Mechanisms of epileptogenic effects of some convulsants on the neuronal activity in the neocortex

The data obtained in the studies of neurophysiological aspects of epileptogenesis in the brain cortex, which have been carried out in our laboratory for many years, are used for the analysis of epileptogenic effects of a few convulsants (penicillin, strychnine, and d-tubocurarine) on the activity of neocortical neurons. It has been demonstrated that the development of the epileptiform activity in the cortex is accompanied by suppression of IPSP, and the above convulsants directly influence the mechanisms of postsynaptic inhibition. Epileptogenic effects of strychine and penicillin are based on blocking of chloride ion channels and depend on the direction of chloride currents. The role of excitatory and inhibitory synaptic interactions among neurons in generation of the epileptiform activity is discussed.     

Conformational properties of Pro-Pro sequences. I. Crystal structures of two dipeptides with L-Pro-L-Pro and L-Pro-D-Pro sequences

The crystal structures of Bu^tCO-L -Pro-L -Pro-NHMe, H₂O (1: monoclinic; P2₁; a = 6.662, b = 11.067, c = 12.205 Å; β = 96.28°) and Bu^tCO-L -Pro-D -Pro-NHMe (2: monoclinic; P2₁; a = 10.770, b = 15.039, c = 11.325 Å; β = 110.00°) have been solved by x-ray diffraction. Structure 1 accommodates an open disposition with intermolecular interactions involving the water molecule, while 2 is βII-folded by an intramolecular i + 3 → i hydrogen bond. In both derivatives, small thermal parameters are indicative of fairly fixed conformations for the proline rings. Comparison between conformations of either isolated or adjacent L -Pro residues in the crystal structures of unstrained oligopeptides shows that the conformational properties of L -Pro-L -Pro sequences are probably a simple combination of those found for isolated L -Pro residues.     

Mathemathical model of Ca2+ diffusion and buffering in extracellular solution after Ca2+ extrusion from a spherical cell

A mathematical model evaluating the changes in the concentration of free extracellular calcium ([Ca²⁺]) as a function of time and radial distance from a spherical cell following uniform calcium release across its external membrane was developed. The space of extracellular solution was represented as concentric shells of uniform thickness. Two simplified approaches were used, which allowed us to describe calcium diffusion and buffering in each extracellular shell by only a single differential equation. The possibility of simulating the spatio-temporal [Ca²⁺] distribution in the case of uniformly located calcium pumps was demonstrated. Developed for pancreatic acinar cells, our model can be also used for studying ion release from other cells under similar experimental conditions and for reconstruction of ion fluxes in the presence of different fluorescent dyes.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 243–249, November–December, 1996.     

Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli

 The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2⁴ factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 g l^-1 to 1.9 g l^-1 (dry weight) and from 10 units ml^-1 to 210 units ml^-1 within 24 h at 30°C in shake flasks. Received: 26 July 1995/Received revision: 22 January 1996/Accepted: 29 January 1996     

Synthesis and conformational analysis of AzAsx-containing oligopeptides

Summary For the sake of improving synthetic methods and evaluating the conformational perturbation induced by the substitution of AzAsx for the Asx residue in the cognate dipeptide R-CO-Asx-Pro-NHR, we prepared the AzAsx-dipeptide sequence by making use of the crystalline triphosgene. This reagent allows, in situ and under very mild experimental conditions, both the carbonylation and the activation of the properly substituted and N-protected hydrazine before coupling with the proline partner. With regard to conformational behaviour, the azadipeptide sequence displays a -fold, unlike the cognate dipeptide which adopts an Asx-turn.     

Expression of bak in S. pombe results in a lethality mediated through interaction with the calnexin homologue Cnx1

Expression studies in the yeast S. pombe have been utilised to establish the basis for a genetic analysis designed to identify the lethal partners of the pro-apoptotic proteins bak and bax. Bak expression in S. pombe is lethal and this lethality is rescued by co-expression of bcl-2 or bcl-x(L). S. pombe cells expressing bak have a terminal phenotype in which the majority of cells are blocked in the G1 phase of the cell cycle while the remainder of cells, unable to complete M-phase, mis-coordinate the timing of subsequent events in the cell cycle. Although bax expression in S. pombe gives rise to a slow growth phenotype, not a lethality, bax expressing cells display the same cell cycle phenotypes described for bak. Electron microscopy of cells expressing bak reveals a dramatic accumulation of large vesicular structures. A two-hybrid screen designed to identify S. pombe proteins which interact with bak, isolated the S. pombe calnexin homologue cnx1. Genetic analysis demonstrates that the Cnx1 domain which binds to bak in two-hybrid experiments, is necessary for bak lethality in S. pombe. This report identifies a lethal interacting partner for bak and the observations suggest a model for bak mediated lethality which can be tested in higher cells.     

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.