Purification, characterization and antifungal properties of a lipid-transfer protein from sunflower (Helianthus annuus) seeds

An antifungal protein from Helianthus annuus L. seeds (Ha-AP10) has been purified to homogeneity and characterized. Ha-AP10 purification was performed by gel filtration, cation exchange chromatography and reverse phase HPLC. Its molecular mass was estimated to be 10 kDa and western blot analyses suggest that it has an extracellular location. The N-terminal sequence of Ha-AP10 showed strong homology to some plant lipid-transfer proteins (LTPs). Antifungal tests have demonstrated that Ha-AP10 exerts a fungistatic effect. It completely inhibits the germination of spores of the fungal pathogen Fusarium solani f. sp. eumartii at a concentration of 40 μg ml⁻¹ and produces a 50% growth inhibition at 6.5 μg ml⁻¹ (0.65 μ M ). These data place Ha-AP10 among the most potent antifungal LTPs described so far.     

A potent antifungal protein from Helianthus annuus flowersis a trypsin inhibitor

A 16-kDa protein was isolated from Helianthus annuus flowers by its ability to inhibit the germination of fungal spores. This protein, SAP16, displays an associated activity of trypsin inhibitor and was further purified to apparent homogeneity by affinity chromatography on trypsin-agarose. SAP16 causes the complete inhibition of Sclerotinia sclerotiorum ascospores germination at a concentration of 5 μg·mL^–1 (0.31 μM) and a clear reduction of mycelial growth at lower concentrations, indicating a strong antifungal potency against this natural pathogen of sunflower. Our data suggest that the antifungal ability of SAP16 would not be the result of the inhibition of a fungal protease. This study contributes to the characterization of the emerging family of antifungal proteins with an associated activity of trypsin inhibition and emphasizes their role in plant resistance against fungal attack.     

Glutamate Activates Phospholipase D in Hippocampal Slices of Newborn and Adult Rats

Abstract: Phospholipase D (PLD) is activated by many neuro-transmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD) time- and concentrationdependently enhanced the formation of [³H]phosphatidylpropanol ([³H]PP) and of [³H]phosphatidic acid in the presence of 2% propanol. N -MethylD-aspartate and kainate (both 1 m M ) caused small, but significant increases (∼50%). whereas α-amino-3-hydroxy-5-methylisoxazole-4-propionate (100 μ M ) was ineffective. Maximally effective concentrations of glutamate (1 m M ) and of 1 S ,3 R -ACPD (300 μ M ) increased the PLD activity to almost 300% of basal activity; the EC₅₀ values were 199 and 47 μ M , respectively. Glutamate receptor antagonists, such as DL-2-amino-3-phosphonopropionic acid (AP3). DL-2-aminc-5-phosphonovalenic acid, and kynurenate (all 1 m M ) did not inhibit the glutamate-evoked increase of PP formation. In slices of adult rats, the response to 1 S ,3 R -ACPD was significant, but small, whereas glutamate was effective only in the presence of the glutamate uptake inhibitor L-aspartate-β-hydroxarnate. It is concluded that glutamate activates PLD in rat hippocampus through an AP3-resistant metabotropic receptor. This effect is subject to ontogenetic development, with one important factor being glutamate uptake.     

Susceptibility of <Emphasis Type="Italic">Candida</Emphasis> biofilms to histatin 5 and fluconazole

Candida-associated denture stomatitis has a high rate of recurrence. Candida biofilms formed on denture acrylic are more resistant to antifungals than planktonic yeasts. Histatins, a family of basic peptides secreted by the major salivary glands in humans, especially histatin 5, possess significant antifungal properties. We examined antifungal activities of histatin 5 against planktonic or biofilm Candida albicans and Candida glabrata. Candida biofilms were developed on poly(methyl methacrylate) discs and treated with histatin 5 (0.01–100 μM) or fluconazole (1–200 μM). The metabolic activity of the biofilms was measured by the XTT reduction assay. The fungicidal activity of histatin 5 against planktonic Candida was tested by microdilution plate assay. Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50% reduction in the metabolic activity; biofilm) of 4.6 ± 2.2, 6.9 ± 3.7 and 1.7 ± 1.5 μM, and IC₅₀ (planktonic cells) of 3.0 ± 0.5, 2.6 ± 0.1 and 4.8 ± 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were less susceptible to histatin 5, with 50% RMA of 31.2 ± 4.8 and 62.5 ± 0.7 μM, respectively. Planktonic C. glabrata was insensitive to histatin 5 (IC₅₀ > 100 μM). Biofilm-associated Candida was highly resistant to fluconazole in the range 1–200 μM; e.g. at 100 μM only ~20% inhibition was observed for C. albicans, and ~30% inhibition for C. glabrata. These results indicate that histatin 5 exhibits antifungal activity against biofilms of C. albicans and C. glabrata developed on denture acrylic. C. glabrata is significantly less sensitive to histatin 5 than C. albicans.     

Cyclosporine A from a nonpathogenic Fusarium oxysporum suppressing Sclerotinia sclerotiorum

AIMS: To evaluate the antagonistic activity of Fusarium oxysporum nonpathogenic fungal strain S6 against the phytopathogenic fungus Sclerotinia sclerotiorum and to identify the antifungal compounds involved. METHODS AND RESULTS: The antagonistic activity of Fusarium oxysporum strain S6 was determined in vitro by dual cultures. The metabolite responsible for the activity was isolated by chromatographic techniques, purified and identified by spectroscopic methods as cyclosporine A. The antifungal activity against the pathogen was correlated with the presence of this metabolite by a dilution assay and then quantified. Cyclosporine A caused both growth inhibition and suppression of sclerotia formation. In a greenhouse assay, a significant increase in the number of surviving soybean (Glycine max) plants was observed when S. sclerotiorum and F. oxysporum (S6) were inoculated together when compared with plants inoculated with S. sclerotiorum alone. CONCLUSION: Fusarium oxysporum (S6) may be a good fungal biological control agent for S. sclerotiorum and cyclosporine A is the responsible metabolite involved in its antagonistic activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyclosporine A has not been previously described as an inhibitor of S. sclerotiorum. Its minimum inhibitory concentration (MIC) of 0.1 microg disc(-1) makes it suitable to use as a biofungicide. In vivo experiments showed that F. oxysporum (S6) is a good candidate for the biocontrol of S. sclerotiorum in soybean.     

Inhibition of Methanosarcina barkeri strain 227 by cadmium

Abstract The effect of cadmium (Cd) on methane formation from methanol and/or H₂–CO₂ by Methanosarcina barkeri was examined in a defined growth medium and in a simplified buffer system containing 50 mM Tes with or without 2 mM dithiothreitol (DTT). No inhibition of methanogenesis by high concentrations of cadmium was observed in growth medium. Similarly, little inhibition of methanogenesis by whole cells in the Tes buffer system was observed in the presence of 430 μM Cd or 370 μM mercury (Hg) with 2 mM DTT. When the concentration of DTT was reduced to 0.4 mM, almost complete inhibition of methanogenesis from H₂–CO₂ and methanol by 600 μM Cd was observed. In the absence of DTT, 150 μM Cd inhibited methanogenesis from H₂–CO₂ completely and from methanol by 97%. Methanogenesis from H₂–CO₂ was more sensitive to Cd than that from methanol.     

Production of macrosphelide A by the mycoparasite Coniothyrium minitans

Coniothyrium minitans, a mycoparasite of sclerotia of Sclerotinia sclerotiorum and Sclerotium cepivorum, produced four closely related metabolites inhibitory to fungal growth. The major metabolite, identified as macrosphelide A, had IG(50) values (the concentration of metabolite to inhibit growth by 50%) of 46.6 and 2.9 microgram ml(-1) against S. sclerotiorum and S. cepivorum, respectively. This is the first report of both antifungal activity due to macrosphelide A as well as isolation of macrosphelide A from C. minitans.     

A Comparison of the Effects of Ethanol and the Competitive Glycine Antagonist 7-Chlorokynurenic Acid on N-Methyl-d-Aspartic Acid-Induced Neurotransmitter Release from Rat Hippocampal Slices

Abstract: N -Methyl- d -aspartate (NMDA; 500 μ M ) stimulated the net release of preloaded tritiated norepinephrine from rat hippocampal slices. Both ethanol and the competitive glycine antagonist 7-chlorokynurenic acid (7-CK) dose-dependently inhibited NMDA-stimulated release without affecting basal, nonstimulated efflux. These inhibitory effects were readily reversed upon washout of the drugs. Over the concentration range tested (25–200 m M ), ethanol inhibited ∼65% of NMDA-stimulated release with an estimated IC₅₀ of ∼70 m M . In contrast, 7-CK fully inhibited release (>95%) at a concentration of 30 μ M with half-maximal inhibition occurring at ∼2 μ M . The combination of 7-CK (1–30 μ M ) and ethanol (25–100 m M ) had an additive inhibitory effect on NMDA-stimulated release but did not alter the inhibitory potency of 7-CK. Calculated IC₅₀values for 7-CK in the presence of 25, 50, or 100 m M ethanol were (mean × SEM; μ M ) 2.33 (0.11), 2.38 (0.23), and 1.99 (0.30), respectively. 7-CK (3 μ M ) inhibited NMDA-stimulated [³H]norepinephrine release by ∼50%. This inhibition was fully attenuated by the addition of the glycine agonistserine with complete reversal occurring at 30 μ M d -serine. Increasing the 7-CK concentration to 10 μ M shifted the d -serine dose-effect curve to the right in a parallel fashion as expected for a competitive antagonist. In contrast, the inhibitory effects of ethanol or the combination of 7-CK (3 μ M ) and ethanol (25 or 50 m M ) were not reversed by the addition of d -serine (0.1–1,000 μ M ). Together, these results suggest that ethanol's inhibition of NMDA-stimulated [³H]norepinephrine release from hippocampal slices is not due to a simple competitive interaction with the glycine site on the NMDA receptor.     

(R,S)-α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid (AMPA) Receptors Mediate a Calcium-Dependent Inhibition of the Metabotropic Glutamate Receptor-Stimulated Formation of Inositol 1,4,5-Trisphosphate

Abstract: l -Glutamate (3-1,000 μ M ) and (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid (1S.3R-ACPD; 10-1,000 μ M ), a selective agonist for the metabotropic glutamate receptor, stimulated the formation of inositol 1,4,5-trisphosphate in a concentration-dependent manner. l -Glutamate was half as efficacious as 1S,3R-ACPD. N -methyl- d -aspartate (nMDA; 1 n M to 1 m M ) did not significantly influence the response to a maximally effective concentration of 1S,3R-ACPD (100 μ M ). On the other hand, coapplication of (R,S)-α-amino-3-hydroxy-5-methylisoxa-zole-4-propionic acid (AMPA; 1-300 n M ) produced a concentration- and time-dependent inhibition of the 1S,3R-ACPD effect, with a maximal inhibition (97%) at 100 n M . Ten micromolar 6-cyano-7-nitroquinoxaline-2,3-dione. an antagonist of the AMPA receptor, blocked the inhibitory effect of AMPA. Reduced extracellular calcium concentration, as well as 10 μ M nimodipine, an l -type calcium channel antagonist, inhibited the AMPA influence on the 1S,3R-ACPD response. W-7, a calcium/calmodulin antagonist, prevented the inhibition by AMPA. whereas H-7. an inhibitor of protein kinase C, had no effect. These data suggest that activation of AMPA receptors has an inhibitory influence on inositol 1,4,5-trisphosphate formation mediated by stimulation of the metabotropic glutamate receptor. The mechanism of action involves calcium influx through l -type calcium channels and possible activation of calcium/calmodulin-dependent enzymes.     

Biochemical characterisation of ketoconazole inhibitory action on Aspergillus fumigatus

Abstract The effect of ketoconazole on growth, sterol composition, in vitro sterol biosynthesis and P450-CO complex formation and its interaction with microsomal P450 was determined. On solid medium and in liquid medium ketoconazole inhibited Aspergillus fumigatus growth completely at 5 × 10⁻⁵ M and 50% of the growth at 1.3 × 10⁻⁵ M and 2.1 × 10⁻⁵ M respectively. A close relationship between accumulation of 14α-methyl sterols (eburicol, obtusifoliol and 14α-methyl fecosterol) and depletion of ergosterol with growth arrest was observed in ketoconazole treated cultures. The half inhibitory concentration for in vitro ergosterol biosynthesis and half saturating concentration for type II binding spectrum of ketoconazole were calculated as 73.8 ± 6.3 nM and 0.13 ± 0.04 μM respectively. CO displacement studies revealed inhibition of CO-P450 complex formation by ketoconazole.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Antifungal effect of gaseous nitric oxide on mycelium growth, sporulation and spore germination of the postharvest horticulture pathogens, Aspergillus niger, Monilinia fructicola and Penicillium italicum

Aim:  To evaluate the antifungal activity of nitric oxide (NO) against the growth of the postharvest horticulture pathogens Aspergillus niger , Monilinia fructicola and Penicillium italicum under in vitro conditions.
Methods and Results:  Different volumes of NO gas were injected into the Petri dish headspace to obtain the desired concentrations of 50–500  μ l l⁻¹ . The growth of the fungi was measured for 8 days of incubation in air at 25°C . All concentrations of NO were found to produce an antifungal effect on spore germination, sporulation and mycelial growth of the three fungi, with the most effective concentration for A. niger and P. italicum being 100 and 500  μ l l⁻¹ for M. fructicola .
Conclusions:  Short-term exposure to a low concentration of NO gas was able to inhibit the subsequent growth of A. niger , M. fructicola and P. italicum .
Significance and Impact of the Study:  NO gas has potential use as a natural fungicide to inhibit microbial growth on postharvest fruit and vegetables.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

Cell culture density dependent toxicity and chromatin changes upon cadmium treatment in murine pre-B-cells

Murine pre-B-cells grown in the presence of lower (1 μM) or higher (5 μM) concentration of cadmium chloride were separated into 13 fractions by centrifugal elutriation. The rate of DNA synthesis after cadmium treatment determined in permeable cells was dependent on cell culture density during cadmium treatment. Cell cycle analysis revealed a shift in the profile of DNA synthesis from replicative to repair DNA synthesis upon cadmium treatment. The study of the relationship between cell culture density and cell diameter at lower and higher cell densities in the presence of 1 μM cadmium chloride concentration showed that a. at 5×10⁵ cell/ml or lower densities cells were shrinking indicating apoptotic changes, b. at higher cell culture densities the average cell size increased, c. the treatment of cells with low CdCl₂ concentration (1 μM) at higher cell culture density (>5×10⁵ cell/ml) did not change significantly the average cell diameter. At 5 μM cadmium concentration and higher cell culture densities (>5×10⁵ cell/ml) the average cell size decreased in each elutriated fraction. Most significant inhibition of cell growth took place in early S phase (2.0–2.5 C value). Apoptotic chromatin changes in chromatin structure after cadmium treatment were seen as large extensive disruptions, holes in the nuclear membrane and stickiness of incompletely folded chromosomes.     

An antifungal peptide from Phaseolus vulgaris cv. brown kidney bean

A 5.4-kDa antifungal peptide, with an N-terminal sequence highly homologous to defensins and inhibitory activity against Mycosphaerella arachidicola (IC(50)= 3 μM), Setospaeria turcica and Bipolaris maydis, was isolated from the seeds of Phaseolus vulgaris cv. brown kidney bean. The peptide was purified by employing a protocol that entailed adsorption on Affi-gel blue gel and Mono S and finally gel filtration on Superdex 75. The antifungal activity of the peptide against M. arachidicola was stable in the pH range 3-12 and in the temperature range 0°C to 80°C. There was a slight reduction of the antifungal activity at pH 2 and 13, and the activity was indiscernible at pH 0, 1, and 14. The activity at 90°C and 100°C was slightly diminished. Deposition of Congo red at the hyphal tips of M. arachidicola was induced by the peptide indicating inhibition of hyphal growth. The lack of antiproliferative activity of brown kidney bean antifungal peptide toward tumor cells, in contrast to the presence of such activity of other antifungal peptides, indicates that different domains are responsible for the antifungal and antiproliferative activities.     

Influence of copper on proton efflux from Saccharomyces cerevisiae and the protective effect of calcium and magnesium

Abstract The inhibitory effect of Cu on glucose-dependent H⁺ efflux from Saccharomyces cerevisiae was manifest at low (micromolar) concentrations, with the time period between the addition of glucose and commencement of H⁺ efflux, H⁺ efflux rate and duration all being affected with increasing Cu concentration (5–100 μM). Ca, at a concentration of 0.5 mM, completely removed the inhibitory effect of Cu at concentrations up to 50 μM and considerably reduced it at higher concentrations (up to 150 μM). Mg exhibited a similar but weaker protective effect against the influence of Cu. The protective effect of Ca against 50 μM Cu was evident at low Ca concentrations (2.5–5 μM), whereas Mg was effective at ≥50 μ M. In order to prevent the inhibitory effect of Cu, it was necessary to add Ca or Mg to the cell suspension before Cu addition. It is concluded that the protective effect of Ca and Mg is mediated by competitive and stabilizing interactions at the cell surface as well as physiological functions of Ca and Mg.     

The mechanism of cobalt toxicity in mung beans

The effects of cobalt on the growth and nutrient balance of mung beans were investigated. Inhibition of seedling growth occurred at 5 μ M Co and was associated with chlorosis of the younger leaves. Analysis of nutrient concentrations in root and leaf tissue of mung beans treated with 5 μ M Co showed that none of the macronutrients and only two of the micronutrients, Mn and Fe, were significantly affected. The Mn concentration in roots was reduced by 55% and the Fe concentration in the leaves by 80%. Uptake of Fe into roots was not inhibited by Co but transport of Fe to the shoot was greatly reduced. It was shown that the effect of Co on growth was additive to that of Fe deficiency, which argues against Co-induced Fe deficiency as the primary cause of growth inhibition by Co. Rather, it was considered that the high concentrations of Co in the roots and leaves compared with essential micronutrient cations can disrupt a range of metabolic processes due to competitive interactions. Comparison of the toxic effects of Co with those of other toxic trace metals Cd, Cu, Ni and Hg showed that at an applied concentration of 5 μ M , there were obvious differences in both the visual symptoms and in nutrient concentrations. The main difference between Co and the other metals was that only Co stimulated the uptake of S into the plant and its transport to the shoots, where the S concentration in the leaves was increased 2-fold. The common feature of all the trace metals examined was the strong inhibition of Fe transport to the shoot. A possible mechanism for the interaction of other trace metals with Fe transport is discussed.     

A comparison of the chitin synthase-inhibitory and antifungal efficacy of nucleoside-peptide antibiotics: structure-activity relationships

Abstract Using Mucor rouxii , the chitin synthase (ChS)-inhibitory and antifungal activity was determined of 6 nucleoside-peptide antibiotics (NPAs) representing pairs of structural analogues, each consisting of a dipeptide (DP) and a corresponding tripeptide (TP). These were the nikkomycins X and I (X, I), the nikkomycins Z and J (Z, J), and the polyoxins D and A (D, A). Although all were very good ChS-inhibitors (X and A being best, with K _i approx. 0.5 μM), only X and Z elicited a strong response in vivo as determined by the degree of inhibition exerted on N -acetylglucosamine (GlcNAc) incorporation into the chitin fraction, the survival rate, and the minimum inhibitory concentration (MIC). The MIC values were about 2 μM (for X and Z) and 100 μM (for I, J, D and A). Certain DPs and TPs reduced the antifungal activity of X, the effect being much more pronounced with DPs. It is suggested that uptake of NPAs involves the transpeptidase reaction of the γ-glutamyl cycle, the observed antagonism thus resulting from competition for a common carrier.     

A comparison between 3,5-diiodo-4-hydroxybenzoic acid and 2,3,5-triiodobenzoic acid. I. Effects on growth and gravireaction of maize roots

A comparison between the effects of DIHB and TIBA on growth and gravireaction of 15 mm primary maize ( Zea mays L. cv. LG 11) roots is presented. Intact roots were pretreated in the dark for 1 h with buffered solutions (pH 5.0 or 6.0) containing DIHB (10, 50, 100 μ M ). The plantlets were then maintained either vertically or horizontally in the dark or the light, and growth and gravireaction were recorded using a macrophotographic technique. Pretreatment with DIHB slightly inhibited growth and delayed gravireaction. These effects were most marked with DIHB at 100 μ M and were enhanced when DIHB was applied at pH 5.0. Similar effects were observed in roots pretreated with TIBA, but at a lower concentration (1 μ M ). The similarities between DIHB and TIBA as regards both chemical structure and the inhibition of gravireaction and growth, lead us to suggest that a major mode of action of DIHB, like TIBA, is the inhibition of indol-3yl-acetic acid transport.     

Identification and Function of Glycine Receptors in Cultured Cerebellar Granule Cells

Abstract: Poly(A)⁺ mRNA was isolated from cultured mouse cerebellar granule cells and injected into Xenopus oocytes. This led to the expression of receptors that evoked large membrane currents in response to glycine. Current-responses were also obtained after application of β-alanine and taurine, but these were very low relative to that of glycine (maximal β-alanine and taurine responses were 8 and 3% of that of glycine, respectively). The role of glycine receptors on K⁺-evoked transmitter release in cultured cerebellar granule cells was also assayed. Release of preloaded d -[³H]aspartate evoked by 40 m M K⁺ was dose dependently inhibited by glycine, and the concentration producing half-maximal inhibition was 50 μ M. Taurine, β-alanine, and the specific GABA_A receptor agonist isoguvacine also inhibited K⁺-evoked release, and the maximal inhibition was similar for all agonists (˜40%). The EC₅₀ value was 200 μ M for taurine, 70 μ M for β-alanine, and 4 μ M for isoguvacine. Bicuculline (150 μ M ) antagonized the inhibitory effect of isoguvacine (150 μ M ) but not that of glycine (1 m M ). In contrast, strychnine (20 μ M ) antagonized the inhibitory effect of glycine (1 m M ) but not that of isoguvacine (150 μ M ). The pharmacology of the responses to β-alanine and taurine showed that these agonists activate both glycine and GABA_A receptors. The results indicate that cultured cerebellar granule cells translate the gene for the glycine receptor and that activation of glycine receptors produces neuronal inhibition.