High-performance liquid chromatographic determination of indalpine,a new non-tricyclic antidepressant,in human plasma : Identification and simultaneous measurement of its major plasma metabolite

Indalpine or 4-[2-(3-indolyl)ethyl]piperidine, a selective inhibitor of 5-hydroxytryptamine uptake in central monoamine neurons, has proved to be an effective agent in the treatment of chronically ill depressed patients. We have developed a rapid high-performance liquid chromatographic method for the simultaneous determination of indalpine and its major metabolite in human plasma. Isolation by high-performance liquid chromatography and identification of this metabolite by mass spectrometry are also described.     

Metabolism of 22-oxacalcitriol by a vitamin D-inducible pathway in cultured parathyroid cells.

Catabolism of 22-oxacalcitriol (OCT) in parathyroid cells was compared to that of the parent hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Catabolism of both compounds was greatly accelerated by pretreatment of the cells with 1,25-(OH)2D3 or OCT. The rate of degradation of OCT was slightly greater than that of 1,25-(OH)2D3. Excess unlabeled OCT or 1,25-(OH)2D3 inhibited metabolism of both tritiated substrates. Ketoconazole, a cytochrome P450 inhibitor, blocked catabolism of both compounds. The major OCT metabolite appeared to be 1,20-dihydroxy-22,23,24,25,26,27-hexanor-vitamin D3 which was not active in suppressing PTH secretion. We conclude that OCT appears to be metabolized by the same vitamin D-inducible side chain oxidation pathway that catabolizes other vitamin D compounds and that its higher than expected suppression of PTH secretion is not due to slower cellular metabolism.     

The metabolism of sodium cortisone 27-[35S]-sulphate in the rat

1. The metabolism of sodium cortisone 21-[(35)S]sulphate was investigated in rats. 2. Quantitative and qualitative experiments showed that substantial amounts of (35)SO(4) (2-) appeared in the urine of free-ranging rats receiving the ester. 3. Whole-body radioautograms indicated considerable biliary elimination of (35)S and also pointed to the liver as the site of metabolism. 4. When female rats with bile-duct cannulae received sodium cortisone 21-[(35)S]sulphate approx. 70% of the dose appeared in the bile as a doubly conjugated steroid (metabolite I). This metabolite was identified as 3alpha-(beta-d-glucopyranuronosido)- 17alpha-hydroxy-21-[(35)S]sulpho-oxy-5alpha-pregnane-11,20-dione. 5. When metabolite I was administered to a rat with a bile-duct cannula 90% of the dose appeared in the bile unchanged. After the administration (intraperitoneally or orally) of metabolite I to free-ranging rats considerable amounts of (35)SO(4) (2-) appeared in the urine. 6. The route by which (35)SO(4) (2-) might be produced from cortisone [(35)S]sulphate in free-ranging animals is discussed.     

Simultaneous determination of clobazam and its major metabolite in human plasma by a rapid HPLC method

A rapid and specific HPLC method has been developed and validated for simultaneous determination of clobazam, the anticonvulsant agent, and its major metabolite in human plasma. The sample preparation was a liquid-liquid extraction with tuloene yielding almost near 100% recoveries of two compounds. Chromatographic separation was achieved with a Chromolith Performance RP-18e 100 mm x 4.6mm column, using a mixture of a phosphate buffer (pH 3.5; 10mM)-acetonitrile (70:30, v/v), in isocratic mode at 2 ml/min at a detection wave-length of 228 nm. The calibration curves were linear (r(2)>0.998) in the concentration range of 5-450 ng ml(-1). The lower limit of quantification was 5 ng ml(-1) for two compounds studied. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 0.89-9.1% and 2.1-10.1% R.S.D., respectively. The developed procedure was applied to assess the pharmacokinetics of clobazam and its major metabolite following administration of a single 10mg oral dose of clobazam to healthy volunteers.     

Extracellular fibrinogenolytic enzyme of Aspergillus fumigatus: substrate-dependent variations in the proteinase synthesis and characterization of the enzyme

Abstract To get a better understanding of the role of the previously reported fibringenolytic enzyme of Aspergillus fumigatus , we investigated the in vitro conditions of enzyme synthesis and attempted to characterize it. Modification of the nitrogen source did not influence the extracellular serine-proteinase profile, but resulted in important quantitative differences in the yields in batch cultures. The enzyme synthesis appeared to be an inducible phenomenon in A. fumigatus since it was initiated exclusively in the presence of proteins or protein hydrolysate. Free amino acids or inorganic nitrogen compounds could not promote significant enzyme production. Moreover, peptone at a concentration of 0.1% appeared to be the best inducer of enzyme synthesis. Conversely, modification of the carbon source did not affect fungal growth or enzyme synthesis. However, the production of chymotrypsin was highly sensitive to the carbohydrate level in the culture medium and, with peptone as nitrogen source, highest yields were obtained in the presence of 0.3 or 0.5% glucose. Culture filtrates of A. fumigatus CBS 113.26 grown with peptone or nitrate as nitrogen source were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the protein patterns suggested for the proteinase a molecular mass of 33 kDa which was confirmed by chromatographic purification of the enzyme through (Nα-CBZ)- d -phenylalanine agarose.     

Synthesis and antimicrobial activity of 3-arylamino-1-chloropropan-2-ols

A series of nine 3-arylamino-1-chloropropan-2-ols 2a-2i were synthesized and their anti-fungal activity against pathogenic strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Candida albicans, and antibacterial activity against four pathogenic bacterial strains of Salmonella typhi, Pseudomonas aeruginosa, Streptococcus pneumonae and Staphylococcus aureus were evaluated using different assay systems. 1-Chloro-3-(4'-chlorophenylamino)-propan-2-ol was found to be the most active anti-fungal compound against three pathogenic strains under study, i.e., A. fumigatus, A. flavus and A. niger; the compound showed more than 90% inhibition of growth of A. fumigatus at a concentration of 5.85 microg/ml in disc diffusion assay. Interestingly, 1-chloro-3-(4'-chlorophenylamino)-propan-2-ol did not show any toxicity up to a concentration of 4000 microg/ml. Although 1-chloro-3-(4'-chlorophenylamino)-propan-2-ol was about 8 times less active than the standard compound amphotericin B, its toxicity was many more fold less than the toxicity of amphotericin B. Further, 1-chloro-3-(2',6'-dichlorophenylamino)-propan-2-ol and 1-chloro-3-(3',5'-dichlorophenylamino)-propan-2-ol were found to be the most active compounds against C. albicans. In the anti-microbial assay, 1-chloro-3-(2',4'-dichlorophenylamino)-propan-2-ol and 1-chloro-3-(3',5'-dichlorophenylamino)-propan-2-ol were found to be the most active compounds against Salmonella typhi and 1-chloro-3-(3',4'-dichlorophenylamino)-propan-2-ol was found to be the most active compound against P. aeruginosa. Although, the activities of 1-chloro-3-(2',4'-dichlorophenylamino)-propan-2-ol and 1-chloro-3-(3',5'-dichlorophenylamino)-propan-2-ol are about half the activity of the standard anti-bacterial compound tetracycline, these compounds also were many fold less toxic than the standard drug.     

A new class of 2-(4-cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6-piperazinyl (piperidinyl)-1,3,5-triazine analogues with antimicrobial/antimycobacterial activity

This study presents the synthesis and in vitro pharmacological evaluations of novel 2-(4-cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6-piperazinyl (piperidinyl)-1,3,5-triazines. The title compounds were assayed for their in vitro antimicrobial activity against eight bacteria (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Shigella flexneria) and four fungi (Aspergillus niger, Aspergillus fumigatus, Aspergillus clavatus, Candida albicans) using paper disc diffusion and agar streak dilution method as well as against Mycobacterium tuberculosis H37Rv strain using BACTEC MGIT and Lowenstein-Jensen MIC method. The bioassay results indicate that nine compounds namely 5d, 5h, 5n, 5p, 5q, 5r, 5s, 5t and 5u could be considered as possible potential agents with dual antimicrobial and antimycobacterial activities. The structures of the compounds were elucidated with the aid of IR, (1)H NMR, (13)C NMR, (19)F NMR spectroscopy and CHN analysis.     

Simultaneous determination of 1,5-dicaffeoylquinic acid and its active metabolites in human plasma by liquid chromatography-tandem mass spectrometry for pharmacokinetic studies

1,5-Dicaffeoylquinic acid (1,5-DCQA), a potent HIV-1 integrase inhibitor, is undergoing an evaluation as a promising novel HIV therapeutic agent. Here, we report a simple, rapid and robust LC-MS/MS method for simultaneous determination of 1,5-DCQA and its two active metabolites, 1-caffeoyl-5-feruoylquinic acid (1,5-CFQA) and 1,5-O-diferuoylquinic acid (1,5-DFQA) in human plasma. The quantitation of the target compounds was determined by selected reaction monitoring (SRM) mode using electrospray ionization (ESI). Good linearity was obtained in the 3-500 ng/ml range for each analyte and the analytical method was validated in terms of specificity, precision, accuracy, recovery, stability and matrix effect. These assays gave R.S.D.% values for precision always lower than 13.8% and R.E.% values for accuracy between -8.9 and 0.9%. In addition, the specificity, extraction recovery, stability and matrix effect were satisfactory too. Using the measured plasma concentrations of 1,5-DCQA and its active metabolites in five healthy volunteers, pharmacokinetic profiles of 1,5-DCQA and its active metabolites were evaluated, which supported the clinical pharmacokinetic studies successfully. Due to its high sensitivity, specificity and simplicity, the method could be used for pharmacokinetic studies of both 1,5-DCQA and its active metabolite, and for routine monitoring of their levels in human plasma.     

Synthesis and biological activity of tricyclic analogues of 9-[[cis-1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine

The base moiety of the potent antiherpetic agent 9-[[cis-1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine 3 was transformed into that of the tricyclic 3,9-dihydro-9-oxo-6-R-5H-imidazo[1,2-a]purine system. The tricyclic analogues 5a-d were evaluated for their activity against herpes viruses as well as for cytostatic activity against HSV-1 thymidine kinase (TK) gene-transduced human osteosarcoma tumor cells. Marked activity was found against VZV. The 6-phenyl-substituted fluorescent analogues 5c and d were comparable to that of parent 3 in activity against the VZV strain YS and were 3-fold less active against the VZV strain OKA. The compounds 5a-d also showed marked activity against HSV-1 (KOS) and HSV-2 (G)-against the former generally approximately comparable to that of acyclovir 1a and one order of magnitude lower than 3; against the latter comparable to that of 1a and approximately 6- to 30-fold lower than that of 3. The most pronounced cytostatic activity (5-fold lower than that of 3) was exhibited by compounds 5c and d. Tricyclic analogues with pseudosugar moieties are intrinsically bio-active.     

Direct and fast capillary zone electrophoretic method for the determination of Gleevec and its main metabolite in human urine

A capillary zone electrophoretic (CZE) method was investigated for the determination of Gleevec and its main metabolite (N-demethylated piperazine derivative) in human urine using a fused-silica capillary (75 microm I.D.x60 cm total length, 10 cm effective length). The separation was performed with an hydrodynamic injection time of 10 s (0.5 p.s.i.) a voltage of -25 kV, a capillary temperature of 25 degrees C and a 100 mM phosphoric acid adjusted to pH 2 with the addition of triethanolamine. Under these conditions, the analysis takes about 5 min. A linear response over the 0.4-30.0 mg l(-1) concentration range was investigated for two compounds. A dilution of the sample was the only step necessary before the electrophoresis analysis. Detection limits of 0.1 mg l(-1) for Gleevec and its metabolite (S/N=3) were obtained. The developed method is easy, rapid and sensitive and has been applied to determine Gleevec and its main metabolite in clinical urine samples.     

Synthesis and structure-antifungal activity relationships of 3-aryl-5-alkyl-2,5-dihydrofuran-2-ones and their carbanalogues: further refinement of tentative pharmacophore group

Two series of 3-(substituted phenyl)-5-alkyl-2,5-dihydrofuran-2-ones related to a natural product, (-)incrustoporine, were synthesized and their in vitro antifungal activity evaluated. The compounds with halogen substituents on the phenyl ring exhibited selective antifungal activity against the filamentous strains of Absidia corymbifera and Aspergillus fumigatus. On the other hand, the influence of the length of the alkyl chain at C(5) was marginal. The antifungal effect of the most active compound against the above strains was higher than that of ketoconazole, and close to that of amphotericin B. In order to verify the hypothesis about a possible relationship between the Michael-accepting ability of the compounds and their antifungal activity, a series of simple carbanalogues, 2-(substituted phenyl)cyclopent-2-enones, was prepared and subjected to antifungal activity assay as well.     

Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid

We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C(18) SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C(18) column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml(-1) for cyclo(L-Phe-L-Pro) and 7.5 mg ml(-1) for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.     

Analysis of Volatile Fingerprints for Monitoring Anti-Fungal Efficacy Against the Primary and Opportunistic Pathogen Aspergillus fumigatus

The aims of this study were to use qualitative volatile fingerprints obtained using a hybrid sensor array system to screen anti-fungals for controlling the important lung infecting fungus, Aspergillus fumigatus, especially in immunocompromised patients. SIFT-MS was also used to try and identify key volatiles produced by A. fumigatus. Initial studies were carried out to identify the ED(50) and ED(90) (effective dose) for inhibiting growth of A. fumigatus using three anti-fungal compounds, benomyl, tebuconazole and fluconazole. Subsequent studies involved inoculation of malt extract agar plates with spores of A. fumigatus (25 and 37°C) over periods of 24-72 h to examine the headspace volatile fingerprints generated from the sample treatments using the hybrid sensor array system to compare controls and ED(50)/ED(90) concentrations. The sensor responses showed discrimination between treatments after 48-h incubation when benomyl and tebuconazole were used against A. fumigatus at 37°C using Principal Components Analysis and Cluster Analysis. SIFT-MS analysis showed that methyl pentadiene, ethanol, isoprene and methanol were key biomarker volatiles produced by A. fumigatus in the presence of anti-fungal compounds. This may also be a good approach for the development of rapid screening of anti-microbial compounds and potentially useful for monitoring the possible build up of resistance to specific drug types. Volatile fingerprints produced by patient samples could also be used to evaluate whether lung infections are caused by bacteria or specific fungi to facilitate early diagnosis and enable the right drug treatment to be prescribed.     

Preadipocyte differentiation in vitro: identification of a highly active adipogenic agent

A highly active adipogenic agent was identified in an in vitro adipose conversion system. This agent, ADD 4743 (or ADD), was synthesized by Takeda Chemical Industries (Osaka) as a 3-hydroxy derivative of an oral antidiabetic agent, ciglitazone, and has been presumed to be an active metabolite of the latter substance. When ST 13 mouse preadipose cells were treated with micromolar concentrations of ADD they rapidly and uniformly converted into lipid-accumulating adipocytelike cells within 8-11 days after cell seeding. The degree of adipose conversion and lipid accumulation induced by ADD far exceeded those of the previously known inducing agents such as indomethacin plus insulin. The highly potent adipogenic activity of ADD was confirmed with two other preadipose cell lines (3T3 L1 and RMT rat preadipose cells). In addition to adipogenic activity, ADD inhibited cell proliferation of preadipose cells specifically. Activity of ADD induced lipid accumulation and growth inhibition of ST 13 cells, exhibiting very similar dose-response relationships. Cell proliferation or triacylglycerol content of nonadipocytic mesenchymal cells or epithelial cells were not affected by ADD. These observations strongly suggest that ADD-induced growth inhibition is not due to the nonspecific toxicity of the drug but is tightly associated with the adipocytic character of the treated cells. The present observation provides evidence that ADD would be a powerful agent in studies that involve preadipocyte differentiation.     

Determination of antifungal activities in serum samples from mice treated with different antifungal drugs allows detection of an active metabolite of itraconazole

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.     

In vivo kinetics with rapid perturbation experiments in Saccharomyces cerevisiae using a second-generation BioScope

We present a robust second-generation BioScope: a system for continuous perturbation experiments. Firstly, the BioScope design parameters (i.e., pressure drop, overall oxygen (O2) and carbon dioxide (CO2) mass transfer, mean residence time distribution and plug flow characteristics) were evaluated. The average overall mass transfer coefficients were estimated to be 1.8E-5 m s(-1) for O2 and 0.34E-5 m s(-1) for CO2. It was determined that the O2/CO2 permeable membrane accounted for 75% and 95% of the overall resistance for O2 and CO2, respectively. The Peclet number (Pe) of the system was found to be >500 for liquid flow rates between 1 and 4 ml min(-1), ensuring plug flow characteristics. Secondly, steady-state intracellular metabolite concentrations obtained using direct rapid sampling from the fermentor were compared with those obtained by rapid sampling via the pre-perturbation sample port of the BioScope. With both methods the same metabolite levels were obtained. Thirdly, glucose perturbation experiments were carried out directly in the fermentor as well as in the BioScope, whereby steady-state Saccharomyces cerevisiae cells from a glucose/ethanol limited chemostat were perturbed by increasing the extracellular glucose concentration from 0.11 to 2.8 mM. Intracellular and extracellular metabolite levels were measured within a time window of 180 s. It was observed that the dynamic metabolite concentration profiles obtained from both perturbations were nearly the same, with the exception of the C4 metabolites of the TCA cycle, which might be due to differences in culture age.     

Metabolism of [14C] amitraz in larvae of Boophilus microplus

Amitraz, 1, 5-di(2, 4-dimethylphenyl)-3-methyl-1, 3, 5-triazapenta-1, 4-diene, labelled with 14C in the 2-methyl groups was applied to B. microplus larvae by an immersion technique. The chemical penetrated readily but never appeared in large amounts internally due to rapid cleavage to N-2, 4-dimethylphenyl-N'-methylformamidine. The expected complementary cleavage product 2, 4-dimethylformanilide was not produced in equivalent quantity. However, large amounts of polar metabolite(s) were produced. Small quantities of 2, 4-dimethylaniline and an unidentified non-polar metabolite were also produced. Of the identified chemicals only amitraz and N-2, 4-dimethylphenyl-N'-methylformamidine were toxic to larvae. Piperonyl butoxide applied simultaneously with amitraz had only a slight effect on metabolism but had a three-fold synergistic effect. SKF 525-A similarly applied had a negligible effect on both metabolism and toxicity.     

Isolation and identification of Aspergillus fumigatus mycotoxins on growth medium and some building materials

Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of A. fumigatus and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions.     

The bioavailability and metabolism of trilostane in normal subjects, a comparative study using high pressure liquid chromatographic and quantitative cytochemical assays

High pressure liquid chromatographic (HPLC) analysis of plasma taken over 8 h from ten normal male subjects medicated with 120 mg of trilostane revealed that the drug is rapidly metabolised into at least one metabolite, 17-keto trilostane. Both compounds were detected in the blood stream at concentrations greater than 2 X 10(-7) M within an hour and were cleared from the blood by 6-8 h. Approximately 3 times the concentration of metabolite was detected compared to the parent compound in most samples analysed. There were large subject to subject variations in the handling of drug. Standard curves of pure 17-keto trilostane and trilostane were parallel as assessed by cytochemical bioassay. This assay is based upon the inhibition of 3 beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of the dioestrous rat ovary. The relative potency of the metabolite compared to trilostane was 1.71 (95% confidence 1.5-2.0) over the dose range 0.15-1.5 microM. Thus, the metabolite may be the major active agent when trilostane is administered for clinical purposes. In a further 4 volunteers, who also received 120 mg trilostane and were sampled over an 8 h period, plasma was analysed independently by HPLC and cytochemical assays. In the majority of cases the bioactivity recorded (relative to a trilostane standard curve) was substantially higher than the molar sum of circulating trilostane and 17-keto-trilostane (as assessed by HPLC). However, if the relative potency of 17-keto-trilostane is taken into consideration, correlation between the two assays was excellent (r = 0.947, n = 18, P less than 0.001). This also suggests that no further active metabolites were present in the plasma samples. The drug profiles seen in the second study were essentially the same as described for the first 10 volunteers. The combination of a bioassay, which detects trilostane-like bioactivity, and HPLC, which reveals the type of metabolism, should aid our understanding of the clinical value of this potentially important drug.     

Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system.

The enzymes for luminescence in Vibrio fischeri are induced by the accumulation of a species-specific metabolite (autoinducer) in the culture medium. Tritium-labeled autoinducer was used to study the mechanism of autoinduction. When 3H-autoinducer was added to suspensions of V. fischeri or Escherichia coli, cellular concentrations equaled external concentrations. For V. fischeri, equilibration of 3H-autoinducer was rapid (within 20 s), and greater than 90% of the cellular tritium remained in unmodified autoinducer. When V. fischeri or E. coli cells containing 3H-autoinducer were transferred to autoinducer-free buffer, 85 to 99.5% of the radiotracer escaped from the cells, depending on the strain. Concentrations of autoinducer as low as 10 nM, which is equivalent to 1 or 2 molecules per cell, were sufficient for induction, and the maximal response to autoinducer occurred at about 200 nM. If external autoinducer concentrations were decreased to below 10 nM after induction had commenced, the induction response did not continue. Based on this study, a model for autoinduction is described wherein autoinducer association with cells is by simple diffusion and binding of autoinducer to its active site is reversible.