Mutant GABA(A) receptor gamma 2-subunit in childhood absence epilepsy and febrile seizures

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants(1,2). We have found a mutation in a gene encoding a GABA, receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS), There is a recognized genetic: relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Truncation of the GABA(A)-receptor gamma 2 subunit in a family with generalized epilepsy with febrile seizures plus

Recent findings from studies of two families have shown that mutations in the GABA(A)-receptor gamma2 subunit are associated with generalized epilepsies and febrile seizures. Here we describe a family that has generalized epilepsy with febrile seizures plus (GEFS(+)), including an individual with severe myoclonic epilepsy of infancy, in whom a third GABA(A)-receptor gamma2-subunit mutation was found. This mutation lies in the intracellular loop between the third and fourth transmembrane domains of the GABA(A)-receptor gamma2 subunit and introduces a premature stop codon at Q351 in the mature protein. GABA sensitivity in Xenopus laevis oocytes expressing the mutant gamma2(Q351X) subunit is completely abolished, and fluorescent-microscopy studies have shown that receptors containing GFP-labeled gamma2(Q351X) protein are retained in the lumen of the endoplasmic reticulum. This finding reinforces the involvement of GABA(A) receptors in epilepsy.

Altered kinetics and benzodiazepine sensitivity of a GABA(A) receptor subunit mutation [gamma(2)(R43Q)] found in human epilepsy

The gamma-aminobutyric acid type A (GABA(A)) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA(A) receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABA(A) gamma(2)-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABA(A) receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from alpha(1)beta(2)gamma(2) or alpha(1)beta(2)gamma(2)(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imiclazopyricline zolpidem. These results provide evidence of impaired GABA(A) receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6') common to both the glycine receptor (GlyR) and gamma-aminobutyric acid, type A receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA(A)Rs have divergent open state pore structures at the 6' position. When both the human alpha1(T6'C) homomeric GlyR and the rat alpha1(T6'C)beta1(T6'C) heteromeric GABA(A)R were expressed in human embryonic kidney 293 cells, their 6' residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA(A)R pore structure at the 6' level may vary between different expression systems.

Differential expression of the GABA transporters GAT-1 and GAT-3 in brains of rats, cats, monkeys and humans

The homeostasis of GABA is critical to normal brain function. Extracellular levels of GABA are regulated mainly by plasmalemmal gamma-aminobutyric acid (GABA) transporters. Whereas the expression of GABA transporters has been extensively studied in rodents, validation of this data in other species, including humans, has been limited. As this information is crucial for our understanding of therapeutic options in human diseases such as epilepsy, we have compared, by immunocytochemistry, the distributions of the GABA transporters GAT-1 and GAT-3 in rats, cats, monkeys and humans. We demonstrate subtle differences between the results reported in the literature and our results, such as the predominance of GAT-1 labelling in neurons rather than astrocytes in the rat cortex. We note that the optimal localisation of GAT-1 in cats, monkeys and humans requires the use of an antibody against the human sequence carboxyl terminal region of GAT-1 rather than against the slightly different rat sequence. We demonstrate that GAT-3 is localised mainly to astrocytes in hindbrain and midbrain regions of rat brains. However, in species such as cats, monkeys and humans, additional strong immunolabelling of oligodendrocytes has also been observed. We suggest that differences in GAT distribution, especially the expression of GAT-3 by oligodendrocytes in humans, must be accommodated in extrapolating rodent models of GABA homeostasis to humans.

Developmental changes in expression of GABA(A) receptor-channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was similar to 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

The crosslinking mechanism in gamma irradiation of polyarylsulfone: Evidence for Y-links

Measurements of molecular weights, soluble fractions and examination of NMR spectra of bisphenol-A polysulfone, after gamma irradiation in vacuum at 150 degrees C were used to elucidate the mechanism of crosslinking. Excellent agreement was obtained between G(S) and G(X) determined from measurements above and below the gel dose when a Y-linking mechanism was assumed, whereas poor agreement was obtained when an H-link mechanism was assumed, which is the mechanism normally believed to be responsible for crosslinking. New resonances were observed in the C-13 NMX spectra of the irradiated polymer which were consistent with the formation of Y-links involving phenylene units in the backbone chain. (C) 1998 John Wiley & Sons, Ltd.

An ESR and NMR study of the gamma radiolysis of a bisphenol-A based polycarbonate and a phthalic acid ester

A study of the gamma-radiolysis of the commercial polymers U-polymer, UP (Unitake) and polycarbonate, PC, (Aldrich) has been undertaken using ESR spectroscopy. The G-value of radical formation at 77 K has been found to be 0.31 +/- 0.01 for UP and 0.5 +/- 0.02 for PC. By using thermal annealing and spectral subtraction, the paramagnetic species formed on irradiation has been assigned. The effect of radiation on the chemical structure of UP and PC has been investigated at ambient temperature and at 423 K. The NMR results show that a new phenol type chain end is formed in the polymers on exposure to gamma-radiation. The G-value of formation of the new phenol ends was estimated to be 0.7 for PC (423 K) and 0.4 for UP (300 K). (C) 1998 John Wiley & Sons, Ltd.

Volatile products and new polymer structures formed on Co-60 gamma-radiolysis of poly(lactic acid) and poly(glycolic acid)

A gas product analysis has been conducted on gamma-irradiated samples of poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) by means of gas chromatography. The major volatile products have been identified to be CO, CO2, CH4 and C2H6 for PLA, and CO and CO2 for PGA. In addition, the yield of evolved gases for PLA has been found to be 1.81 for CO2, 0.98 for CO, 0.026 for CH4 and 0.012 for C2H6; and that for PGA to be 1.70 for CO2 and 0.42 for CO. The new chain ends formed due to gamma-induced bond cleavage in PLA have been assigned to CH3-CH2-CO-O- and CH3-CH2-O-CO-, and the G values for formation of these chain ends were found to be 1.9 and 0.6, respectively. The G value for chain scission reported previously of 2.3 is comparable with that for the formation of the propanoic acid end group. (C) 1997 Elsevier Science Limited.

Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: Prevalence of latent STAT1 in high-molecular weight complexes

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA, The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell Lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immune-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor ct-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) nonactivated STAT 1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

Thermogravimetric analysis of carbon deposition over Ni/gamma-Al2O3 catalysts in carbon dioxide reforming of methane

Carbon formation on Ni/gamma-Al2O3 catalysts and its kinetics during methane reforming with carbon dioxide was studied in the temperature range of 500-700 degrees C using a thermogravimetric analysis technique. The activation energies of methane cracking, carbon gasification in CO2, as well as carbon deposition in CO2-CH4 reforming were obtained. The results show that the activation energy for carbon gasification is larger than that of carbon formation in methane cracking and that the activation energy of coking in CO2-CH4 reforming is also larger than that of methane decomposition to carbon. The dependencies of coking rate on partial pressures of CH4 and CO2 indicate that methane decomposition is the main route for carbon deposition. A mechanism and kinetic model for carbon deposition is proposed.

Effect of temperature on the gamma-radiolysis of poly(tetrafluoroethylene-co-perfluoromethyl vinyl ether)

The effect of irradiation temperature on the polymer properties was investigated for the fluoroelastomer poly(tetrafluoroethylene-co-perfluoromethylvinyl ether) (TFE/PMVE). TFE/PMVE samples were gamma-irradiated to 150 kGy at temperatures ranging from 77 K to 373 K. Analysis of the sol/gel behaviour, tensile properties, and glass transition temperatures indicated that crosslinking commenced in the temperature range 195 to 263 K, for a dose of 150 kGy. The latter temperature was 13 K below the glass transition temperature. Crosslinking remained relatively constant to higher temperatures. Chain scission reactions were found to occur well below the glass transition temperature and increased at higher temperatures. The optimum temperature for the radiation crosslinking of TFE/PMVE, for the temperatures investigated, was 263 K. (C) 1998 Elsevier Science Ltd. All rights reserved.

The use of crosslinking promoters in the gamma-radiolysis of poly(tetrafluoroethylene-co-perfluoromethylvinyl ether). I

Incorporation of 1 wt % of triallyl isocyanurate (TAIC) significantly enhanced the radiation crosslinking of the perfluoroelastomer, poly(tetrafluoroethylene-co-perfluoromethylvinyl ether) (TFE/PMVE). The dose for gelation was lowered by 70% with the presence of TAIC. The additive also improved the tensile properties of TFE/ PMVE both before and after crosslinking by irradiation. Higher radical yields were obtained with the presence of TAIC at 77 K, indicating the crosslinking promoter was acting as a radical trap. ESR studies showed that radiolysis of TAIC and subsequent photobleaching cleaved an allyl branch from the ring structure. Upon thermal annealing, an allyl radical on the TAIC molecule was observed. (C) 1999 John Wiley & Sons, Inc.

A comprehensive study on carbon dioxide reforming of methane over Ni/gamma-Al2O3 catalysts

Carbon dioxide reforming of methane into syngas over Ni/gamma-Al2O3 catalysts was systematically studied. Effects of reaction parameters on catalytic activity and carbon deposition over Ni/gamma-Al2O3 catalysts were investigated. It is found that reduced NiA1204, metal nickel, and active species of carbon deposited were the active sites for this reaction. Carbon deposition on Ni/gamma Al2O3 varied depending on the nickel loading and reaction temperature and is the major cause of catalyst deactivation. Higher nickel loading produced more coke on the catalysts, resulting in rapid deactivation and plugging of the reactor. At 5 wt % Ni/gamma-Al2O3 catalyst exhibited high activity and much lesser magnitude of deactivation in 140 h. Characterization of carbon deposits on the catalyst surface revealed that there are two kinds of carbon species (oxidized and -C-C-) formed during the reaction and they showed different reactivities toward hydrogenation and oxidation. Kinetic studies showed that the activation energy for CO production in this reaction amounted to 80 kJ/mol and the rate of CO production could be described by a Langmuir-Hinshelwood model.

A comparative study of the effects of UV- and gamma-radiation on copolymers of acrylonitrile/butadiene

The radiolysis of nitrile rubbers with different acrylonitrile/butadiene composition and the homopolymers, poly(butadiene) (PBD) and poly(acrylonitrile) (PAN) has been investigated and compared with the photolysis of the same polymers. A significantly different mechanism of degradation was found for the two types of radiation. The results obtained by ESR, FTIR and measurements of soluble fractions of irradiated samples, indicated that the acrylonitrile units of the nitrile rubbers are more sensitive units to gamma-radiation, with the effects of irradiation increasing with the acrylonitrile content. The reactions observed were consumption of double bonds, crosslinking, and cyclization with the formation of conjugated double bonds. No chain-scission reactions were detected. In contrast to gamma-irradiation, the effects of photolysis were centred at the butadiene units, and increases in the acrylonitrile content resulted in a proportional decrease in the sensitivity of the copolymers. Crosslinking and chain scission were identified as the main effects of photolysis of NBR rubbers. (C) 1999 Society of Chemical Industry.