Production of tylosin in solid-state fermentation by Streptomyces fradiae NRRL-2702 and its gamma-irradiated mutant (γ-1)

Aims:  To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant γ-1 of Streptomyces fradiae NRRL-2702 and its parent strain.
Methods and Results:  Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 μg of tylosin g⁻¹ substrate by mutant γ-1 against parent strain (300 μg tylosin g⁻¹ substrate). The tylosin yield was further improved to 4500 μg g⁻¹ substrate [70% moisture, 10% inoculum (v/w), pH 9·2, 30°C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 μg of tylosin g⁻¹ substrate) in SSF even after optimization of process parameters.
Conclusion:  The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant γ-1.
Significance and Impact of the Study:  This study proved to be very useful and resulted in 6·87 ± 0·30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.     

Development and characterization of a lux-modified 2,4-dichlorophenol-degrading Burkholderia sp. RASC

lux -marked biosensors for assessing the toxicity and bioremediation potential of polluted environments may complement traditional chemical techniques. lux CDABE genes were introduced into the chromosome of the 2,4-dichlorophenol (2,4-DCP)-mineralizing bacterium, Burkholderia sp. RASC c2, by biparental mating using the Tn 4431 system. Experiments revealed that light output was constitutive and related to cell biomass concentration during exponential growth. The transposon insertion was stable and did not interrupt 2,4-DCP-degradative genes, and expression of lux CDABE did not constitute a metabolic burden to the cell. A bioluminescence response was detectable at sublethal 2,4-DCP concentrations: at < 10.26 μg ml⁻¹, bioluminescence was stimulated (e.g. 218% of control), but at concentrations > 60 μg ml⁻¹ it declined to < 1%. Investigating the effect of [14C]-2,4-DCP concentration on the evolution of ¹⁴CO₂ revealed that, for initial concentrations of 2.5–25 μg ml⁻¹, ≈55% of the added ¹⁴C was mineralized after 24 h compared with < 1% at 50 and 100 μg ml⁻¹. Inhibition of 2,4-DCP mineralization between 25 and 50 μg ml⁻¹ corresponded well to the EC₅₀ value (33.83 μg ml⁻¹) obtained from bioluminescence inhibition studies. lux -marked RASC c2 may therefore be used as a functionally (i.e. 2,4-DCP degrader) and environmentally relevant biosensor of toxicity and biodegradation inhibition.     

Characterization and toxicity of Bacillus thuringiensis isolates from warehouses to Spodoptera exigua (Lep., Noctuidae)

Bacillus thuringiensis was isolated from 122 of 413 samples obtained from warehouses. Eighty-seven (71.31%) of these B. thuringiensis isolates were toxic to Spodoptera exigua , causing more than 60% mortality. Twenty-seven isolates were highly toxic to S. exigua , causing more than 95% mortality. Isolates 133, 47 and 58, which belonged to serotype H₇, H₄, H₄, respectively, were more active than the other isolates and their 50% lethality concentration (LC₅₀) values were 17.93, 14.78 and 15.55  μ g/ml, respectively. The isolate 133, 47 and 58 were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and polymerase chain reaction. The results showed that they contained ~135 and 65 kDa crystal proteins, that were similar with reference strain HD-1. Isolate 133 contained the cryIA(a) , cryIA(b) , cryIA(c) , cryIE and cryII genes whereas both isolate 47 and 58 contained the cryIA(b) , cryIA(c) , cryIE and cryII genes; but they did not contain cryIII gene.     

Effect of CO2-enrichnient on seedling physiology and growth of two tropical tree species

Seedlings of two tree species from the Atlantic lowlands of Costa Rica, Ochroma la-gopus Swartz, a fast-growing pioneer species, and Pentaclethra macroloba (Willd.) Kuntze, a slower-growing climax species, were grown under enriched atmospheric CO₂ in controlled environment chambers. Carbon dioxide concentrations were maintained at 350 and 675 μl 1⁻¹ under photosynthetic photon flux densities of 500 μol m⁻² s⁻¹ and temperatures of 26°C day and 20°C night. Total biomass of both species increased significantly in the elevated CO₂ treatment; the increase in biomass was greatest for the pioneer species, O. lagopus . Both species had greater leaf areas and specific leaf weights with increased atmospheric CO₂. However, the ratio of non-pho-tosynthetic tissue to leaf area also increased in both species leading to decreased leaf area ratios. Plants of both species grown at 675 μl 1⁻¹ CO₂ had lower chlorophyll contents and photosynthesis on a leaf area basis than those grown at 350 μl 1⁻¹. Reductions in net photosynthesis occurred despite increased internal CO₂ concentrations in the CO₂-enriched treatment. Stomatal conductances of both species decreased with CO₂-enrichment resulting in significant increases in water use efficiency.     

Insecticidal and synergistic effects of Majorana hortensis essential oil and some of its major constituents

The essential oil from leaves of Majorana hortensis Moench (Lamiaceae) was isolated by hydrodistillation with a yield of 1.6% (wt/wt). The insecticidal activity of the oil was evaluated against fourth instars of Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) and adults of Aphis fabae L. (Hemiptera: Aphididae). The oil showed a remarkable toxic effect against S. littoralis in a topical application assay (LD₅₀ = 2.48 μg per larva) and in a residual film assay (LC₅₀ = 3.14 g/l). The oil of M. hortensis also exhibited a pronounced toxic effect against A. fabae adults with LC₅₀ values of 1.86 and 2.27 g/l in rapid dipping and residual film assays, respectively. Gas chromatography-mass spectrometry analyses of M. hortensis essential oils revealed the presence of 31 compounds and the main components were terpinen-4-ol (30.0%), γ-terpinene (11.3%), and trans -sabinene hydrate (10.8%). Repeated column chromatography of M. hortensis oil on silica gel led to the isolation of two major constituents, which were characterized based on ¹H-nuclear magnetic resonance and mass spectrometric data, as terpinen-4-ol and γ-terpinene. These two components were examined for their insecticidal and synergistic activities towards S. littoralis and A. fabae . Terpinen-4-ol and γ-terpinene exhibited a significant insecticidal activity against both insects, but γ-terpinene was more toxic than terpinen-4-ol. When tested in a binary mixture with the synthetic insecticides profenofos and methomyl, it was found that both compounds enhanced the insecticidal activity of these insecticides by two- to threefold. These results show that terpinen-4-ol and γ-terpinene have a synergistic effect on the insecticidal activities of synthetic insecticides profenofos and methomyl.     

Larvicidal toxicity of Japanese Bacillus thuringiensis against the mosquito Anopheles stephensi

Japanese isolates of Bacillus thuringiensis were screened for larvicidal activity against the mosquito Anopheles stephensi , the urban malaria vector of the Indian subcontinent. Among more than 30 strains identified, larvicidal activity causing >80% mortality in 72 h was demonstrated for 41/1449 (2.8%) isolates. The majority of strains and isolates (97.2%) exhibited little or no larvicidal activity. Anopheles -active strains belonged to more than 12 H serotypes, especially H3ade (serovar fukuokaensis ) and H44 (serovar higo ). SDS-PAGE profiles of inclusion proteins showed 4 distinct types among 6 active strains examined. The most active Japanese isolates were H20 strain 89-T-34-14 (LC₅₀ 4.4 μg/ml) and H44 serovar higo strain 74-E-45-24 (LC₅₀ 7.6 μg/ml), respectively, 13-fold and 23-fold less active than the international standard H14 serovar israelensis (LC₅₀ 0.33 μg/ml).     

Nicotine-induced and depolarisation-induced glutamate release from hippocampus mossy fibre synaptosomes: two distinct mechanisms

Hippocampus mossy fibre terminals activate CA3 pyramidal neurons via two distinct mechanisms, both quantal and glutamatergic: (i) rapid excitatory transmission in response to afferent action potentials and (ii) delayed and prolonged release following nicotinic receptor activation. These processes were analysed here using rat hippocampus mossy fibres synaptosomes. The relationships between synaptosome depolarisation and glutamate release were established in response to high-KCl and gramicidin challenges. Half-maximal release corresponded to a 52 mV depolarisation step. KCl-induced release was accompanied by transient dissipation of the proton gradient across synaptic vesicle membrane. Nicotine elicited a substantial glutamate release from mossy fibre synaptosomes (EC₅₀ 3.14 μM; V _max 12.01 ± 2.1 nmol glutamate/mg protein; Hill's coefficient 0.99). However, nicotine-induced glutamate release was not accompanied by any change in the membrane potential or in the vesicular proton gradient. The effects of acetylcholine (200 μM) were similar to those of nicotine (25 μM). Nicotinic α7 receptors were evidenced by immuno-cytochemistry on the mossy fibre synaptosome plasma membrane. Therefore, the same terminals can release glutamate in response to two distinct stimuli: (i) rapid neurotransmission involving depolarisation-induced activation of voltage-gated Ca²⁺ channels and (ii) a slower nicotinic activation which does not involve depolarisation or dissipation of the vesicular proton gradient.     

Neuropeptide Y stimulates retinal neural cell proliferation--involvement of nitric oxide

Neuropeptide Y (NPY) is a 36 amino acid peptide widely present in the CNS, including the retina. Previous studies have demonstrated that NPY promotes cell proliferation of rat post-natal hippocampal and olfactory epithelium precursor cells. The aim of this work was to investigate the role of NPY on cell proliferation of rat retinal neural cells. For this purpose, primary retinal cell cultures expressing NPY, and NPY Y₁, Y₂, Y₄ and Y₅ receptors [Álvaro et al. , (2007) Neurochem. Int., 50, 757] were used. NPY (10–1000 nM) stimulated cell proliferation through the activation of NPY Y₁, Y₂ and Y₅ receptors. NPY also increased the number of proliferating neuronal progenitor cells (BrdU⁺/nestin⁺ cells). The intracellular mechanisms coupled to NPY receptors activation that mediate the increase in cell proliferation were also investigated. The stimulatory effect of NPY on cell proliferation was reduced by l -nitroarginine-methyl-esther ( l -NAME; 500 μM), a nitric oxide synthase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ; 20 μM), a soluble guanylyl cyclase inhibitor or U0126 (1 μM), an inhibitor of the extracellular signal-regulated kinase 1/2 (ERK 1/2). In conclusion, NPY stimulates retinal neural cell proliferation, and this effect is mediated through nitric oxide–cyclic GMP and ERK 1/2 pathways.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.     

Symbiotic effectiveness, rate of respiration and glutamine synthetase activity of sodium azide-resistant strains of Rhizobium leguminosarum biovar trifolii

Nitrogen fixing efficiency of sodium azide-resistant strains of Rhizobium leguminosarum bv. trifolii was studied in symbiosis with berseem clover plants in chillum jars. Rate of respiration and glutamine synthetase activity were tested in cultured cells and nodules, respectively. It was observed that shoot dry weight and percentage shoot nitrogen were maximum in plants inoculated with strains resistant to 15 μg ml⁻¹ sodium azide. Rate of respiration in cultured cells was lowest in strains resistant to 15 μg ml⁻¹ sodium azide and highest in strains resistant to 5 μg ml⁻¹ sodium azide. A negative correlation was observed between rate of respiration (in cultured cells) and shoot dry weight of host plants. Glutamine synthetase activity was maximum in nodule extracts of host plants inoculated with strains resistant to 5 and 10 μg ml⁻¹ sodium azide, whereas it was minimum for strains resistant to 15 μg ml⁻¹ sodium azide. Hence, resistance to low doses (15 μg ml⁻¹) of sodium azide, together with lower respiratory and glutamine synthetase activities, could be used as a potential method for isolating the symbiotically effective strains of Rh. leguminosarum bv. trifolii.     

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (−)nicotine on the function of the NMDA-mediated modulation of [³H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [³H]DA release in rats chronically treated with vehicle (14 days) with an EC₅₀ of 13.1 ± 2.0 μM. The NMDA-evoked overflow of the [³H]DA in PFC nerve endings of rats treated with (−)nicotine was significantly lower (−43%) than in vehicle treated rats. The EC₅₀ was 9.0 ± 1.4 μM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [³H]DA overflow with an EC₅₀ of 14.5 ± 5.5 μM. This effect was significantly enhanced in chronically treated animals. The EC₅₀ was 10.5 ± 0.5 μM. The K⁺-evoked release of [³H]DA was not modified by the (−)nicotine administration. Both the changes of the NMDA-evoked [³H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (−)nicotine differentially affects the NMDA-mediated [³H]DA release in the PFC and NAc of the rat.     

Isolation of Bacillus thuringiensis subsp. israelensis from diseased field-collected larvae of the saturniid moth, Hylesia metabus, in French Guiana

Abstract An isolate of Bacillus thuringiensis subsp. israelensis (BTI) was obtained from field-collected larvae of the saturniid moth, Hylesia metabus . This isolate was shown to belong to serotype H 14, and to produce a spherical parasporal body identical to that of the type strain of BTI. With an LC₅₀ of 0.764 μg cm⁻², this isolate was more toxic to H. metabus than the HD-1 strain of B. thuringiensis subsp. kurstaki (HD-1). These results demonstrate that BTI can be active against lepidopterous insects, a wider spectrum of activity than previously realized.     

Cationic amino acid transporter 1-mediated l-arginine transport at the inner blood–retinal barrier

The purpose of this study was to identify the transporter mediating l -arginine transport at the inner blood–retinal barrier (BRB). The apparent uptake clearance of [³H] l -arginine into the rat retina was found to be 118 μL/(min·g retina), supporting a carrier-mediated influx transport of l -arginine at the BRB. [³H] l -Arginine uptake by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), used as an in vitro model of the inner BRB, was primarily an Na⁺-independent and saturable process with Michaelis-Menten constants of 11.2 μM and 530 μM. This process was inhibited by rat cationic amino acid transporter (CAT) 1-specific small interfering RNA as well as substrates of CATs, l -arginine, l -lysine, and l -ornithine. The expression of cationic amino acid transporter (CAT) 1 mRNA was 25.9- and 796-fold greater than that of CAT3 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of CAT1 protein was detected in TR-iBRB2 cells and immunostaining of CAT1 was observed along the rat retinal capillaries. In conclusion, CAT1 is localized in retinal capillary endothelial cells and at least in part mediates l -arginine transport at the inner BRB. This process seems to be closely involved in visual functions by supplying precursors of biologically important molecules like nitric oxide in the neural retina.     

Polygalacturonase biosynthesis by Lactobacillus plantarum: effect of cultural conditions on enzyme production

Lactobacillus plantarum was found to produce extracellular polygalacturonase (EC 3.2.1.15.). Maximum enzyme production was obtained in a medium containing 0.5% glucose and 1.5% low methyl-pectin as inducer at 27°C at an initial pH of 6.8. Enzyme production was strongly inhibited by 5 μmol/l NiCl₂, 5 μmol/l CoCl₂, 5 μmol/l CuSO₄, and 10 μmol/l ZnCl₂. MnSO₄ and MgSO₄ at 200 μmol/l and 50 μmol/l respectively seemed to enhance enzyme biosynthesis. The optimal pH and temperature for enzyme activity were 4.5 and 30°C respectively. Enzyme production in batch culture accompanied growth.     

Anti-bacterial activity of synthetic N-heterocyclic oxidizing compounds

Synthetic chlorochromate derivatives of pyridine and quinoline were active in vitro against type cultures of Escherichia coli (ATCC 128), Staphylococcus aureus (ATCC 14775), Pseudomonas aeruginosa (ATCC 10145) and Bacillus subtilis (NCTC 8236). The minimum inhibitory concentrations (MIC) were 125–250 μg ml⁻¹ and 250–500 μg ml⁻¹ for pyridinium chlorochromate and quinolinium chlorochromate, respectively. An established derivative of quinoline (Perfloxacin) had an MIC of 125–250 μg ml⁻¹. The extinction time for 10⁵ cfu in broth was 90 min for pyridinium chlorochromate and 120 min for quinolinium chlorochromate, except for B. subtilis which survived up to about 180 min and 360 min. A combination of the two compounds produced an antagonistic effect. The 50% lethal dose (LD₅₀ toxicity) in mice was estimated at 76 μg g⁻¹ and 33 μg g⁻¹ body weight for the quinolinium and pyridinium chlorochromates. The compounds also exhibited some potential for suppressing a simulated staphylococcal infection in mice at the dosage levels of ca 22 μg g⁻¹ for pyridinium chlorochromate and 45 μg g⁻¹ for quinolinium chlorochromate.     

Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis

Aims:  To elaborate an effective electroporation protocol for large plasmids and wild type strains of Bacillus thuringiensis .
Methods and Results:  The effect of DNA desalting, wall-weakening agency, cell growth conditions, electroporation solutions, and electric fields on electroporation efficiency was evaluated to optimize electroporation conditions for B. thuringiensis . By using this improved method, the greatest efficiency was reached 2 × 10¹⁰ CFU  μ g⁻¹ with pHT304, which is 10⁴ times higher than previously reported. Four large plasmids (29·1, 44·9, 58 and 60 kb) were successfully transferred into the acrystalliferous B. thuringiensis strain BMB171; these results have not been achieved with previous protocols. Three wild type B. thuringiensis strains which could not be transformed previously were also transferred successfully.
Conclusions:  This improved method is more efficient for small plasmids; it is also appropriate for large plasmids and wild type B. thuringiensis strains which were not transformed by previous procedures.
Significance and Impact of the Study:  The present study established an effective electroporation protocol for large plasmids and wild type strains of B. thuringiensis . This method is well suited for the cloning and expression of huge DNA fragments such as gene clusters in B. thuringiensis . It also can be used as a reference method for other Bacillus strains that are refractory to electroporate.     

Expression of mel gene improves the UV resistance of Bacillus thuringiensis

Aims:  To improve ultraviolet (UV) resistance of Bacillus thuringiensis for increasing the duration of the Bt product applied in the field, a genetically engineered strain Bt TD841 that produced both melanin and Cry1A protein was constructed, and its UV resistance was evaluated in the laboratory.
Methods and Results:  Melanin quantitative analysis revealed that the recombinant strain Bt TD841 could synthesize 0.15 mg melanin ml⁻¹ sporulated culture. Atomic force microscopy confirmed the production of diamond crystal and SDS-PAGE results showed the expression of the 130 kDa Cry1A protein. Bioassay results demonstrated that the LC₅₀ value of Bt TD841 was 3.69 μl ml⁻¹ against Helicoverpa armigera and the UV resistance of this recombinant was enhanced 9.7-fold compared to its parental strain Bt HC42 after 4-h UV irradiation.
Conclusion:  Expression of the mel gene can significantly increase UV resistance of B. thuringiensis.
Significance and Impact of the Study:  This is the first report on genetically engineered Bt strain with co-expression of melanin and the insecticidal crystal proteins gene, and the results may offer a practical solution for improving the photoprotection of Bt products in field application.     

Relations between carbohydrate, water status and adventitious root formation in leafy pea cuttings rooted under various levels of atmospheric CO2 and relative humidity

Three levels of atmospheric CO₂ and 2 levels of relative humidity (RH) during the rooting period were tested for their effect on several factors presumed to influence adventitious root formation in leafy pea ( Pisum sativum L. cv. Alaska) cuttings. Compared to normal CO₂ levels (350 μl l⁻¹), neither 1800 nor 675 μl l⁻¹ CO₂ affected the rooting percentage or the number of roots per cutting. However, 1800 μl l⁻¹ CO₂ increased root and shoot dry weight, root length, carbohydrate levels in the base of the cuttings and water potential (Ψ_w) of cuttings compared to normal levels of CO₂. Compared to 87% RH. 55% RH decreased all of the above parameters, including the number of roots per cutting. A polyvinyl chloride antitranspirant (which partially blocks stomata and slows photosynthesis) applied simultaneously with 87% RH increased Ψ_w and root length but lowered all of the other above parameters, compared to 87% RH without antitranspirant. Increasing current photosynthate (products of photosynthetic activity after excision), carbohydrate, or Ψ_w either alone or together was associated with increased root system size but not necessarily with increased rooting percentage or root number. The data are consistent with a hypothesis that the number of roots per cutting increased with increasing current photosynthate and carbohydrate until some other factor became limiting. Also, the effect of Ψ_w on rooting percentage and root number was mediated through its effect on current photosynthate and carbohydrate.     

Endogenous cannabinoid anandamide inhibits nicotinic acetylcholine receptor function in mouse thalamic synaptosomes

The effects of the endogenous cannabinoid anandamide [arachidonylethanolamide (AEA)] on the function of nicotinic acetylcholine receptor (nAChR) were investigated using the ⁸⁶Rb⁺ efflux assay in thalamic synaptosomes. AEA reversibly inhibited ⁸⁶Rb⁺ efflux induced by 300 μM ACh with an IC₅₀ value of 0.9 ± 2 μM. Pre-treatment with the cannabinoid (CB₁) receptor antagonist SR141716A (1 μM), the CB₂ receptor antagonist SR144528 (1 μM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA, suggesting that known CB receptors are not involved in AEA inhibition of nAChRs. AEA inhibition of ⁸⁶Rb⁺ efflux was not reversed by increasing acetylcholine (ACh) concentrations. In radioligand binding studies, the specific binding of [³H]-nicotine was not altered in the presence of AEA, indicating that AEA inhibits the function of nAChR in a non-competitive manner. Neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor, indomethacin, (5 μM) affected AEA inhibition of nAChRs, suggesting that the effect of AEA is not mediated by its metabolic products. Importantly, the extent of AEA inhibition of ⁸⁶Rb⁺ efflux was significantly attenuated by the absence of 1% fatty acid free bovine serum albumin pre-treatment, supporting previous findings that fatty acid-like compounds modulate the activity of nAChRs. Collectively, the results indicate that AEA inhibits the function of nAChRs in thalamic synaptosomes via a CB-independent mechanism and that the background activity of these receptors is affected by fatty acids and AEA.