Inhibition of Brain Glutamate Decarboxylase Activity Is Related to Febrile Seizures in Rat Pups

Because previous work showed that in the newborn brain, but not in the adult brain, glutamate decarboxylase (GAD) is notably susceptible to heat, we have studied the possible involvement of GAD inhibition in febrile convulsions and the related changes in gamma-aminobutyric acid (GABA) content. Rats of different ages were subjected to hyperthermia, and GAD activity was determined in brain homogenates by measuring the release of 14CO2 from labeled glutamate and by measuring the formation of GABA. The latter method gave considerably lower values than the former in the youngest rats, and was considered more reliable. With this method, we found a 37-48% inhibition of GAD activity in rat pups 2-5 days old, which showed febrile seizures at progressively higher body temperatures, whereas in 10- and 15-day-old animals, which did not show convulsions, GAD activity was not affected by hyperthermia. Whole-brain GABA levels, however, did not change at any age. In contrast to GAD, choline acetyltransferase and lactic dehydrogenase activities were not altered by hyperthermia at any of the ages studied. These results suggest that a decreased efficiency of the inhibitory neurotransmission mediated by GABA, consequent to the inhibition of GAD activity, may be a factor related to febrile convulsions.     

INFLORESCENCE SIZE AND FRUIT DISTRIBUTION AMONG INDIVIDUALS IN THREE ORCHID SPECIES

Fruit distribution among individuals in populations of three orchid species was analyzed. Fruit set was low in the three species. The distribution of fruits in the non-autogamous species was skewed to the right, with high variance: mean ratios, and a moderate to high proportion of fruitless plants. Larger inflorescence size did not necessarily result in an increase in percent fruit set but tended to increase the probability to produce at least one fruit. Pollinator-mediated selection on inflorescence size through fruit production may be limited by the low overall level of visitation and the resulting uncertainty of pollination at the individual level.     

Selective Stimulation of Neurotransmitter Release from Chick Retina by Kainic and Glutamic Acids

The excitatory action of kainic and glutamic acids in chick whole retina was demonstrated as an immediate stimulation of the release of labeled gamma-aminobutyric acid (GABA) and glycine in a superfusion system. This stimulatory effect was 3-10 times greater than that produced by a depolarizing K+ concentration; in addition, it was independent of Ca2+ in the medium, but notably inhibited when Na+ was omitted from the medium. Under identical experimental conditions, neither kainic nor glutamic acid had any effect on the release of labeled dopamine or alpha-aminoisobutyric acid, thus indicating that their effect is not unspecific or due to cell damage. Similar although less marked stimulation of labeled GABA and glycine release by kainic acid was obtained in subcellular retinal fractions, particularly in fraction P1, which contained photoreceptor terminals and outer segments. This stimulation was also Ca2+ independent and greatly reduced when Na+ was omitted from the medium. It is suggested that the stimulation of GABA release by kainic and glutamic acids is probably due to a Na+-dependent, carrier-mediated mechanism that responds to the entry of Na+ produced by the interaction of glutamic and kainic acids with retinal membranes. In cortical or striatal slices from mouse brain, these acids had a negligible stimulatory effect on GABA and dopamine release.     

Outward sodium and potassium cotransport in human red cells

Summary This paper reports some kinetic properties of Na–K cotransport in human red cells. All fluxes were measured in the presence of 10^–4 M ouabain. We measured Na and K efflux from cells loaded by the PCMBS method to contain different concentrations of these ions into a medium that contained neither Na nor K (MgCl₂-sucrose substitution) in the absence and presence of furosemide. Furosemide inhibited 30–60% of the total efflux depending on the internal ion concentration and the individual subject. We took the furosemide-sensitive fluxes to be a measure of Na–K cotransport. The ratio of Na to K cotransport was 1 over the entire range of internal Na and K concentrations studied. When Na was substituted for K as the only internal cation, cotransport was maximally activated when the Na and K concentrations were between 20 and 90 mmol/liter cells. The concentration of internal Na required to produce half-maximal cotransport was about 13±4 mmol/liter cells (n=4), while the comparable concentration of K was somewhat lower. The activation curve was definitely sigmoid in character, suggesting that at least two Na ions are involved in the transport process. The maximum of Na–K cotransport was about 0.5±0.15 mmol/liter cells × hr (n=5); it had a flat maximum in the medium at about pH 7.0, decreasing in both the acid and alkaline sides. furosemide-resistant effluxes were found to be linear functions of internal Na and K concentrations and to yield rate coefficients of 0.019±0.002 hr^–1 and 0.014±0.002 hr^–1 (n=7), respectively. These values are of the same order of magnitude expected of ions moving across phospholipid bilayers.Charge de Recherches CNRS.     

Differences in some properties of newborn and adult brain glutamate decarboxylase

Some properties of glutamate decarboxylase (EC 4.1.1.15) activity in brain of newborn and adult mouse were studied comparatively. It was found that glutamate decarboxylase of the newborn brain was strongly inactivated by homogenization in hypotonic medium, centrifugation of isotonic sucrose homogenates, preincubation at 37°C or the addition of Triton-X-100, whereas the adult brain enzyme was practically unaffected by any of these conditions. It was also found that the newborn glutamate decarboxylase was less activated by pyridoxal 5′-phosphate and less inhibited by pyridoxal 5′-phosphate oxime-O-acetic acid, than the adult enzyme. These differences do not exist for brain dihydroxyphenylalanine decarboxylase (EC 4.1.1.26) and are not due to the release of inhibitors from the newborn brain. On the basis of the results obtained it is postulated that two forms of glutamate decarboxylase exist in brain: a newborn form, which is unstable and has high affinity for pyridoxal 5′-phosphate, and an adult form, which is much more stable and has low affinity for pyridoxal 5′-phosphate. The possible implications of these findings in the establishment of the σ-aminobutyric acid dependent synaptic inhibitory mechanisms during development are discussed.     

Molecular cloning and functional expression of a novelNeurospora crassa xylose reductase inSaccharomyces cerevisiae in the development of a xylose fermenting strain

The development of a xylose-fermentingSaccharomyces cerevisiae yeast would be of great benefit to the bioethanol industry. The conversion of xylose to ethanol involves a cascade of enzymatic reactions and processes. Xylose (aldose) reductases catalyse the conversion of xylose to xylitol. The aim of this study was to clone, characterise and express a cDNA copy of a novel aldose reductase (NCAR-X) from the filamentous fungusNeurospora crassa inS. cerevisiae. NCAR-X harbours an open reading frame (ORF) of 900 nucleotides. This ORF encodes a protein (NCAR-X, assigned NCBI protein accession ID: XP_956921) consisting of 300 amino acids, with a predicted molecular weight of 34 kDa. TheNCAR-X-encoded aldose reductase showed significant homology to the xylose reductases ofCandida tenuis andPichia stipitis. WhenNCAR-X was expressed under the control of phosphoglycerate kinase I gene (PGK1) regulatory sequences inS. cerevisiae, its expression resulted in the production of biologically active xylose reductase. Small-scale oxygen-limited xylose fermentation with theNCAR-X containingS. cerevisiae strains resulted in the production of less xylitol and at least 15% more ethanol than the strains transformed with theP. stipitis xylose reductase gene (PsXYL1). TheNCAR-X-encoded enzyme produced byS. cerevisiae was NADPH-dependent and no activity was observed in the presence of NADH. The co-expression of theNCAR-X andPsXYL1 gene constructs inS. cerevisiae constituted an important part of an extensive research program aimed at the development of xylolytic yeast strains capable of producing ethanol from plant biomass.     

Structural and ecophysiological adaptations to forest gaps

To survive new microclimatic conditions of a forest gap environment, plant species must physiologically and structurally adjust. A morpho-anatomical, ultrastructural and ecophysiological study was performed at three different times in a forest gap that was created by illegal selective logging. The study followed the early successional Actinostemon verticillatus and the late-successional Metrodorea brevifolia, to elucidate the adaptive strategies of acclimation to gaps. Additionally, Schinus terebinthifolius was included in the study in order to test the plasticity of a pioneer species that grows on forest edges, where this species had higher values of leaf thickness, leaf mass area and succulence. M. brevifolia had succulent leaves, high leaf area and a thin cuticle. A. verticillatus presented the densest leaves and was the only species to show leaf morpho-anatomical plasticity. Ultrastructural and physiological differences were observed only in A. verticillatus and M. brevifolia leaves from the gap: increase in the stroma volume, oil droplets, plastoglobuli, photochemical and non-photochemical quenching. Photosynthetic efficiency showed that the early stages of gap formation are the most critical. Acclimation strategies of A. verticillatus suggest this species invests in the efficiency of photosynthesis by increasing its leaf thickness, leaf mass area and in water content maintenance by increasing the density of its leaves, at the expense of gas exchange, was compensated by a high density of stomata. M. brevifolia compensates for the higher cost of leaves and lower leaf plasticity with ultrastructural changes that are used to adjust the photosynthetic process, which promotes a shorter leaf payback time.     

Long-term treatment with fluoxetine induces desensitization of 5-HT4 receptor-dependent signalling and functionality in rat brain

The mode of action of antidepressant drugs may be related to mechanisms of monoamines receptor adaptation, including serotonin 5-HT₄ receptor subtypes. Here we investigated the effects of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine for 21 days (5 and 10 mg/kg, p.o., once daily) on the sensitivity of 5-HT₄ receptors by using receptor autoradiography, adenylate cyclase assays and extracellular recording techniques in rat brain. Fluoxetine treatment decreased the density of 5-HT₄ receptor binding in the CA1 field of hippocampus as well as in several areas of the striatum over the doses of 5–10 mg/kg. In a similar way, we found a significant lower response to zacopride-stimulated adenylate cyclase activity in the fluoxetine 10 mg/kg/day treated group. Furthermore, post-synaptic 5-HT₄ receptor activity in hippocampus-measured as the excitatory action of zacopride in the pyramidal cells of CA1 evoked by Schaffer collateral stimulation was attenuated in rats treated with both doses of fluoxetine. Taken together, these results support the concept that a net decrease in the signalization pathway of 5-HT₄ receptors occurs after chronic selective serotonin reuptake inhibitor treatment: this effect may underlie the therapeutic efficacy of these drugs.     

Affinity labeling of residues within Hv2 of guinea pig anti-azobenzenearsonate antibodies of different isotypes and from different strains

Anti-p-azobenzenearsonate (ARS) antibodies of IgG1 and IgG2 isotypes produced in inbred strain 13 and strain 2 guinea pigs were affinity labeled with N-(bromoacetyl)-3-[(p-arsonophenyl)azo]-L-tyrosine (BAAT) or N-(bromoacetyl)-p-arsanilic acid (BAA). BAAT was shown to modify approximately 50% of the binding sites specifically and BAA approximately 30%. Both reagents preferentially modified residues in the heavy (H) chain to the extent that it contained over 80% of the affinity label associated with the native molecule. At least 80% of label borne by the variable domain of the H chain (VH) was found in the second hypervariable region (Hv2). BAAT labeled all anti-ARS antibodies exclusively at position N-59, which contains a lysyl residue. BAA labeled predominantly tyrosine at N-57 and, to a lesser extent, lysine-59 and tyrosine-50. Comparison of Hv2 sequences in anti-ARS and in antibodies reactive with other haptens has shown that tyrosine at N-50 and N-57 as well as lysine at N-59 is distinctive of antibodies with anti-ARS specificity, thus implying their involvement in antigen binding. The predominant sequence of Hv2 was identical in anti-ARS IgG1 and IgG2 molecules induced in either inbred guinea pig strain following either carrier priming or conventional immunization. Although limited variability does occur among the various populations of anti-ARS antibodies in certain residue positions in Hv2, no significant differences in the binding affinities or in the indexes of heterogeneity were seen among the various kinds of anti-ARS antibodies.