Traffic of botulinum toxins A and E in excitatory and inhibitory neurons

Botulinum neurotoxins (BoNTs), proteases specific for the SNARE proteins, are used to study the molecular machinery supporting exocytosis and are used to treat human diseases characterized by cholinergic hyperactivity. The recent extension of the use of BoNTs to central nervous system (CNS) pathologies prompted the study of their traffic in central neurons. We used fluorescent BoNT/A and BoNT/E to study the penetration, the translocation and the catalytic action of these toxins in excitatory and inhibitory neurons. We show that BoNT/A and BoNT/E, besides preferentially inhibiting synaptic vesicle recycling at glutamatergic relative to Gamma-aminobutyric acid (GABA)-ergic neurons, are more efficient in impairing the release of excitatory than inhibitory neurotransmitter from brain synaptosomes. This differential effect does not result from a defective penetration of the toxin in line with the presence of the BoNT/A receptor, synaptic vesicle protein 2 (SV2), in both types of neurons. Interestingly, exogenous expression of SNAP-25 in GABAergic neurons confers sensitivity to BoNT/A. These results indicate that the expression of the toxin substrate, and not the toxin penetration, most likely accounts for the distinct effects of the two neurotoxins at the two types of terminals and support the use of BoNTs for the therapy of CNS diseases caused by the altered activity of selected neuronal populations.     

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron-conditioned astrocytes. K+ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 micromol/L) increased endogenous glutamate or [3H]D-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca2+ channel opening; they were instead abolished by 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K+-induced Ca2+ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.     

Time- and Region-Specific Variations in Somatostatin Release Following Amygdala Kindling in the Rat

Abstract: Somatostatin biosynthesis is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon translates into enhanced release of the peptide and whether it is involved in kindling maintenance. A marked increase in somatostatin-like immunoreactivity (somatostatin-LI) was observed in hilar interneurons of the hippocampus and in their presumed projections to the outer molecular layer 1 week, but not 1 month, after the last kindled seizure. No overt changes were observed in the striatum or in the cortex. Compared with sham-stimulated controls, (a) in the hippocampus, high-K⁺-evoked somatostatin-LI release was unchanged in synaptosomes taken from rats killed 7 days after the last kindled seizure but was bilaterally reduced after 30 days; (b) in the striatum, it was increased (mainly ipsilaterally to stimulation) 7, but not 30, days after the last seizure; and (c) in the cortex, somatostatin-LI release was bilaterally increased in synaptosomes taken from kindled rats 30, but not 7, days after the last seizure. This study shows that distinct changes occur in synaptosomal somatostatin-LI release after kindling acquisition, depending on the brain area analyzed and on the time elapsed from the last generalized seizure.     

Molecular data exclude current hybridization between iguanas <Emphasis Type="Italic">Conolophus marthae</Emphasis> and <Emphasis Type="Italic">C. subcristatus</Emphasis> on Wolf Volcano (Galápagos Islands)

Natural hybridization may influence population fitness and responsiveness to natural selection, in particular in oceanic island systems. In previous studies, interspecific hybridization was detected between the Galápagos iguana species Amblyrhynchus cristatus and Conolophus subcristatus. Further, possible hybridization was also suggested to occur between C. subcristatus and C. marthae at Wolf Volcano on Isabela Island. In this work, we investigated the level of hybridization between C. subcristatus and C. marthae using a large set of microsatellite markers. Results indicated strong differentiation between species and, while we cannot rule out hybridization in the past, there is no evidence of ongoing hybridization between C. marthae and C. subcristatus. These findings have great importance for the design of management actions and conservation plans, in particular for the purposes of a head start program. However, because potential for hybridization may change under different environmental and demographic conditions, genetic characterization of newly marked individuals of C. marthae and C. subcristatus in Wolf Volcano should not be interrupted.     

Chemical probing of a virginiamycin M-promoted conformational change of the peptidyl-transferase domain.

Previous findings suggest the location of the central loop of domain V of 23S rRNA within the peptidyltransferase domain of ribosomes. This enzymatic activity is inhibited by some antibiotics, including type A (virginiamycin M or VM) and type B (virginiamycin S or VS) synergimycins, antibiotics endowed with a synergistic action in vivo. In the present work, the ability of VM and VS to modify the accessibility of 23S rRNA bases within ribosomes to chemical reagents has been explored. VM afforded a protection of rRNA bases A2037, A2042, G2049 and C2050. Moreover, when ribosomes were incubated with the two virginiamycin components, the base A2062, which was protected by VS alone, became accessible to dimethyl sulphate (DMS). Modified reactivity to chemical reagents of different rRNA bases located either in the central loop of domain V or in its proximity furnishes experimental evidence for conformational ribosome alterations induced by VM binding.     

Inhibition of polypeptide synthesis in cell-free systems by virginiamycin S and erythromycin. Evidence for a common mode of action of type B synergimycins and 14-membered macrolides

Macrolides, lincosamides and type B synergimycins are powerful inhibitors of protein synthesis in vivo, but many of them were found to be inactive in vitro. In the present work, we confirm that virginiamycin S (a type B synergimycin) and erythromycin (a 14-membered macrolide) have no effect on poly(U)-directed poly(Phe) synthesis. However, the amino-acid polymerization reactions directed by poly(U,G), poly(U,C), poly(A,G) and poly(A,C) were increasingly inhibited (20-50%) by both antibiotics. The action of these inhibitors proved to be template-dependent and favored by the incorporation of proline and of basic amino acids into peptides. Under these conditions, virginiamycin S and erythromycin markedly stimulated a release of peptidyl-tRNA from the ribosomes. In the poly(A,C) model system, these antibiotics produced a 50% inhibition of amino-acid incorporation into total peptides, a 70% release of ribosome-bound peptidyl-tRNA, and a 95% repression of the synthesis of long peptide chains. The production of equivalent effects at saturating concentrations of these antibiotics in the four model systems examined is suggestive of a similarity in their mode of action. Our results indicate that 14-membered macrolides and type B synergimycins can act on ribosomes during the whole elongation process. The functional block produced by both antibiotics is usually reversible, but may result in a premature release of peptidyl-tRNA when the stability of ribosomal complexes is lowered by the incorporation of basic amino acids.     

Effects of re‐oligotrophication and climate change on lake thermal structure

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Mature Erythrocytes of Iguana iguana (Squamata,Iguanidae) Possess Functional Mitochondria

Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψ_m)-sensitive probe MitoTracker Red indicated that mitochondria i) widely occur in erythrocytes, ii) are polarized, and iii) seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation.     

Ligand-based virtual screening and ADME-tox guided approach to identify triazolo-quinoxalines as folate cycle inhibitors

In the process of drug discovery the lead-identification phase may be critical due to the likely poor safety profile of the candidates, causing the delay or even the abandonment of a certain project. Nowadays, combining molecular modeling and in vivo cellular evaluation can help to identify compounds with an enhanced safety profile. Previously, two quinoxalines have been identified as inhibitors of the folate-dependent proteins belonging to the thymidylate synthase cycle. Unfortunately, cytotoxic activity against a panel of cisplatin(cDDP)-sensitive ovarian carcinoma cell lines and their resistant counterparts was coupled with toxicity to non-tumorigenic Vero cells. Here we describe the application of a ligand-based virtual screening, and several [1,2,4]triazolo[4,3-a]quinoxalines were optimized to improve their ADME-tox profile. The resulting 4-(trifluoromethyl)-1-p-tolyl-[1,2,4]triazolo[4,3-a]quinoxaline (24), which interferes intracellularly with DHFR and TS reducing the protein levels like 5-FU, but without inducing TS ternary complex formation, was 2-times less toxic in vitro than cisplatin and 5-FU.     

Mechanisms of glutamate release elicited in rat cerebrocortical nerve endings by 'pathologically' elevated extraterminal K+ concentrations

Extracellular [K+] can increase during some pathological conditions, resulting into excessive glutamate release through multiple mechanisms. We here investigate the overflow of [3H]D-aspartate ([3H] D-ASP) and of endogenous glutamate elicited by increasing [K+] from purified rat cerebrocortical synaptosomes. Depolarization with [K+] 15 mmol/L were prevented by the glutamate transporter inhibitors DL-threo-beta-benzyloxyaspartate (DL-TBOA) and dihydrokainate. Differently, the overflows of endogenous glutamate provoked by [K+] > 15 mmol/L were insensitive to both inhibitors; the external Ca2+-independent glutamate overflow caused by 50 mmol/L KCl was prevented by bafilomycin, by chelating intraterminal Ca2+, by blocking the mitochondrial Na+/Ca2+ exchanger and, for a small portion, by blocking anion channels. In contrast to purified synaptosomes, the 50 mmol/L K+-evoked release of endogenous glutamate or [3H]D-ASP was inhibited by DL-TBOA in crude synaptosomes; moreover, it was external Ca2+-insensitive and blocked by DL-TBOA in purified gliosomes, suggesting that carrier-mediated release of endogenous glutamate provoked by excessive [K+] in CNS tissues largely originates from glia.