Penicillin production by mutants ofPenicillium chrysogenum resistant to polyene macrolide antibiotics

Summary Polyene-resistant mutants ofPenicillium chrysogenum Wis. 54–1255 have been obtained by stepwise selection in increasing concentrations of polyene antibiotics. From the parent strain, sensitive to 10 g/ml of polyene antibiotics, mutants resistant to fungimycin (1.3 mg/ml), amphotericin B (0. 5 mg/ml), or nystatin (167 g//ml) were obtained. Their penicillin production is different from that of the parent strain and in particular some of the fungimycin-resistant mutants produce higher levels of penicillin.     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

Reversal of cerulenin inhibition ofMycobacterium avium by octanoate

Mycobacterium avium is an opportunistic pathogen that may cause either localized or, in persons with acquired immune deficiency syndrome (AIDS), disseminated disease. Under certain conditionsM. avium exhibits a requirement for fatty acid for maximum production of colony-forming units (CFU). However, cerulenin, a drug that inhibits fatty acid synthetase, inhibited the growth ofM. avium LM1 (serovar 1) with a minimal inhibitory concentration of 10 g/ml regardless of oleic acid supplementation at 50 g/ml. Cerulenin at 10 g/ml also inhibited the growth of five other strains ofM. avium isolated from AIDS patients. Octanoate, oleate, and palmitate, individually or in two-way combinations, were tested for ability to reverse the effect of cerulenin. Octanoate at 50 g/ml could reverse the bactericidal effect of cerulenin at 10 g/ml and the bacteriostatic effect of the drug at 5 g/ml. Because cerulenin when effectively inhibiting production of CFU also prevented cell elongation, the data suggest that octanoate, or a metabolic product, is critical to cell division ofM. avium.     

Ultrastructure of Sporothrix schenckii treated with iodine-potassium iodide solution

The ultrastructural changes produced by iodine-potassium iodide solution on yeast cells of Sporothrix schenckii were investigated by transmission electron microscopy in order to clarify the mechanism of oral potassium iodide therapy for sporotrichosis. Yeast cells were dipped with solutions containing various concentrations of iodine. The rate of germination decreased markedly between the range of iodine concentrations from 0.63 g/ml to 5.0 g/ml. No significant ultrastructural changes were seen at the concentration of the iodine of 1.25 g/ml (80% germination) or less. In the concentration of 2.5 g/ml (50% germination), normal cells and degenerated cells coexisted. When the cells were treated with 5.0 g of iodine per ml (0% germination) or more, their interior structures were completely destroyed. It is assumed that iodine treatment of the organism causes rapid destruction in the whole cell.     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

Evaluation of acetylator phenotype,renal function and serum sulfadiazine levels in patients with paracoccidioidomycosis treated with cotrimazine (a combination of sulfadiazine and trimethoprim)

The authors evaluated the relationships among renal function, acetylator phenotype and serum sulfadiazine levels in 22 patients with paracoccidioidomycosis treated with 1 tablet of cotrimazine (a combination of 820 mg sulfadiazine and 180 mg trimethoprim) administered orally every 12 hours.Fifteen patients (68.18%) presented free sulfadiazine levels above 50 g/ml, 6(27.28%) presented serum levels above 40 g/ml, and 1(4.54%), levels lower than 40 g/ml, this being the patient in which treatment failed. The highest free sulfadiazine levels were obtained in slow acetylator patients with reduced renal function. One patient with neuroparacoccidioidomycosis presented free sulfadiazine levels in cerebrospinal fluid corresponding to 55% of the serum levels.Finally, the authors consider cotrimazine to be an important therapeutic alternative for neuroparacoccidioidomycosis and conclude that administration every 12 hours can provide therapeutic sulfadiazine levels. They also suggest that when the sulfadiazine-trimethoprim combination is used, the therapeutic levels of sulfadiazine should be above 40 g/ml.     

Effects of insecticides on cultures of insect cells

Serial dilutions of 20 insecticides were examined for their effects on the growth of insect cells cultivated in vitro. No differences in susceptibility were found for cells derived from the moth Antheraea eucalypti and the mosquito Aedes aegypti.Rotenone was the most effective inhibitor investigated, decreasing the rate of cell division at 0.001 g/ml. Malathion and diazinon first showed effects at 12 g and 112/ml respectively. Toxicants first effective at 10 g/ml included pp-DDT, dieldrin, pyrethrins and sodium arsenate; at 100 g/ml they included lindane and carbaryl; at 1000 g/ml only nicotine sulphate.The majority of insecticides tested (principal exception rotenone) were very much more toxic to last instar A. aegypti larvae than to the insect cells, suggesting that the functions of highly organized tissues are more readily interfered with than those of individual cell types comprising them.

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Zusammenfassung Verdünnungsserien von 20 Insektiziden wurden auf ihren Effekt auf das Wachstum von in vitro kultivierten Insektenzellen untersucht. Die untersuchten Zellen stammten aus Kulturen von Ovariolen von Antheraea eucalypti-Puppen und von Gewebe von Aedes aegypti-Larven. Rotenon erwies sich als das wirksamste Insektizid: es verlangsamte die Zellteilung in einer Konzentration von 0.001 g/ml. Malathion wurde erst in einer Konzentration von 12 g/ml wirksam, Diazinon bei 112 g/ml. Mehrere Insektizide zeigten erste Wirksamkeit bei 10 g/ml; diese waren: pp-DDT, pp-DDD, pp-DDE, Methoxychlor, Aldrin, Dieldrin, Pyrethrine, Allethrin und Natriumarsenat. Insektizide mit einer ersten Wirksamkeit bei 100 g/ml waren Lindan, Isolan, Dimetilan, Carbaryl, DNOC und Piperonylbutoxid. Nikotinsulfat war erst bei 1 mg/ml oder bei höheren Konzentrationen wirksam. Zwischen den Antheraea- und Aedes-Zellen wurde kein Unterschied in der Empfindlichkeit gegen die verschiedenen Insektizide gefunden. Bei niedrigen Konzentrationen zeigten Malathion und Natriumarsenat erst nach dem 4. Tag bedeutendere Effekte. Ein schwacher synergistischer Effekt wurde beim Mischen von Pyrethrinen mit Piperonylbutoxid in niedrigen Konzentrationen beobachtet, nicht aber bei hohen Konzentrationen.Bei der Mehrzahl (17 von 19) der Insektizide waren die Zellen in 10 bis 10.000 mal höheren Insektizid-Konzentrationen zu überleben fähig als A. aegypti-Larven im letzten Stadium. Rotenon war das einzige Insektizid, dessen Toxizität auf Zellen stärker war (10.000 Mal) als auf intakte Larven.

     

Effects of a new pyrazolo [3, 4-d]pyrimidine on growth and morphology of Candida albicans

A new pyrazolo [3, 4-d]pyrimidine derivative was synthesized and its antifungal activity evaluated in vitro against mycelial and yeast cells of Candida albicans. The most striking ultrastructural changes following treatment with 10–30 g/ml (mycelia) and 25–75 g/ml (yeasts) consisted in the deterioration of the organelle membranes and in aberrant thickenings of the cell wall. The complete disorganization of the cytoplasmic structures seemed to be the final event.     

Activation of immunoglobulin control elements in trasgenic mice

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EkCAT, contains the Ig heavy chain enhancer (E) and the light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, EkCAT, CAT is under the control of the promoter alone. E and relative activity were assessed by CAT assay. In EkCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In EkCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon- (IFN-) increased CAT expression to varying extents in cells derived from EkCAT mice but not in spleen cells prepared from EkCAT mice. Thus, the presence of E, in addition to the promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EkCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN- caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.This work has been submitted in partial fulfillment of the requirements for the doctoral degree from the George Washington University.     

The effect of antibiotics on the inhibition of callus induction and plant regeneration from cotyledons of sugarbeet (Beta vulgaris L.)

Callus induction and plantlet regeneration from cotyledonary expiants of sugarbeet was observed utilizing two media formulations, MS and a modified MS termed RVIM both supplemented with 1.0 g/ml BAP as the sole growth regulator. Callus induction was genotype dependent The USDA line 8787 produced the highest response for callus induction followed by Betaseed 4587 and the USDA line C600. This order was conserved on both media formulations. Shoot induction was consistently higher averaging 32% from the RVIM formulation over the 3 genotypes compared to 25% from MS. The antibiotics geneticin, gentamycin, hygromycin, kanamycin and phleomycin were screened with the modified RV system utilizing Betaseed 4587. Callus growth was inhibited by levels of 50 g/ml geneticin, 150 g/ml gentamycin, 10 g/ml hygromycin, 150 g/ml kanamycin and 20 g/ml phleomycin. The results indicate that the concentrations of antibiotics used to inhibit callus induction will be sufficient for use as selectable markers in transformation experiments with Beta vulgaris.Abbreviations B₅ basal medium (Gamborg et al, 1968) - BAP N⁶-Benzylaminopurine - IBA Indole-3-butanoic acid - MS basal medium (Murashige and Skoog 1968) - RVIM modified MS basal medium (Freytag et al, 1988) - MES (2[N-Morpholino] ethanesulfonic acid     

Search for A-factor-Dependent Variants in Actinomycete Populations

A study of 28 nocardia-like, asporogenous, and oligosporous spontaneous morphological variants belonging to 23 species of streptomycetes revealed five strains producing regulators of the A-factor group. Streptomyces griseus 1439, which forms aerial mycelium and spores only in the presence of exogenous A-factor, was used as the test strain. Among the 28 spontaneous variants, three new A-factor-dependent strains were revealed, which represented the species Streptomyces griseus, S. citreofluorescens, and S. viridovulgaris subsp. albomarinus. These weakly differentiated variants did not produce A-factor and behaved as its recipients, responding by changes in their morphological characteristics at a concentration of this regulator in the medium of 0.01 g/ml or higher. The original collection strains in whose populations the variants were selected produced substances of the A-factor group. The A-factor-dependent variants differed in the level of the regulator required for maximal expression of the morphological characteristics: it was necessary to introduce the A-factor at a concentration of 1 g/ml for S. citreofluorescens and S. viridovulgaris subsp. albomarinus and at 10 g/ml for S. griseus.     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Determination of mannitol in ectomycorrhizal fungi and ectomycorrhizas by enzymatic micro-assays

Two sensitive methods for the enzymatic determination of mannitol are described which were applied to fungal and mycorrhizal extracts. Both methods are based on the oxidation of mannitol by mannitol dehydrogenase from Agaricus hortensis and the fluorometric determination of the NADPH produced in this reaction. The detection limits are 125 pmol for the direct fluorometric assay and 100 fmol, when enzymatic cycling of NADPH is included. The levels of mannitol detected were 123 pmol/g dry wt (mycelia from Cenococcum geophilum, cultivated on malt medium), below 0.3 or about 2.4 pmol/g dry wt (mycelia from Amanita muscaria, dependent on carbon source in the cultivation medium), and between 1.9 and 5.2 pmol/g dry wt in mycorrhizal short roots of Picea abies/Amanita muscaria.     

ATPase-dependent energy spilling by the ruminal bacterium,Streptococcus bovis

When the ruminal bacterium Streptococcus bovis was grown in batch culture with glucose as the energy source, the doubling time was approximately 21 min and the rate of bacterial heat production was proportional to the optical density (1.72 W/g protein). If exponentially growing cultures were treated with chloramphenicol, there was a decline in heat production, but the rate was greater than 0.30 W/g protein even after growth ceased. Since there was no heat production after glucose depletion, this growth-independent energy dissipation (spilling) was not simply due to endogenous metabolism. Stationary cells which were washed and incubated in nitrogen-free medium containing an excess of glucose produced heat at a rate of 0.17 W/g protein. Monensin and tetrachlorosalicylanilide (TCS), compounds which facilitate an influx of protons, caused a more than 2-fold increase in heat production. Dicyclohexylcarbodiimide (DCCD) virtually eliminated growth-independent heat production regardless of the mode of growth inhibition. Because DCCD had little effect on the glucose phosphotransferase system, it appeared that the combined action of proton influx and the membrane bound F₁F₀ proton ATPase was responsible for energy spilling.Abbreviations DCCD dicyclohexylcarbodiimide - TCS tetrachlorosalicylanilide     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Angiogenesis and myogenesis as two facets of inflammatory post-ischemic tissue regeneration

Recently Hippophae rhamnoides has been reported to render chromatin compaction and significantly inhibit radiation induced DNA strand breaks. To investigate the mechanism of action of RH-3, a preparation of Hippophae rhamnoides, in this connection, present study was undertaken. Chromatin compaction induced by RH-3 (100 g/ml or more) was maximum at alkaline pH but was completely negated by acidic pH (< 6) or presence of free radical scavengers like glycerol, DMSO etc. In a concentration dependent manner, RH-3 inhibited the intercalation of ethidium ions from Et Br into calf thymus DNA and also increased the precipitation of DNA-protein cross-links (DPC) in thymocytes. Chromatin compaction caused by RH-3 treatment did not permit the separation of proteins from DNA even after treatment with 2 M NaCl solution. SDS-PAGE profiles also revealed that RH-3 in a dose dependent manner compacted the chromatin organization, induced DPC and inhibited the extraction of both histone and non-histone matrix proteins from chromatin maximally at 80 g/ml. More than 80 g/ml of RH-3, though extracted low molecular weight histones but did not separate non-histone proteins. The RH-3 mediated DPCs were resistant even to 1% SDS, 4 M NaCl and 3.8 M hydroxyl amine hydrochloride but were prone to both urea (8 M) and guanidine hydrochloride (6 M) indicating covalent bonding between DNA and proteins (serine/threonine). RH-3 in a concentration dependent manner induced superoxide anions and the phenomenon was dependent upon nature of medium, presence of metal ions and pH. RH-3 at concentrations up to 100 g/ml in presence of 50 M copper sulfate inflicted significant damage to extraneously added 2-deoxyribose molecules and maximum TBARS were formed at a concentration of 100 g/ml. Higher concentrations of RH-3 more than 100 g/ml quenched free radicals and inhibited 2-deoxyribose degradation. RH-3 also induced strand breaks in plasmid DNA at concentrations lower than 100 g/ml but completely inhibited at concentrations higher than 250 g/ml, indicating bimodal function. Strand breaks induced by lower concentrations of RH-3 (up to 100 g/ml) were inhibited by antioxidants like GSH, DFR etc. RH-3, in a concentration dependent mode also inhibited the relaxation of supercoiled plasmid DNA (PBR322) by topoisomerase I.Present study indicated that RH-3 caused compaction of reversible (< 100 g/ml) and irreversible (> 100 g/ml) nature which was related to the magnitude of DNA-protein cross-links formed. Maintenance of chromatin organization, induction of hypoxia, hydrogen atom donation, free radical scavenging and blocking of cell cycle at G2-M phase by interfering with topoisomerase I activity seem to contribute towards the radioprotective efficacy of RH-3.     

Ethambutol potentiates extracellular and intracellular activities of clarithromycin,sparfloxacin, amikacin,and rifampin againstMycobacterium avium

Intracellular bactericidal activities of the antituberculosis drugs rifampin and amikacin, as well as those of newly described drugs clarithromycin (a macrolide) and sparfoxacin (a difluoroquinolone), were assessed against three strains of theMycobacterium avium complex (MAC) growing in two different in vitro macrophage systems, namely, mouse bone marrowderived macrophages (BMMØ) and human peripheral blood monocyte-derived macrophages (human MØ). All the infected macrophages were fed reported C_max concentrations of the drugs, i.e., 15g/ml for rifampin, 20 g/ml for amikacin, 4g/ml for clarithromycin, and 1.5g/ml for sparfloxacin. Further potentiation of drug activity in the presence of C_max level of ethambutol (6g/ml) during 9 days of intracellular growth (measured by lysing the macrophages and making bacterial counts) was assessed. Our results showed that all four drugs were active against the strains used in this study and that the addition of ethambutol (which had no significant intracellular activity against the bacteria in this system) further potentiated the bactericidal effect of the drugs. When the same drug combinations were tested at their sublethal concentrations by BACTEC^® radiometric methodology, a good correlation between the drug enhancement data in extracellular and intracellular systems was found. We conclude that ethambutol may serve as an essential component in effective anti-M. avium chemotherapy and that the effective drug combinations may be routinely screened by the Bactec radiometric methodology.     

Enhancement of cationic liposome-mediated transfection by lactoferrin

Lactoferrin(Lf) at 3 g/ml increased transfection about 3 fold with cationic liposomes, which is a similar enhancing effect with transferrin(Tf). However, the mechanism of enhancement by Lf was different from that by Tf in that the concentration of Lf for the peak luciferase activity was 3 g/ml while that of Tf was 30 g/ml and furthermore, when Lf and Tf were supplemented together in the liposome-DNA complex, luciferase activity was increased additively. © Rapid Science Ltd. 1998     

Effects of GABA antagonists,SR 95531 and bicuculline,on GABAA receptor-regulated chloride flux in rat cortical synaptoneurosomes

Synaptoneurosomes isolated from cerebral cortices of male Sprague-Dawley rats were used for studying GABA_A receptor-regulated chloride influx. The in vitro effects of GABA antagonists, SR 95531 (a pyridazinyl GABA derivative) and bicuculline, on pentobarbital-stimulated, muscimol-stimulated or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied. The chloride uptake was determined at 30°C, for 5 sec. Pentobarbital and muscimol produced a maximal stimulation of chloride uptake in cortical synaptoneurosomes at 500 M and 50M, respectively. SR 95531 as well as bicuculline had no effect on the basal uptake of chloride. Whereas, SR 95531 (0.3–30 M) and bicuculline (0.1–100 M), when added 5 min before muscimol (50 M), produced a significant concentration-dependent inhibition of muscimol (50 M)-stimulated chloride uptake (IC₅₀ s of 0.89±0.11 M and 13.45±2.10M, respectively). In studies of the inhibitory effects of SR 95531 and bicuculline on pentobarbital (500 M)-stimulated chloride uptake, the IC₅₀ s were 0.81±0.12 M and 3.86±1.14 M, respectively. SR 95531 exhibited a more potent inhibitory effect than bicuculline on flunitrazepam-enhanced, muscimol-stimulated chloride uptake. The results revealed that SR 95531 has a more potent antagonistic effect than bicuculline on GABA_A-regulated chloride flux.