Journal de physiologie最新文献

Diphtheria, tetanus, botulinum, and anthrax toxin are multipartate toxins, one of the domains of which is (or is presumed to be) an enzyme. Cell intoxication requires that the enzymatic portion gain access to the cytosol via endocytosis into an acidic vesicle compartment of the cell. Translocation of the enzyme across the vesicular membrane is dependent on the low pH of the vesicle and involves another domain of the toxin; for each of these toxins, that domain is capable of forming channels in phospholipid bilayer membranes. These channels are large (greater than 12 A diameter) and voltage-gated, and the pH conditions required for their formation in lipid bilayers are similar to those existing in acidic vesicles and required for cell intoxication.

We have employed a new technique in Drosophila that allows in vivo detection of genomic regulatory elements using a beta-galactosidase reporter gene. A translational fusion of the reporter gene to the P-transposase gene, which is encoded by the P-transposon of Drosophila, places the expression of beta-galactosidase under the control of the weak P-transposase promoter. Flies carrying single insertions of this P-element construct at different locations in the Drosophila genome frequently stain for beta-galactosidase activity in a temporally and spatially restricted fashion in embryos, larvae and adult ovaries, reflecting the influence of nearby genomic regulatory elements on the P-transposase promoter. This technique is a powerful tool as it can be used to produce very many different cell markers and to isolate developmentally regulated genes in Drosophila. We discuss the implications of our results and the applications of the technique to further the study of Drosophila development.

Down syndrome (DS), the phenotypic expression of human trisomy 21, is presumed to result from overexpression of certain genes residing on chromosome 21 at the segment 21q22-the Down locus. The "housekeeping" enzyme CuZn-superoxide dismutase (CuZnSOD) is encoded by a gene from that region and its activity is elevated in DS patients. To investigate the possible involvement of CuZnSOD gene dosage in the etiology of the syndrome we have developed both cellular and animal models which enabled us to investigate the physiological consequences resulting from overexpression of the CuZnSOD gene. 1. Rat PC12 cells expressing elevated levels of transfected human CuZnSOD gene were generated. These transformants (designated PC12-hSOD) closely resembled the parental cells in their morphology, growth rate, and response to nerve growth factor, but showed impaired neurotransmitter uptake. The lesion was localized to the chromaffin granule transport mechanism. We found that the pH gradient (delta pH) across the membrane, which is the main driving force for amine transport, was diminished in PC12-hSOD granules. These results show that elevation of CuZnSOD activity interferes with the transport of biogenic amines into chromaffin granules. Since neurotransmitter uptake plays an important role in many processes of the central nervous system, CuZnSOD gene-dosage may contribute to the neurobiological abnormalities of Down's syndrome. 2. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human CuZnSOD gene have been prepared. These animals express the transgene in a manner similar to that of humans, with 0.9 and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable of forming human-mouse enzyme heterodimers. CuZnSOD activity is increased from 1.6 to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. 3. To investigate the possible involvement of CuZnSOD gene dosage in the neuropathological symptoms of Down's syndrome, we analyzed the tongue muscle of the transgenic mice that express elevated levels of human CuZnSOD. The tongue neuromuscular junctions (NMJ) in the transgenic animals exhibited significant pathological changes, namely, withdrawal and destruction of some terminal axons and the development of multiple small terminals. The ratio of terminal axon area to postsynaptic membrane decreased, and secondary folds were often complex and hyperplastic. The morphological changes in the transgenic NMJ were similar to those previously seen in muscles of aging mice and rats as well as in tongue muscle of patients with Down's syndrome.(ABSTRACT TRUNCATED AT 400 WORDS)

mRNAs encoding the light chain of tetanus and botulinum neurotoxins were transcribed, in vitro, from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic identified cholinergic neurons of the buccal ganglia of Aplysia californica. The size of the current response measured in the voltage clamped postsynaptic neuron was taken as indicator of the quantity of acetylcholine released. Depression of neurotransmitter release similar to that observed when native light chains of the two toxins were injected but needing an additional delay of 30 to 40 minutes, demonstrated a successful expression of a foreign mRNA injected into a neuron in situ.

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.

Botulinum neurotoxin appears to undergo structural alterations after synthesis and also before it inhibits neurotransmitter release. Discussions and conjectures are presented in this context: 1. At what sites on the 150 kDa neurotoxin does posttranslational proteolytic processing occur? 2. Does neurotransmitter inhibition depend on separation of a segment of the neurotoxin from the rest of the molecule? 3. At what step in the intoxication pathway does activation of neurotoxin (enhanced lethality following limited proteolysis) manifest? 4. Can the receptor binding parameters (based on bovine brain synaptosome and lipid membrane), channel forming property (lipid bilayer membrane) and intracellular inhibitory activity (based on permeabilized chromaffin and PC 12 cells) provide clues to differences in the lethal potency between the neurotoxin serotypes? In addition, the following issues are considered: 5. The spontaneous fragmentation of isolated 50 kDa light chain, after its separation from 100 kDa heavy chain, 6. Effect of specific chemical modification of Arg, His, Lys, Trp, Tyr and Asp/Glu residues of types A, B and E neurotoxins on lethality and antigenicity, and 7. Development of second generation toxoids.

1. The specificity of synaptogenesis of identified adult neurons of Helisoma was determined in cell culture. Cholinergic neuron B5 indiscriminately forms the presynaptic element of chemical connections with novel cholinoceptive target neurons and muscle. By contrast, cholinergic neuron B19 is selective and discriminates between novel and appropriate target cells. Neuron B19 forms chemical connections with appropriate muscle targets only. 2. The acquisition of presynaptic properties independent of target contact was studied for both identified neurons. Functional connections form between neuron B5 and novel targets within seconds of contact, indicating that this cell has synthesized the presynaptic apparatus before target contact. In contrast, neuron B19 showed no evidence of possessing the ability to release neurotransmitter. 3. To further study the development of presynaptic properties, a model system of giant synaptic terminals was developed. The soma of neuron B5, acutely isolated from the nervous system is non-secretory. In conditions that prevent the extension of neurites, somata gain the ability to release neurotransmitter. This experimentally tractable system was used to study the calcium currents of presynaptic neuron B5. Acutely-isolated non-secretory somata contain two types of calcium currents: low-voltage-activated (LVA) and high-voltage-activated (HVA). The types of calcium currents in the soma change when B5 gains its secretory capacity. Secretory somata contain HVA calcium current only. 4. Neuron B5 was also plated in conditions which permit the extension of neurites. LVA and HVA calcium currents were maintained in its soma (non-secretory) but HVA calcium current only was maintained in its growth cones (secretory). Thus, B5 differentially regulates the presence of specific calcium currents in its membrane in relation to local secretory capacity without target-derived cues. 5. These data suggest that neuron B5 has an intrinsic program which generates presynaptic calcium channels and secretory apparatus prior to target contact. This autonomy of initial presynaptic development may underlie the lack of target cell discrimination exhibited by B5 in synaptogenesis.

1. From denervation studies the trophic influence of the motor nerve on the muscle cell is well documented while little is known about the influence of the muscle on the nerve. Sectioning the axon invariably destroys the nerve terminals and produces nerve degeneration products which themselves may affect nerve and muscle properties. With regard to those difficulties we believe that the botulinal neurotoxins (BoTx) are valuable complements to denervation since they selectively interrupt impulse transmission across the synapse without damaging its morphology. 2. Paralysis of mouse or rat skeletal muscle in vivo with BoTx type A causes marked growth of motor nerve terminals. The sprouting terminals are rich in large dense-core synaptic vesicles containing various neuropeptides and they spontaneously release large quanta of ACh. Thus, it appears that paralysis by BoTx is a strong stimulus for motor nerve growth and the delivery of "trophic" substances to the nerve terminals. 3. Postsynaptically, in extrajunctional areas, paralysis by BoTx induces all the changes observed following denervation, i.e. atrophy, appearance of extra-junctional ACh receptors, TTX-resistant action potentials, a fall of resting membrane potential, fibrillation potentials and the disappearance of extrajunctional acetylcholinesterase activity. Endplate properties are, however, largely maintained. 4. BoTx blockade delays and prevents the retraction of polyneuronal innervation and motoneurone death during development. This supports the suggestion that the paralysed muscle secretes factors essential for growth and for the survival of motoneurones. 5. Like denervated muscle, BoTx paralysed ones, express a high endocytotic activity restricted to a segment in the endplate region.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetanus and botulinum neurotoxins (TeNT and BoNT) bind strongly and specifically to the nervous tissue, as it can be inferred from their potency and from their effects restricted to the nervous system. The molecular basis of these properties are presently unknown. As a first approach, we have investigated the interaction of TeNT and BoNT with model membranes by photolabelling with phospholipid analogues carrying the photoreceptor group at different positions of the lipid molecule in order to probe different membrane regions. We found that at neutral pH TeNT and BoNTs (type A, B and E) adsorb onto the surface of negatively charged liposomes. Polysialogangliosides increase this interaction only slightly thus suggesting that they provide a minor contribution to toxin lipid binding. On this basis we propose that clostridial neurotoxins bind to lipids via both a predominant unspecific interaction with negatively charged lipids (including gangliosides) and a specific, but weaker, interaction with polysialogangliosides. At acidic pH values both chains of these neurotoxins are labelled strongly by photogroups located in the hydrophobic milieu of the membrane with a pH dependence that overlaps the range of pH values reached in the endosomal lumen. This result is consistent with their insertion into the lipid bilayer in agreement with the idea that clostridial neurotoxins may penetrate into cells via intracellular low pH compartments.

Cells of the neurogenic ectoderm of insects have to decide between a neural and an epidermal fate. In Drosophila, this decision id mediated by cellular interactions. The products of two different groups of genes, i.e., the neurogenic genes and the genes of the achaete-scute complex and daughterless, seem to provide the molecular basis for the elements of a signal chain that permits the commitment of the cells to a given fate.