Philip Lazarovici

Sequencing and synthesis of pardaxin, a polypeptide from the Red Sea Moses sole with ionophore activity

Pardaxin, an amphipathic polypeptide secreted by the Red Sea flatfish Pardachirus marmoratus whose sequence is NH2‐G‐F‐F‐A‐L‐I‐P‐K‐I‐I‐S‐S‐P‐L‐F‐K‐T‐L‐L‐S‐A‐V‐G‐S‐A‐L‐S‐S‐S‐G‐G‐Q‐E, was synthesized by the solid‐phase method. The structure was verified by sequencing. The synthetic polypeptide changed the resistance of lipid bilayers by forming pores. At 10−7−10−8 M, the synthetic pardaxin increased the frequency of the spontaneous release of quanta of acetylcholine at the neuromuscular junction by up to 100‐fold, resembling the native product. Synthetic pardaxin seems to be a suitable tool for investigating the molecular structures underlying channel selectivity.

Nerve growth factor (NGF)-induced calcium influx and intracellular calcium mobilization in 3T3 cells expressing NGF receptors

The neurotrophins have been implicated in the acute regulation of synaptic plasticity. Neurotrophin-stimulated presynaptic calcium uptake appears to play a key role in this process. To understand the mechanism of neurotrophin-stimulated calcium uptake, the regulation of calcium uptake and intracellular mobilization by nerve growth factor (NGF) was investigated using NIH 3T3 cells stably transfected with either the high affinity NGF receptor p140 trk (3T3-Trk) or the low affinity NGF receptor p75NGFR(3T3-p75). In 3T3-Trk cells, NGF increased both calcium uptake and intracellular calcium mobilization. In 3T3-p75 cells, NGF increased calcium uptake but not intracellular calcium mobilization. K-252a alone increased intracellular calcium in 3T3-Trk cells but not in 3T3-p75 cells. Nifedipine, an inhibitor of calcium uptake throughl-type calcium channels, inhibited the action of NGF on both 3T3-Trk cells and 3T3-p75 cells, indicating that both p140 trk and p75NGFR receptors are linked to nifedipine-sensitive l-type calcium channels. These studies show that either NGF receptor will support increases in intracellular calcium but that p140 trk does so by increasing both uptake and mobilization, whereas p75NGFR does so by increasing uptake only.

Both p140trk and p75NGFR Nerve Growth Factor Receptors Mediate Nerve Growth Factor-stimulated Calcium Uptake

Human p140trk and p75NGFR were transfected separately into 3T3 cells. Nerve growth factor stimulates calcium uptake into both transfectants but not into untransfected 3T3 cells. p140trk cells were stimulated maximally by 25 ng/ml; 100 ng/ml was submaximal for p75NGFR cells. K-252a inhibits the effect of NGF on p140trk cells but not on p75NGFR cells; brain-derived neurotrophic factor stimulates calcium uptake in p75NGFR cells but not in p140trk cells. The data suggest that both nerve growth factor receptors could be involved in the nerve growth factor-mediated actions of calcium on its target cells: neuronal survival, neuronal protection, and synaptic plasticity.

Down-regulation of Epidermal Growth Factor Receptors by Nerve Growth Factor in PC12 Cells Is p140trk-, Ras-, and Src-dependent

Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level measured by 125I-EGF binding, tyrosine phosphorylation, and Western blotting. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of [35S]methionine-labeled EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140trk NGF receptor. However, in PC12nnr5 cells stably transfected with p140trk, the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140trk-, Ras- and Src-dependent mechanism of NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells.

Expression of human p140trk receptors in p140trk‐deficient, PC12/endothelial cells results in nerve growth factor‐induced signal transduction and DNA synthesis

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140trk. These biological effects of NGF depend upon the signal‐mediating function of p140trk substrates which are likely to differ from cell to cell. To define p140trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full‐length human p140trk cDNA sequence. Two stably transfected clones, one expressing p140trk with higher affinity (PC12EN‐trk3; Kd 57.4 pM, Bmax 9.7 pmole/mg) and one expressing p140trk with a lower affinity (PC12EN‐trk1; Kd 392.4 pM, Bmax 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140trk receptors show slow NGF‐dissociation kinetics, are resistant to trypsin or Triton X‐100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140trk receptors. NGF stimulates p140trk tyrosine phosphorylation in a dose‐ (0.01‐10 ng/ml) and time‐ (5‐120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200‐1,000 nM K‐252a. NGF‐induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70S6k, SNT, and phospholipase Cγ, demonstrating that the major NGF‐stimulated signalling pathways found in other cells are activated in PC12EN‐trk cells. Staurosporine (5‐50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN‐trk cells. Rather, NGF treatment in low‐serum medium stimulated a 1.3‐ and 2.3‐fold increase in DNA synthesis measured by [3H]thymidine incorporation in PC12EN‐trk1 and PC12EN‐trk3, respectively. These data highlight the functionality of the transfected p140trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140trk receptor substrates. J. Cell. Biochem. 66:229‐244.

Preparation of affinity-purified, biotinylated tetanus toxin, and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 1∶1 to 20∶1 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using125I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these “primed” cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.

Pardaxin induces aggregation but not fusion of phosphatidylserine vesicles

The effects on membranes of pardaxin, an amphipathic polypeptide, purified from the gland secretion of the Red Sea Moses sole flatfish Pardachirus marmoratus are dose‐dependent and range from formation of voltage‐gated, cation‐selective pores to lysis. We have now investigated the interactions of pardaxin with small unilamellar liposomes. Light scattering showed that pardaxin (10−7–10−9M) mediated the aggregation of liposomes composed of phosphatidylserine but not of phosphatidylcholine. Aggregation of phosphatidylserine vesicles was impaired by vesicle depolarization. Furthermore, pardaxin‐mediated aggregation between fluorescent‐labeled PS vesicles was accompanied by leakage of the vesicle contents, and not by fusogenic process within the aggregates. We suggest that pardaxin is a unique polypeptide, that induces vesicle aggregation and membrane destabilization, but not membrane fusion; the mechanism of the aggregation activity of pardaxin is related to its amphipathic properties.

Staphylococcus aureus α-toxin. 1. Effect on protein phosphorylation in myelin

It has previously been demonstrated that staphylococcal alpha-toxin can selectively induce disruption of myelin sheaths in the central nervous system, albeit the exact mechanism is not known. In this report we show for the first time that the staphylococcal alpha-toxin could stimulate the endogenous phosphorylation of several proteins, including myelin basic protein (Mr = 18,500) in purified guinea pig brain myelin. This stimulatory effect does not require the presence of calcium and is distinct from those modulated by calcium and phospholipids. In vitro phosphorylation of isolated myelin basic protein by the purified catalytic subunit of cAMP-dependent protein kinase was enhanced in the presence of alpha-toxin, whereas the reaction catalyzed by protein kinase C, a Ca2+-activated and phospholipid-dependent protein kinase, was not affected. These results suggest that some of the toxic effects of staphylococcal alpha-toxin on myelin may be mediated through post-translation covalent modification, such as phosphorylation of specific proteins.

Preparation of a cell-free translation system from PC12 cells

The postmitochondrial fraction (S10) contains the cellular components essential for translation, and a high-salt wash (HSW) of the ribosomes is enriched in eukaryotic initiation factors. This report describes the preparation of a cell-free translation system utilizing an S10 extract from PC12 cells. The products synthesized from either firefly luciferase mRNA or PC12 cell poly(A) RNAs in the PC12-S10 extract were increased by the addition of the HSW from PC12 cells. Increases in the translation of luciferase mRNA by the addition of PC12-HSW were dose-dependent and also dependent on the time of incubation. The translation of human epidermal growth factor receptor (hEGFR) mRNA could also be detected in the PC12-S10 extract translation system by immunoprecipitation.N-linked glycosylation of the translation products also was observed. The efficiency of translation was altered by the addition of Mg2+ or K+, and optimization of the concentrations of these ions was necessary for each mRNA. The translation system made from PC12 cells, then, is capable of the synthesis of proteins of relatively high molecular weight and should be useful for analyzing mechanisms of translational control during proliferation and differentiation of cells from a neuronal lineage.

Staphylococcus aureus α-toxin. 2. Reduction of epidermal growth factor receptor affinity in PC12 cells

Staphylococcus aureus alpha-toxin, at sub-cytotoxic concentrations, inhibits both the 125I-labeled epidermal growth factor (EGF) binding and autophosphorylation properties of EGF-receptors in PC12 cells. This inhibition occurred only in intact cells and is probably due to a decrease in the affinity of the receptor for EGF. Streptolysin S and parcelsin could mimic the alpha-toxin effect below cytotoxic concentrations, as measured by a 51Cr release assay. In contrast, other membrane perturbing toxins with different lipid specificity, such as tetanolysin and cobra direct lytic factor, inhibited [125I]EGF binding only at cytotoxic concentrations. Staphylococcal alpha-toxin also stimulated 3-fold the specific binding of a radioactive tumor-promoting phorbol ester (PDBu) to PC12 cells at concentrations similar to those required for the inhibition of [125I]EGF binding. Although the exact mechanism for the inhibition of EGF binding by alpha-toxin has not been established, our results suggest that protein kinase C may be involved in this time-dependent process.