The high affinity binding site for chlorpromazine is present only as a single copy per cholinergic receptor molecule and is shared by four polypeptide chains].

3H chlorpromazine binds to acetylcholine receptor-rich membrane fragments prepared from Torpedo marmorata electric organ in three different manners: (1) at the level of high affinity sites from which it is displaced by perhydrohistrionicotoxin, (2) at the level of low affinity sites insensitive to this toxin, and (3) in a non saturable manner to a presumably lipidic phase. Binding of chlorpromazine to the first two categories of sites independently stabilizes the "desensitized" high affinity state of the receptor for cholinergic agonists. There exists one high affinity site per two snake alpha-toxin sites, thus per 250,000 daltons light form of the receptor. Under the conditions of 3H chlorpromazine high affinity binding, ultraviolet irradiation results in the covalent incorporation of this ligand to the four chains of the receptor.

Animals↗
PubMed1981

[Preservation of the allosteric properties of the protein receptor for acetylcholine in detergent solution without addition of lipids].

Solubilization by Na cholate of acetylcholine receptor-rich membrane fragments from Torpedo marmorata, followed by exchange of NA cholate by the neutral detergent "Tween 80" yields the receptor protein in its 9 S soluble light form; under these conditions, without adding lipids, the receptor protein conserves its characteristic binding properties for the fluorescent agonist Dns-C6-Cho as followed by fast kinetic techniques, and the allosteric regulation by non-competitive blockers.

Allosteric Regulation↗