Induction of neuronal apoptosis by Semaphorin3A-derived peptide.

Collapsin-1/Semaphorin3A (Sema3A) belongs to the secreted type III semaphorins family of axon guidance molecules with chemorepulsive activity, and is suggested to play a major role in navigating axonal networks throughout development into their correct destinations. We have previously shown that semaphorins are mediators of neuronal apoptosis and can induce neuronal death in the absence of any other apoptotic trigger. We report here that exposure of neuronal cells to a small conserved peptide derived from Sema3A initiates an apoptotic death process. Administration of this peptide to cultured chick sympathetic and mouse cerebellar granule neurons caused a marked shrinkage of their axonal network and cell death, which was characterized as apoptotic, based on nuclear staining. Attenuation of neuronal cell death was obtained by treatment with antioxidants and by vascular endothelial growth factor. Survival of neurons exposed to this peptide increased by co-treatment with caspase inhibitors. Induction of apoptosis was specific to neuronal cells, similarly to that induced by the full-length Sema3A protein. Our findings therefore suggest active participation of this conserved Sema3A-derived peptide in semaphorin-induced neuronal death process.

Semaphorins as mediators of neuronal apoptosis.

Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up-regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine-induced apoptosis. A synchronized induction of mRNA of the secreted collapsin-1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin-derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin-1, a putative component of the semaphorin III/collapsin-1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III-alkaline phosphatase secreted from 293EBNA cells. Anti-collapsin-1 antibodies were effective in blocking the semaphorin III-induced death process. We therefore suggest that, before their death, apoptosis-destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a "death signal" across specific and susceptible neuronal populations.

Exposure of phosphatidylcholine and phosphatidylinositol in plasma membranes from rat brain synaptosomes treated with phospholipase A2 toxins (beta-bungarotoxin, notexin) and enzymes (Naja nigricollis, Naja naja atra).

Phospholipase A2 (PLA2) toxins act presynaptically to block acetylcholine release and are much more potent and specific in their actions than PLA2 enzymes even though they have lower enzymatic activity. Since their mechanism of action is not completely understood, it was of interest to examine the toxins' effects on phospholipid asymmetry as changes in asymmetry are associated with changes in membrane functioning. Rat brain synaptosomes were treated with the PLA2 toxins beta-bungarotoxin (beta-BuTx) and notexin and with the PLA2 enzymes Naja nigricollis and Naja naja atra under relatively non-disruptive conditions as judged by leakage of lactate dehydrogenase (LDH) and levels of phospholipid hydrolysis. The exposure of phosphatidylcholine (PC) and phosphatidylinositol (PI) on the synaptosomal surface was investigated by means of a specific PC-exchange protein (PCEP) and a PI-specific phospholipase C (PI-PLC), respectively. Treatment of the synaptosomes with N. nigricollis PLA2, beta-BuTx and notexin did not affect the availability of PC to exchange by PCEP, but significantly increased the exposure of PI to hydrolysis by PI-PLC. In contrast, N. n. atra PLA2 slightly decreased the exposure of PC and did not affect that of PI. The differences between N. n. atra PLA2, on the one hand, and N. nigricollis PLA2, beta-BuTx and notexin, on the other hand, parallel differences in their pharmacological activities. Our earlier studies showed that PLA2 enzymes, and possibly PLA2 toxins, have a pharmacological site separate from the enzymatic site. Since in the present study the effect on PI was abolished by EDTA, the presence of an enzymatic site in addition to the pharmacological site may be required or alternatively divalent cations may be required for the effects on PI asymmetry independent of the inhibition of PLA2 by EDTA.

The asymmetric distribution of phosphatidylcholine in rat brain synaptic plasma membranes.

The distribution of phosphatidylcholine between inner and outer monolayers of rat brain synaptic plasma membrane was investigated by means of a phosphatidylcholine specific exchange protein. About 70% of the total membranal phosphatidylcholine was in the outer leaflet, 33% of which was exposed and readily exchanged in intact synaptosomes while the remainder was exchangeable following osmotic shock. Permeabilization of the synaptic plasma membranes by overnight incubation in buffer or by saponin (< 0.08%) exposed an additional 30% of phosphatidylcholine to exchange, presumably from the inner cytoplasmic leaflet. Phosphatidylcholine is therefore asymmetrically distributed in the synaptosomal plasma membrane, as it is in other plasma membranes.

An EDTA.Ca2+ complex inhibits the enzymatic activity but not the lethality of beta-bungarotoxin.

An EDTA.Ca2+ complex inhibits the phospholipase A2 activity of the presynaptic neurotoxin beta-bungarotoxin without affecting its lethal potency. The EDTA.Ca2+ complex induces a conformational change in the enzymatic active site region of beta-BuTx, as indicated by the suppression of the 340 nm tryptophan fluorescence peak. Modification of the enzymatic site without loss of toxicity supports the presence of separate loci for the two activities.

Inhibitory effect of EDTA.Ca2+ on the hydrolysis of synaptosomal phospholipids by phospholipase A2 toxins and enzymes.

Phospholipases A2 (PLA2) are Ca2(+)-dependent enzymes that are inhibited by EDTA; this inhibition would be expected to be reversed by restoring the Ca2+ concentration. By examining the hydrolysis of synaptosomal phospholipids by PLA2 enzymes, Naja naja atra and Naja nigricollis, and by toxins with PLA2 activity, beta-bungarotoxin (beta-BuTX) and notexin, we demonstrated a novel inhibitory action of EDTA manifested in the presence of excess Ca2+. We postulate the formation of an EDTA.Ca2+ complex which inhibits PLA2 activity in a concentration-dependent manner. Synaptosomes in which phospholipids are hydrolyzed by PLA2 have membranal damage expressed by increased acetylcholine (ACh) release and decreased osmotic activity. Addition of EDTA.Ca2+, which inhibits phospholipid hydrolysis, also reversed the PLA2 effect on ACh release, but not its effect on osmotic activity. The inhibition of PLA2 was observed on membranal phospholipids as well as on an artificial substrate of phospholipid-Triton mixed micelles. Moreover, we found that another enzyme, lactate dehydrogenase, was also inhibited. Our results indicate a non-specific inhibition exerted on the enzyme rather than on the substrate.

Phospholipid hydrolysis and loss of membrane integrity following treatment of rat brain synaptosomes with beta-bungarotoxin, notexin, and Naja naja atra and Naja nigricollis phospholipase A2.

The effects of the phospholipase A2 (PLA2) toxins, beta-bungarotoxin and notexin, and the PLA2 enzymes from Naja naja atra and Naja nigricollis snake venoms on the plasma membrane integrity of synaptosomes were examined. Synaptosomes were isolated from rat brain cerebral cortex, corpus striatum and hippocampus. Osmotic activity, lactate dehydrogenase leakage, and leakage of 2-deoxy-D-(1-3H)-glucose-6-phosphate were monitored (37 degrees C, 10-120 min) following incubation with 0.5, 5 and 50 nM concentrations of toxins and enzymes. Damage to the synaptosomal plasma membrane was time and concentration but not tissue dependent. The potencies of the treatments were as follows: N. n. atra PLA2 greater than or equal to N. nigricollis PLA2 greater than notexin greater than beta-bungarotoxin. Chelation of Ca2+ with 5 mM EDTA completely inhibited plasma membrane disruption caused by beta-bungarotoxin and N. n. atra PLA2. One mg/ml of bovine serum albumin also blocked the disruptive action of N. n. atra PLA2, while 8 mg/ml was required to antagonize beta-bungarotoxin. A correlation between phospholipid hydrolysis and loss of membrane integrity was also observed. The generation of phospholipid hydrolytic products may be critical in the permeabilization of synaptic plasma membranes by these toxins and enzymes, however, they do not explain the presynaptic specificity and potency of beta-bungarotoxin and notexin.

A novel cardiotoxic polypeptide from the venom of Atractaspis engaddensis (burrowing asp): cardiac effects in mice and isolated rat and human heart preparations.

A new cardiotoxic polypeptide isolated from the venom of the snake Atractaspis engaddensis has an LD50 of 15 micrograms/kg body weight in white mice. Intravenous administration in mice of lethal doses of the toxin causes, within seconds, marked changes in the ECG, consisting primarily of a transient slope elevation of the S-T segment, a temporary diminution of the S-wave and an increase in the amplitudes of the R- and T-waves. Concomitantly, and apparently unrelated to these changes, a severe A-V block develops and leads to complete cardiac arrest within a few min. Studies with rat and human isolated heart preparations showed that the toxin exerts a powerful coronary vasoconstriction (rats), and positive inotropic effects (rats and humans).

Interactions of the neurotoxic complex from the venom of the false horned viper (Pseudocerastes fieldi) with rat striatal synaptosomes.

The very high lethal potency of the neurotoxic complex (Cb) from the venom of Pseudocerastes fieldi following direct administration into the lateral ventricle of the brain, as compared with potency following i.v. administration, suggests that the toxin acts on the central nervous system. Rat striatal synaptosomes were selected to study interactions of the toxin with nerve endings. CbII, the toxic phospholipase A2 component of the toxin, as well as the reconstituted complex (CbI + CbII), inhibited the high affinity choline transport into synaptosomes. Fifty per cent inhibition was obtained at 10 nM CbII after 20 min preincubation of the synaptosomes at 37 degrees C. Choline uptake was inhibited under conditions of minimal leakage of lactate dehydrogenase and probably very low phospholipase A2 activity (in the absence of Ca2+ with Sr2+ or with EGTA). The inhibition of choline uptake was irreversible and was evident after a short preincubation at 0 degrees C. CbII also enhanced the release of acetylcholine from synaptosomes preloaded with labelled choline, but this effect was markedly reduced in the presence of the acidic component (CbI) of the complex. Binding of 125I-CbII could be demonstrated with synaptosomes and with erythrocytes, however, the reconstituted complex (CbI + CbII) was bound only by the synaptosomes, though less effectively than free 125I-CbII. An increased specific binding was evident with purified synaptosomes as compared with a crude preparation. These results support the notion that the non-toxic subunit increases the specificity of the toxic phospholipase A2.

[Morphological changes in the cells of Escherichia coli damaged by nitrous yperite]. / Morfologicheskie izmeneniia kletok Escherichia coli, povrezhdennykh azotistym ipritom.

Nitrous yperite supressed division of E. coli cells; as a result, filaments and giant abnormal cells appeared. Apparently, giant forms were caused by defects in the cell wall induced by nitrous ypertie due to a mutation in the mon gene of the strain. These cells gave rise to normal cells as well as to small bacteria resembling mini-cell.