Temperature-Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase-Insensitive Toxin-Receptor Complex

Abstract: Energy-dependent internalization of ¹²⁵I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside G_D1b (1–5 μg/ml) also lowers the amount of cell-associated toxin. The fraction of ¹²⁵I-labeled toxin retained by the cells after exposure to high-salt medium at 0–4°C or after addition of G_D1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.     

Application of the ‘profiles of relationship’ method to distantly related proteins

Summary An analysis is made of the applicability of the recently published profiles of relationship method for establishing evolutionary relatedness among proteins by using the distantly related proinsulin and neurotoxin protein se-quences as a test object. The method is based on a simultaneous group analysis of both the frequency of acceptance of mutations and their genetic code inter-changeability. Regularities in the patterns of the profiles, which reflect decreased similarity with the passage of time, are established for typical cases of closely related, distantly related and unrelated proteins. This makes it possible to distinguish distantly related from unrelated proteins without extensive statistical randomization procedures. New evidence is stated in favour of a previously suggested definition of interchangeability which does not consider the third base in the codon. The applicability of the profiles of relationship method is examined on the distant relationship between proinsulin and the snake and scorpion neurotoxins which has been established previously by means of conventional approaches.     

Monoaminergic neurons of the mudpuppy retina

Summary The mudpuppy retina was investigated with the histofluorescence method of Falck and Hillarp in normal animals and in animals injected intraocularly with -methylnoradrenaline, 5,6-dihydroxytryptamine, or a combination of the two drugs. Catecholaminergic amacrine cells were found to form a thin layer of terminals at the border between the inner nuclear and the inner plexiform layers. Catecholaminergic interplexiform cells were not found. Indoleamine-accumulating amacrine cells were also observed. They are fifteen to twenty times more numerous than the catecholaminergic cells, and their terminals occur diffusely throughout the inner plexiform layer. In a number of eyes the majority of the indoleamine-accumulating terminals were eliminated with intraocular injections of the neurotoxin, 5,7-dihydroxytryptamine, but the reproducibility of this effect was not consistent. Intravitreal injections of 5,6-dihydroxytryptamine were used to label both types of neurons for electron microscopy. They were found to make conventional type synapses on amacrine cells and, less frequently, on bipolar cells.     

Measurement of mitochondrial respiration in permeabilized murine neuroblastoma (N-2alpha) cells, a simple and rapid in situ assay to investigate mitochondrial toxins

Most mitochondria-based methods used to investigate toxins require the use of relatively large amounts of material and hence compromised sensitivity in assay. We adopted procedures from methods initially developed to diagnose mitochondrial encephalomyopathies and unified these into a single assay. Eukaryotic cell membranes are selectively permeabilized with digitonin to render a system in which mitochondrial respiration can be measured rapidly and with considerable sensitivity. Mitochondria remain intact, uninjured, and in their natural environment where mitochondrial respiration can be measured in situ under physiologically relevant conditions. This approach furthermore allows measurement of toxin effects on individual mitochondrial complexes. Numerous compounds at varying concentrations can be screened for mitochondrial toxicity, while the site of mitochondrial inhibition can be determined simultaneously. We used this assay to investigate, in murine neuroblastoma (N-2alpha) cells, the mitochondrial inhibitory properties of the parkinsonian-inducing proneurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its neurotoxic monoamine oxidase-B (MAO-B)-generated metabolite, the 1-methyl-4-phenylpyridinium species (MPP(+)). Within the time frame of each measurement (15 min), MPTP (< or = 1 mM) did not interfere with in situ mitochondrial respiration. As expected, MPP(+) was found to be a potent Complex I inhibitor but surprisingly also found to inhibit Complex IV. Optimized conditions for performing this assay are provided.     

Dopamine-related and caspase-independent apoptosis in dopaminergic neurons induced by overexpression of human wild type or mutant alpha-synuclein

Human wild type (WT) and mutant alpha-synuclein (alpha-syn) genes were overexpressed using a Tet-on expression system in stably transfected dopaminergic MN9D cells. Their overexpression induced caspase-independent and dopamine-related apoptosis not rescued by general caspase inhibitor Z-VAD-FMK. While apoptosis due to overexpression of WT alpha-syn was completely abrogated by a specific tyrosine hydroxylase (TH) inhibitor, alpha-methyl-p-tyrosine (alpha-MT), the inhibitor only partially rescued apoptosis caused by overexpression of alpha-syn mutants. In addition, overexpression of mutants enhanced the toxicity of 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxyldopamine (6-OHDA) to MN9D cells, whereas overexpression of WT protected MN9D cells against MPP+ toxicity, but not against 6-OHDA. We conclude that WT alpha-syn is beneficial to dopaminergic neurons but its overexpression in the presence of endogenous dopamine makes it a potential threat to the cells. In contrast, mutant alpha-syn not only caused the loss of WT protective function but also the gain-of-toxicity which becomes more serious in the presence of dopamine and neurotoxins.     

Effects of the Paratemnus elongatus pseudoscorpion venom in the uptake and binding of the L-glutamate and GABA from rat cerebral cortex

L-Glu is the most important and widespread excitatory neurotransmitter of the vertebrates. Four types of receptors for L-glu have been described. This neurotransmitter modulates several neuronal processes, and its dysfunction causes chronic and acute diseases. L-Glu action is terminated by five distinct transporters. Antagonists for these receptors and modulators of these transporters have anticonvulsant and neuroprotective potentials, as observed with the acylpoliamines and peptides isolated from spiders, solitary and social wasp venoms. On the other hand, the major inhibitory neurotransmitter in mammalian nervous tissue is the GABA. Drugs that enhance GABA neurotransmission comprise effective approaches to protecting the brain against neuronal injury. Is this study, we demonstrate for the first time the inhibition of the [3H]L-glu binding to its specific sites in synaptosomal membranes from rat cerebral cortex, produced by 0.027 U of Paratemnus elongatus venom (EC50). The venom of P. elongatus changes Km and Vmax into the high affinity uptake of the L-glu and decreases Km and Vmax into the parameters of the GABA uptake from rat synaptosomes. This leads us to speculate on the possible presence of selective and specific compounds in this venom that act in L-glu and GABA dynamics, and therefore, that can serve as tools and new drug models for understanding these neurotransmissions.     

Pharmacological and Electrophysiological Characterization of Lithium Ion Flux Through the Action Potential Sodium Channel in Neuroblastoma × Glioma Hybrid Cells

Interaction of Li+ with the voltage-dependent Na+ channel has been analyzed in neuroblastoma X glioma hybrid cells. The cells were able to generate action potentials in media containing Li+ instead of Na+. The uptake of Li+ into the hybrid cells was investigated for the pharmacological analysis of Li+ permeation through voltage-dependent Na+ channels. Veratridine and aconitine increased the uptake of Li+ to the same degree (EC50 30 microM). This increase was blocked by tetrodotoxin (IC50 20 nM). Veratridine and aconitine did not act synergistically; however, the veratridine-stimulated influx was further enhanced by the toxin of the scorpion Leiurus quinquestriatus (EC50 0.06 micrograms/ml). This stimulation was also blocked by tetrodotoxin. Thus, the voltage-dependent Na+ channel of the hybrid cells accepts both Li+ and Na+ in a similar manner.     

Wasp uses venom cocktail to manipulate the behavior of its cockroach prey

The sting of the parasitoid wasp Ampulex compressa is unusual, as it induces a transient paralysis of the front legs followed by grooming behavior and then by a long-term hypokinesia of its cockroach prey. Because the wasp's goal is to provide a living meal for its newborn larva, the behavioral changes in the prey are brought about by manipulating the host behavior in a way beneficial to the wasp and its offspring. To this end, the wasp injects its venom cocktail with two consecutive stings directly into the host's central nervous system. The first sting in the thorax causes a transient front leg paralysis lasting a few minutes. This paralysis is due to the presence of a venom component that induces a postsynaptic block of central cholinergic synaptic transmission. Following the head sting, dopamine identified in the venom appears to induce 30 min of intense grooming. During the long-term hypokinesia that follows the grooming, specific behaviors of the prey are inhibited while others are unaffected. We propose that the venom represses the activity of head ganglia neurons thereby removing the descending excitatory drive to the thoracic neurons.Abbreviations CNS central nervous system - DA dopamine - GI giant interneuron - PSP postsynaptic potential - SEG sub-esophageal ganglion - TI thoracic interneuron     

Biologically Active Polypeptides and Hydrolytic Enzymes in Sea Anemones of Northern Temperate Waters

The content of biologically active polypeptides in aqueous and ethanol extracts of seven sea anemone species collected near Sakhalin Island (Sea of Okhotsk) and in Posyet Bay (Sea of Japan) was analyzed. Water extracts of the sea anemone Cribrinopsis similis showed the highest hemolytic activity, while ethanol extracts proved to have toxic properties. The levels of toxic and hemolytic activity of extracts of sea anemones inhabiting northern temperate waters were 2 to 3 orders of magnitude lower, compared to tropic species. The reason for this is likely to be the differences in the habitat conditions and biological traits of these animals. The water extracts of all species possessed proteolytic, phospholipase A2, and low DNAase activities, except Actinostola sp., whose aqueous extract contained a high activity alkaline DNAase. The species studied contained a wide range of proteinase inhibitors, O-glycosyl hydrolases (glycosidases and polysaccharide hydrolases). Water extracts of C. similis and Stomphia coccinea possessed the highest laminarinase activity. High activity of N-galactopyranosidase was found in water extracts of S. coccinea and Oulactis orientalis.