Ascorbic acid in pollen: Conversion of L-galactono-1,4-lactone to L-ascorbic acid by Lilium longiflorum

The L-ascorbic acid (AA) content of pollen from three cultivars of Lilium longiflorum Thunb. was 260–280 μg/g fresh wt. of pollen. During germination ascorbic acid content gradually decreased reaching 70% of the original value at 6 h. Pollen germinated in media containing 0.29 M D-glucose (an osmoregulator and carbon source) failed to synthesize ascorbic acid but pollen germinated in 0.29 M pentaerythritol (a non-metabolizable osmoregulator) supplemented with L-galactono-1,4-lactone (L-GalAL) did form ascorbic acid, dependent upon the concentration of the lactone. Lycorine inhibited germination but had negligible effect on the conversion of L-GalAL to AA.     

Ascorbic acid in cultured tissue of briar rose,Rosa rugosa Thunb

Ascorbic acid synthesis was studied using suspension cultured cells from the fleshy portion of the fruit of the rose, Rosa rugosa Thunb. Cells cultured in the dark contained ascorbic acid at a level of 13 mg/100 g wet wt. This value increased several fold when the cells were grown under light. Ascorbic acid does not accumulate in the growth medium possibly due to its rapid oxidation. L-galactono-1,4-lactone was readily converted to ascorbic acid whereas L-gulono-1,4-lactone was used to a much lesser degree. D-glucose, D-fructose, D-galactose. D-glucurono-1,4-lactone and myoinositol did not stimulate increased ascorbic acid synthesis. Reducing the sucrose content of the medium reduced the ascorbic acid content of the cells without altering cell yield.     

L-ascorbic acid metabolism in the ascorbate-deficient arabidopsis mutant vtc1.

The biosynthesis of L-ascorbic acid (vitamin C) is not well understood in plants. The ozone-sensitive Arabidopsis thaliana mutant vitamin c-1 (vtc1; formerly known as soz1) is deficient in ascorbic acid, accumulating approximately 30% of wild-type levels. This deficiency could result from elevated catabolism or decreased biosynthesis. No differences that could account for the deficiency were found in the activities of enzymes that catalyze the oxidation or reduction of ascorbic acid. The absolute rate of ascorbic acid turnover is actually less in vtc1 than in wild type; however, the turnover rate relative to the pool of ascorbic acid is not significantly different. The results from [U-14C]Glc labeling experiments suggest that the deficiency is the result of a biosynthetic defect: less L-[14C]ascorbic acid as a percentage of total soluble 14C accumulates in vtc1 than in wild type. The feeding of two putative biosynthetic intermediates, D-glucosone and L-sorbosone, had no positive effect on ascorbic acid levels in either genotype. The vtc1 defect does not appear to be the result of a deficiency in L-galactono-1,4-lactone dehydrogenase, an enzyme able to convert L-galactono-1,4-lactone to ascorbic acid.     

A Biotechnological Approach for Enhancement of Colchicine Accumulation in Iphigenia indica Kunth

An attempt has been made to enhance colchicine biosynthesis in Iphigenia indica L grown in vitro by exogenous supply of precursors and elicitors during culture. Addition of tyrosine at 30 mg l^?1 enhanced colchicine accumulation up to 6.1 mg g^?1 dry wt (control 0.99 mg g^?1 dry wt), while 25 μM NiSO₄ stimulated maximum colchicine accumulation (10.25 fold of control value). The colchicine accumulation was recorded as 16.25 mg g^?1 dry wt when NiSO₄ was added with tyrosine.     

Production of L-ascorbic acid by metabolically engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii

Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme L-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous D-arabinono-1,4-lactone oxidase as well as L-galactose dehydrogenase are capable of producing about 100 mg of L-ascorbic acid liter(-1), converting 40% (wt/vol) of the starting compound L-galactose.     

Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, L-gulono-1,4-lactone dehydrogenase

The last step of the biosynthesis of L-ascorbic acid (vitamin C) in plants and animals is catalyzed by L-gulono-1,4-lactone oxidoreductases, which use both L-gulono-1,4-lactone and L-galactono-1,4-lactone as substrates. L-gulono-1,4-lactone oxidase is missing in scurvy-prone, vitamin C-deficient animals, such as humans and guinea pigs, which are also highly susceptible to tuberculosis. A blast search using the rat L-gulono-1,4-lactone oxidase sequence revealed the presence of closely related orthologs in a limited number of bacterial species, including several pathogens of human lungs, such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, Burkholderia cepacia and Bacillus anthracis. The genome of M. tuberculosis, the etiologic agent of tuberculosis, encodes a protein (Rv1771) that shows 32% identity with the rat L-gulono-1,4-lactone oxidase protein. The Rv1771 gene was cloned and expressed in Escherichia coli, and the corresponding protein was affinity-purified and characterized. The FAD-binding motif-containing Rv1771 protein is a metalloenzyme that oxidizes L-gulono-1,4-lactone (Km 5.5 mm) but not L-galactono-1,4-lactone. The enzyme has a dehydrogenase activity and can use both cytochrome c (Km 4.7 microm) and phenazine methosulfate as exogenous electron acceptors. Molecular oxygen does not serve as a substrate for the Rv1771 protein. Dehydrogenase activity was measured in cellular extracts of a Mycobacterium bovis BCG strain. In conclusion, M. tuberculosis produces a novel, highly specific L-gulono-1,4-lactone dehydrogenase (Rv1771) and has the capacity to synthesize vitamin C.     

Silencing of tomato mitochondrial uncoupling protein disrupts redox poise and antioxidant enzymes activities balance under oxidative stress

Plant mitochondrial uncoupling proteins (pUCPs) play important roles in generation of metabolic thermogenesis, response to stress situation, and regulation of energy metabolism. Although the signaling pathways for the pUCPs-regulated plant energy metabolism and thermogenesis are well studied, the role of pUCPs in the regulation of plant stress tolerance has not been fully substantiated. Here we showed that mitochondrial uncoupling protein was required for effective antioxidant enzymes activities, chlorophyll fluorescence and redox poise in tomato under oxidative stress using virusinduced gene silencing approach. Silencing of LeUCP gene reduced maximal quantum yield of PSII (Fv/Fm) and photochemical quenching coefficient (qP), as well as mitigated activation of antioxidant enzymes and related genes expression. The content of reduced ascorbate and reduced glutathione, redox ratio of ascorbate and L-galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) activity were all decreased in the leaves of LeUCP gene-silenced plant. However, malondialdehyde content was increased under methylviologen (MV) stress. ROS accumulation was increased significantly following MV and heat stress treatments. Meanwhile, LeUCP gene silencing aggravated accumulation of H₂O₂ and O ₂ ^·? in leaves. Taken together, these results strongly suggest that LeUCP gene plays critical role in maintaining the redox homeostasis and balance in antioxidant enzyme system under oxidative stress.     

Involvement of Epicatechin in Cultivar Susceptibility of Avocado Fruits to Colletotrichum gloeosporioides after Harvest1

Avocado cultivars were defined as susceptible and resistant to Colletotrichum gloeosporioides depending upon the length of the incubation period of the disease after fruit softening. In the susceptible cultivars Fuerte, Horshim, Vurtz, Rincon, and Benik, epicatechin concentration of the peel decreased to 60-130 μg.g^?1, fr. wt. at fruit softening and symptoms appeared on the same or one day later. In the resistant cultivars Hass, Nabal, Netaim and Pinkerton, epicatechin concentration was still 632–1740 μg.g^?1 fr. wt. when fruit softening and symptoms appeared only 4-10 days later. When susceptible Fuerte fruits became soft the concentration of the antifungal compound 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15 diene, had decreased to 120 μg.g^?1 fr. wt. and symptoms appeared. In resistant Hass fruits, the antifungal diene was still 238 μg.g^?1 fr. wt. at fruit softening; and it had further decreased to 159 μg.g^?1 fr. wt. when symptoms appeared, four days later. A modified atmosphere and 0.2 M CaCl₂ infiltration both delayed softening of Fuerte fruits; but symptom appearance on these fruits was related to diene decrease and not to fruit softening. The results are discussed in relation to the hypothesis that the susceptibility of avocado cultivars to post-harvest decay by C. gloeosporioides is related to the degradation of the antifungal diene, catalyzed by avocado lipoxygenase, the activity of which is regulated by the decline of its inhibitor epicatechin.     

Biosynthesis of L-ascorbic acid (vitamin C) by Saccharomyces cerevisiae

Saccharomyces cerevisiae cells incubated with D-glucose (D-Glc), D-galactose or D-mannose (D-Man) synthesised D-erythroascorbic acid (D-EAA) but not L-ascorbic acid (L-AA). Accumulation of D-EAA was observed in cells incubated with D-arabinose (D-Ara) whilst accumulation of L-AA occurred in cells incubated with L-galactose (L-Gal), L-galactono-1,4-lactone and L-gulono-1,4-lactone. When S. cerevisiae cells were incubated with D-[U-(14)C]Glc, D-[U-(14)C]Man or L-[1-(14)C]Gal, incorporation of radioactivity into L-AA was observed only with L-[1-(14)C]Gal. Pre-incubation of yeast cells with D-Ara substantially reduced the incorporation of L-[1-(14)C]Gal into L-AA. Our results indicate that, under appropriate conditions, yeast cells can synthesise L-AA via the pathway naturally used for D-EAA biosynthesis.     

Stimulation of transepithelial sodium and chloride transport by ascorbic acid. Induction of Na+ channels is inhibited by amiloride

Ascorbic acid increases the short circuit current (I_sc) across the amphibian cornea when it is present at either surface of this epithelium. These effects were additive. The effect was greater when it was on the tear side. The response returned to baseline levels when the ascorbic acid was washed from the bathing media. The effect of ascorbic acid on I_sc when it was on the aqueous humor side of the cornea could be blocked by bumetanide but that due to the vitamin's presence on the tear side was unchanged. The ascorbic acid could enter the tissue and crossed the cornea at similar rates in either direction. When the cornea was bathed by a Cl^?-free solution or exposed to bumetanide, the rise in I_sc observed with ascorbic acid on the tear side was equivalent to an increased Na⁺ flux from the tear to the aqueous humor side. In normal (Cl^? present) Conway solution the rise in the I_sc seen with ascorbic acid on the aqueous humor side was equal to an increased flux of Cl^? from the aqueous to the tear surface. However, when ascorbic acid was present on the opposite, tear, side the increased I_sc reflected a rise in both Cl^? and Na⁺ transport, aqueous-to-tear side, and tear-to-aqueous side, respectively. Thiol reagents (tear side), including reduced glutathione (10^?5 M), blocked the effect of ascorbic acid (10^?3 M) providing they were added to the bathing solution prior to the vitamin. However, they had no effect once the response had been established. The effect of the reduced glutathione appeared to be of a non-competitive nature. Oxidized glutatione (10^?4 M) (and cystamine) blocked the effect of ascorbic acid (10^?3 M) when present on the tear side prior to the vitamin. However, they also increased the rate of decline of the response when added subsequently to the ascorbic acid. Amiloride (as low as 5·10^?9 M), on the tear side but not the aqueous humor side, prevented the response to ascorbic acid but could not reverse it, once it was established. The possible nature of the effect of ascorbic acid is discussed in relation to its pharmacological interactions with thiol and disulfide reagents and amiloride.     

Studies on nitrate reductase activity in Laminariadigitata (Huds.) Lamour. I. Longitudinal and transverse profiles of nitrate reductase activity within the thallus

The distribution of the enzyme nitrate reductase (NR) within the thallus of the brown alga Laminaria digitata (Huds.) Lamour is described for plants sampled from the east coast of Scotland in May and June when growth rates are at a maximum. Highest NR activities (≈ 0.2 μmol NO₃^? reduced·g^?1 wet wt·h^?1) occurred in the mature blade. NR activities declined towards the basal meristematic region of the blade. Activities in the stipe and holdfast were also low, being between 0.05 and 0.07 μmol NO₃^? reduced·g^?1 wet wt·h^?1. The activities of the enzyme glutamine synthetase (GS), which is important in the assimilation of NH₄⁺, showed a similar distribution within the blade to those of NR.The transverse profile of NR activity in the stipe exhibited a decline from the outer to the inner tissues. Maximum activities (0.13 μmol NO₃^? reduced·g^?1 wet wt·h^?1) occurred in the meristoderm, while those of the cortex and medulla were 0.04 and 0.01 μmol NO₃^? reduced·g^?1 wet wt·h^?1 respectively.These data indicate that most of the NO₃^? assimilation occurs in the mature blade rather than in the meristematic tissue where there is a high nitrogen demand for growth. The data are consistent with the maintenance of meristematic growth by the internal transport of organic nitrogen from the mature blade.     

Engineering ascorbic acid biosynthetic pathway in Arabidopsis leaves by single and double gene transformation

Six genes, which encode enzymes involved in ascorbic acid (AsA) biosynthesis, including guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP), GDP-mannose-3′,5′-epimerase (GME), GDP-galactose guanylyltransferase (GGT), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH) and L-galactono-1,4-lactone dehydrogenase (GLDH) were transformed into Arabidopsis thaliana, to evaluate the contribution of each gene to AsA accumulation. Additionally, two combinations, GGT-GPP and GGT-GLDH, were co-transformed into Arabidopsis with a reliable double-gene transformation system. AsA content of GGT transgenic lines was 2.9-fold higher as compared to the control, and co-transformation led up to 4.1-fold AsA enhancement. These results provided further evidence that GGT is the key enzyme in plant AsA biosynthesis.     

Immobilization of glucoamylase on DEAE-cellulose activated with chloride compounds

Partially purified glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger NRRL 330 has been immobilized on DEAE-cellulose activated with cyanuric chloride in 0.2 m acetate buffer, pH 4.2. In the matrix-bound glucoamylase, enzyme yield was 20 mg g^?1 of support, corresponding to 40 200 units g^?1 of DEAE support. Binding of the enzyme narrows the pH optimum from 3.8–5.2 to 3.6. Thermal stability of the bound glucoamylase enzyme was decreased although it showed a higher temperature optimum (70°C) than the free form (55°C). The rate of reaction of glucoamylase was also changed after immobilization. V_max values for free and bound enzyme were 36.6 and 22.6 μmol d-glucose ml^?1 min^?1 and corresponding K_m values were 3.73 and 4.8 g l^?1 respectively. Free and immobilized enzyme when used in the saccharification process gave 84 and 56% conversion of starch to d-glucose, respectively. The bound enzyme was quite stable and in the batch process it was able to operate for about five cycles without any loss of activity.     

Contents and morphological distribution of 2,4-dihydroxy-l,4-benzoxazin-3-one and 2-benzoxazolinone in Acanthus mollis in relation to protection from larvae of Pseudaletia impuncta

Secondary metabolites 2,4‐dihydroxy‐l,4‐benzoxazin‐3‐one(DIBOA) and 2‐benzoxazolinone (BOA) were quantified in different morphological parts of A. mollis. The highest DIBOA content was determined in the stamens of the flowers (57.0 umol g^?1 fr. wt). Content of DIBOA in leaves decreased from approximately 28 μmol g^?1 fr. wt for younger leaves (less than 4 wk old), to 3.0 μmol g^?1 fr. wt. for the older ones (more than 13 wk of age). The concentration of BOA was lower than that of DIBOA in all parts of the plant (less than 1.5 μmol g^?1 fr. wt) and showed a small variation. Younger leaves exhibit antifeeding activity against the larvae of Pseudaletia impuncta a native moth that feeds on cereals. These results suggest that DIBOA protects A. mollis species from phytophagous insects.     

Nitric oxide is involved in the regulation of ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stress

This study investigated the regulation of ascorbate and glutathione metabolism by nitric oxide in Agropyron cristatum leaves under water stress. The activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), L-galactono-1,4-lactone dehydrogenase (GalLDH) and γ-glutamylcysteine synthetase (γ-ECS), and the contents of NO, reduced ascorbic acid (AsA), reduced glutathione (GSH), total ascorbate and total glutathione increased under water stress. These increases were suppressed by pretreatments with NO synthesis inhibitors N ^G-nitro-L-arginine methyl ester (L-NAME) and 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). However, application of L-NAME and cPTIO to plants sufficiently supplied with water did not affect the activities of above mentioned enzymes and the contents of NO and above mentioned antioxidants. Pretreatments with L-NAME and cPTIO increased the malondialdehyde (MDA) content and electrolyte leakage of plants under water stress. Our results suggested that water stress-induced NO is a signal that leads to the upregulation of ascorbate and glutathione metabolism and has important role for acquisition of water stress tolerance.     

High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Identification of an Antifungal Compound in Unripe Avocado Fruits and its Possible Involvement in the Quiescent Infections of Colletotrichum gloeosporioides1

A new antifungal compound was isolated from peel and flesh of unripe avocado fruits and identified as 1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene. The maximal concentration of the anti-fungal monoene in unripe fruits was about 800 μg. g^?1 fr.wt. During ripening the monoene decreased to 40 μg. g^?1 fr.wt. concomitantly with the appearance of disease symptoms. The concentration of the previously described antifungal diene, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (Prusky et al. 1982), in avocado peel was 1,600 μg. g^?1 fr.wt. in unripe fruits, decreasing during ripening to 120 μg. g^?1 fr.wt. At 750 μg. ml^?1 the inhibition of germ tube elongation of germinated conidia by the antifungal monoene and the antifungal diene was 15 % and 44 %, respectively. A 1: 1 mixture of both antifungal compounds in concentrations ranging from 50 to 750 μg. ml^?1, showed synergistic activity and increased the percent of inhibited germ tubes of germinated conidia up to 15 % over the sum of activities of the separate compounds. The results are discussed in relation to the hypothesis that the antifungal diene and the antifungal monoene are involved in the quiescence of the germinated appressoria of Colletotrichum gloeosporioides in unripe avocado fruits.     

Feeding elicitors and precursors enhance colchicine accumulation in morphogenic cultures of <Emphasis Type="Italic">Gloriosa superba</Emphasis> L.

Morphogenic cultures of Gloriosa superba were initiated on Murashige and Skoog’s medium fortified with 2 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L^?1 naphthaleneacetic acid (NAA), 4% sucrose and 0.1% activated charcoal. To enhance the content of the alkaloid colchicine, morphogenic cultures were treated with different concentrations of abiotic elicitors like signalling compounds, metals, biotic elicitors, precursors and a combination of elicitors. Signalling molecules like acetyl salicylic acid (ASA) and sodium nitroprusside improved the production of colchicine. Abiotic elicitors have markedly (p?≤?0.05 or ≤?0.01) enhanced the colchicine content either at lower or higher concentrations. Among the metals, the highest amount of 11.67 mg of colchicine g^?1 dry wt was noticed at 60 mM rubidium chloride, followed by 60 mM NaCl (11.18 mg g^?1). Contrarily, in the presence of biotic elicitors such as Fusarium oxysporum, Alternaria solani, and Saccharomyces cerevisiae, colchicine content ranged only between 2 and 5.32 mg g^?1, but Bacillus subtilis repressed it. Among the aromatic amino acids, phenylalanine at 500 mg L^?1 influenced the highest accumulation of 19.48 mg g^?1 dry tissue, followed by tryptophan (12.47 mg g^?1), and tyrosine (9.87 mg g^?1), a direct precursor of colchicine biosynthesis, while intact tubers and leaves contained 4.65 and 4.16 mg of colchicine g^?1 dry tissue respectively. A combination of 10 µM AlCl₃ and 50 µM salicylic acid (SA) registered 17.34 mg g^?1 followed by 16.24 mg g^?1 tissue in presence of 1 µM HgCl₂ and 50 µM SA. The results suggest that the elicitor-stimulated colchicine accumulation was a stress response and can be exploited further for commercial production.     

γ-Aminobutyric acid-stimulated chloride permeability in crayfish muscle

γ-Aminobutyric acid selectively increased Cl^? permeability in isolated strips of crayfish abdominal muscle. Muscle fibers incubated in VAn Harreveld's solution at room temperature took up ³⁶Cl^? to the extent of 700 ml/kg wet weight with a halftime of 2.5 min. During 15-s incubations, the control ³⁶Cl^? uptake space was 131 ± 4 ml/kg (n = 60) and this was significantly increased by γ-aminobutyric acid at 200 μM or higher concentrations to 177 ± 4 ml/kg (n = 48, P < 0.05). This effect was specific for chloride since γ-aminobutyric acid did not increase the uptake by crayfish muscle of radioactive sucrose, inositol, or propionate. γ-Aminobutyric acid stimulation of ³⁶Cl^? uptake is mediated by receptor-ionophore function since the process shows pharmacological properties virtually identical to those observed by electrophysiological techniques. The γ-aminobutyric acid stimulation of Cl^? permeability is dose dependent with 50% of the maximal effect at 40 μM γ-aminobutyric acid and the dose vs. response curve is somewhat sigmoid. The γ-aminobutyric acid agonist muscimol causes the same maximal effect on Cl^? uptake as γ-aminobutyric acid, but acts at 5-fold lower concentrations, i.e. is more potent. However, the partial agonist γ-amino, β-hydroxybutyric acid produced little or no stimulation of ³⁶Cl^? flux. The response to γ-aminobutyric acid was blocked by 2 mM β-guanidinopropionate or γ-guanidinobutyrate, 0.5 mM bicuculline, and 10 μM picrotoxinin. Picrotoxinin inhibition was dose dependent with 50% inhibition occurring at 4 μM. Antagonists did not affect control ³⁶Cl^? uptake. These results confirm electrophysiological observations that the postsynaptic response to the inhibitory neurotransmitter γ-aminobutyric acid involves a rapid increase in membrane permeability to Cl^?