In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

Effects of Antibiotics and Bialaphos on the Growth and Development of Embryogenic Callus Cultures of Muscari armeniacum

Effects of 4 potentially selective agents for transformed cells, 3 antibiotics [kanamycin, geneticin (G418) and hygromycin] and bialaphos, as well as 2 antibiotics for eliminating Agrobacterium, carbenicillin and cefotaxime on growth and somatic embryogenesis of embryogenic calli of Muscari armeniacum cv. Blue Pearl were evaluated. Callus growth was completely inhibited by 75 mg dm⁻³ hygromycin or 4 mg dm⁻³ bialaphos, and somatic embryos were never produced on media containing 25 mg dm⁻³ hygromycin or 3 mg dm⁻³ bialaphos. Kanamycin and G418 less inhibited growth and somatic embryogenesis of the calli. On the contrary, carbenicillin and cefotaxime promoted both callus growth and somatic embryogenesis at all concentrations tested. This revised version was published online in July 2006 with corrections to the Cover Date.     

Leaf area estimation in a sugar beet cultivar by linear models

An indirect method of leaf area measurement for Rizor sugar beet cultivar was tested. Leaves were sampled during two growing seasons in a Randomised Complete Block Design experiment. For 2002 samplings, leaf area [cm²] was linearly correlated with maximum leaf width [cm] using all leaf samples (r ² = 0.83, p < 0.001) or using the means of the 8 sampling occasions (r ² = 0.97, p < 0.001). Correlations between leaf area and leaf mid vein length [cm] were weaker (r ² = 0.75, p < 0.001 and r ² = 0.93, p < 0. 001, respectively). For 2003 samplings, the area estimated by the equations was highly correlated to the measured leaf area.     

Role of phosphorus and nitrogen in photosynthetic and whole plant carbon gain and nutrient use efficiency in eastern white pine

Summary In white pine (Pinus strobus) seedlings grown in five forest soils from New York State, net photosynthetic capacity (Amax) plant^-1 was correlated with total foliar N plant^-1 (r ²=0.57), but was more highly correlated with total foliar P plant^-1 (r ²=0.82). There was no relationship (r ²<0.01) between Amax [g leaf]^-1 and foliar N [g leaf]^-1 for the pooled data set, but there was a significant (P<0.001), but weak (r ²=0.20) positive relationship between Amax [g leaf]^-1 and foliar P [g leaf]^-1 across all soils. However, within two of the five soils leaf N concentration was a significant (P<0.05) determinant of photosynthetic capacity. Due to differences in soil nutrient availabilities a large range in foliar P:N ratio (0.02–0.15) was observed, and the proportion of leaf P:N appeared to control Amax [g leaf N]^-1. Whole plant nitrogen (NUE) and phosphorus (PUE) use efficiencies were well correlated with whole plant P:N ratio. In addition, NUE was well correlated with Amax [g leaf N]^-1 and PUE was well correlated with Amax [g leaf P]^-1. However, NUE was not well correlated with PUE, and Amax [g leaf N]^-1 was not well correlated with Amax [g leaf P]^-1. These results indicated that P and/or N limitations were important components of photosynthetic nutrient relations in white pine grown in these five soils and suggest that both P and N and their proportions should be considered in analyses of photosynthesis-nutrient relations.     

The effects of G418 on the growth and metabolism of recombinant mammalian cell lines

It is widely reported that the growth of recombinant bacteria and yeast is adversely affected by increased metabolic load caused by the maintenance of plasmid copy number and recombinant protein expression. Reports suggest that recombinant mammalian systems are similarly affected by increased metabolic load. However, in comparison to bacterial systems relatively little information exists. It was the aim of this study to test the effects of recombinant gene expression on the growth and metabolism of two industrially important cell lines. A BHK and CHO cell line were stably transfected with the human gastric inhibitory peptide (h-GIP)and glucagon receptor respectively. Selection was by way of the neomycin resistance (neo ^r) gene using G418.The growth and metabolism of both cell lines was affected by the presence of G418 in a manner indicative of increased metabolic load and which appeared to be caused by over-expression of the neomycin resistance protein. The two cell lines differed in their metabolic response to G418, which suggested that some cell lines or clones may be better able to tolerate a metabolic load than others. Growth under increased metabolic load was affected by medium composition with serum, insulin and glutamine concentration as influencing factors. Implications for the use of G418 are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transfection of Eastern Oyster (Crassotrea virginica) Embryos

There is a need for research in disease resistance and microbial elimination in the eastern oyster Crassosostrea virginica. Gene transfer may lead to advances in this area, and a means of selecting transfected larvae would be useful. We transfected 3-hour-postfertilization embryos with the bacterial gene aminoglycoside phosphotransferase II (neo ^r ), which confers resistance to neomycin and related antibiotics such as G418. The antibiotic G418 was examined as a potential selective agent. A neutral red assay was used to determine survival after 48 hours of exposure to various concentrations of G418 (0–4 mg/ml). We examined the effects of electroporation and chemically mediated transfection of 3-hour-postfertilization embryos on survival to straight-hinge larvae. DNA alone was found to have no effect on survival (P > .05). For electroporation we found that increased voltage and pulse duration decreased survival (P < .05). Chemically mediated transfection did not significantly affect survival (P= .5172). Transgenic larvae were identified after electroporation and chemically mediated transfection. These larvae were reared for 24 hours and exposed to G418 at 0.3 mg/ml for 48 hours. Significant differences in survival between transfected and nontransfected larvae were detected for electroporation (P= .0147) and chemically mediated transfection (P= .037). Gene transfer was also confirmed with polymerase chain reaction and observation of expression of green fluorescent protein. This study documents the first successful insertion and expression of foreign DNA in eastern oyster larvae. Received September 5, 2000; accepted January 12, 2001.     

High expression of a recombinant human calcitonin precursor peptide in Escherichia coli

Human calcitonin (hCT) is a C-terminus -amidated peptide hormone consisting of 32 amino acids. The amidated structure is essential for its biological activities, and the C-terminal-glycine-extended precursor peptide, hCT[G], is converted to bioactive hCT by a C-terminus--amidating enzyme. An efficient production method is described for the hCT[G] peptide, as a part of the fusion protein consisting of a modified E. coli -galactosidase, linker amino acids and hCT[G]. Stable inclusion bodies of the fusion protein in E. coli were expressed by focusing on the amino acid charge, and the fusion protein was modified by inserting a basic amino acid sequence into its linker region. This modification greatly affected the formation of inclusion bodies. E. coli strain W3110/pG97S4DhCT [G]R4 could produce a large amount of stable inclusion bodies, and the hCT[G] peptide was released quantitatively from the fusion protein by S. aureus V8 protease. This enabled a large-scale production method to be established for the hCT[G] precursor peptide in E. coli to produce mature hCT.     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

Expression of Human Growth Hormone by the Eukaryotic Alga, Chlorella

A method to use Chlorella to express a recombinant heterologous protein that can be recovered from the extracellular medium has been developed. Plasmids are constructed with an extracellular secretion signal sequence inserted between a promoter region and a gene for human growth hormone (hGH). The plasmids also contain a Kan^r region which confers resistance to the antibiotic G418. Protoplasts are prepared by enzymatic treatment, and the plasmid is introduced by incubation of the protoplasts with polyethylene glycol and dimethyl sulfoxide. Cells are then grown in the presence of G418, and the medium is collected from 6 days after transfection. hGH is measured by immunoassay, and values for expressed hGH of about 200–600 ng/ml are obtained. Received: 5 November 1998 / Accepted: 25 January 1999     

The effect of different temperatures on fatty-acid synthesis and polyunsaturation in cell suspension cultures

Cell suspension cultures of Catharanthus roseus G. Don, Glycine max (L.) Merr. and Nicotiana tabacum L. were incubated with [¹⁴C]acetate, [¹⁴C]oleic acid and [¹⁴C]linoleic acid at five different temperatures ranging from 15 to 35° C. When the incubation temperature was increased, [¹⁴C]acetate was incorporated preferentially into [¹⁴C]palmitate, with a concomitant drop in [¹⁴C]oleate formation. Between 15 and 20° C, [¹⁴C]oleic acid accumulated in C. roseus cells. In all cultures, optimum desaturation of [¹⁴C]oleic acid to [¹⁴C]linoleic acid occurred between 20 and 25° C, and in G. max this was also the optimal range for desaturation of [¹⁴C]linoleic acid to [¹⁴C]linolenic acid. Elongation of [¹⁴C]palmitic acid was inhibited when cultures grown at 15° C for 25 h were subsequently incubated with [¹⁴C]acetate at 25° C. [¹⁴C]oleic acid accumulated in G. max and C. roseus cultures grown at 35° C for 25 h and subsequently incubated at 25° C. Desaturation of [¹⁴C]oleic acid increased up to 25° C, but then decreased or leveled off depending on the cell line and on the temperature prior to incubation.     

Heterosis and the metabolism of gibberellin A20 in sorghum

The correlation between gibberellin (GA) metabolism and growth rate was investigated using two Sorghum bicolor inbred lines, Hegari and AT×623, and their heterotic F₁ hybrid. Previous studies have demonstrated that this hybrid is taller and has substantially greater shoot dry weights and leaf areas than either parental inbred. [³H]GA₂₀ was applied to the leaf whorl of seedlings and after 24 hours, plants were harvested and separated into roots, shoot cylinders containing the apical meristems, and leaf blades. Chromatographic analyses of metabolites indicated the conversions of [³H]GA₂₀ to [³H]GA_{1,8 and 29}. The conversion of [²H]GA₂₀ to [²H]GA₁ was demonstrated by gas chromatography-selected ion monitoring (GC-SIM). Putative glucosyl conjugates of all of the [³H]GAs were also produced and GA₈ was identified by GC-SIM following enzymic cleavage of the putative [³H]GA₈ glucosyl conjugate fraction. Comparing the genotypes, [³H]GA₂₀ metabolism was more rapid in the shoot cylinders of the hybrid than in the shoot cylinders from inbreds. In the hybrid samples, there was a three-fold increase in the putative conjugate(s) of [³H]GA₁ which was the principal metabolite, and increased production of [³H]GA₈ and the putative conjugates of [³H]GA₂₉ and [³H]GA₈. Conversely, levels of the remaining precursor, [³H]GA₂₀, and its putative conjugate(s) were reduced in the hybrid. The rate of GA₂₀ metabolism was thus positively correlated with growth rate across these sorghum genotypes. This correlation supports a promotive role of GA in the regulation of shoot growth and in the expression of heterosis (hybrid vigor) in sorghum.     

Purification and characterisation of a novel starch synthase selective for uridine 5′-diphosphate glucose from the red alga Gracilaria tenuistipitata

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-¹⁴C]glucose than with ADP[U-¹⁴C]glucose. The activity with UDP[U-¹⁴C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-¹⁴C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native M_r of 580 kDa and displays kinetic properties similar to other α-glucan synthases such as stimulation by citrate, product (UDP) inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed. Received: 29 January 1999 / Accepted: 11 March 1999     

Regulation of glycolipid synthesis during differentiation of clonal murine muscle cells

The two clonal murine muscle cell lines G7 and G8, originally derived from the M114 line [20], represent unique models for comparative studies of myogenesis. Glycolipid synthesis was examined during differentiation using [³H]-galactose and [³H]-glucosamine as precursors. Upon G7 contact glucosylceramide labeling increased and nLcOse₅Cer labeling stopped. During membrane fusion, glucosylceramide labeling stopped and lactosylceramide became the major synthetic product. G8 cells presented a different pattern, with increased labeling of GbOse₃Cer during myogenesis. The major ganglioside synthesized by both myoblasts was GM3, and more complex structures were observed following completion of myotube formation. Total glycopeptide labeling increased when G8 myoblasts fused and remained elevated in myotubes, whereas no differences during fusion of G7 cells were noted. Upon comparison of the two clonal lines, the only consistent observation was a significant increase in the synthesis of total gangliosides and neutral glycolipid during cell contact and membrane fusion (p < 0.02). The results suggest that changes in the synthesis of specific glycolipid structures during myogenesis are unique to each muscle cell line examined. However, transient increases in synthesis of total myoblast gangliosides and neutral glycolipids may be a more general phenomenon, possibly by curbing proliferation or by altering myoblast membrane fluidity characteristics during differentiation.Abbreviations MG6 VI³NeuAc-V⁴Gal-IV³GlcNAc-nLcOse₄Cer - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - Gal galactose - GlcNH glucosamine - PBS phosphate buffered saline - CK creatine kinase     

3D-QSAR studies of checkpoint kinase 1 inhibitors based on molecular docking and CoMFA

Three-dimensional quantitative structure–activity relationship (3D-QSAR) studies were performed on a series of substituted 1,4-dihydroindeno[1,2-c]pyrazoles inhibitors, using molecular docking and comparative molecular field analysis (CoMFA). The docking results from GOLD 3.0.1 provide a reliable conformational alignment scheme for the 3D-QSAR model. Based on the docking conformations and alignments, highly predictive CoMFA model was built with cross-validated q ² value of 0.534 and non-cross-validated partial least-squares analysis with the optimum components of six showed a conventional r ² value of 0.911. The predictive ability of this model was validated by the testing set with a conventional r ² value of 0.812. Based on the docking and CoMFA, we have identified some key features of the 1,4-dihydroindeno[1,2-c]pyrazoles derivatives that are responsible for checkpoint kinase 1 inhibitory activity. The analyses may be used to design more potent 1,4-dihydroindeno[1,2-c]pyrazoles derivatives and predict their activity prior to synthesis.     

Relationship between specific leaf area,leaf thickness,leaf water content and <Emphasis Type="Italic">SPAD-502</Emphasis> readings in six Amazonian tree species

The aim of this work was to assess the effect of leaf thickness, leaf succulence (L_S), specific leaf area (SLA), specific leaf mass (W_s) and leaf water content (LWC) on chlorophyll (Chl) meter values in six Amazonian tree species (Carapa guianensis, Ceiba pentandra, Cynometra spruceana, Pithecolobium inaequale, Scleronema micranthum and Swietenia macrophylla). We also tested the accuracy of a general calibration equation to convert Minolta Chl meter (SPAD-502) readings into absolute Chl content. On average, SPAD values (x) increased with fresh leaf thickness (FLT [μm] = 153.9 + 0.98 x, r ² = 0.06**), dry leaf thickness (DLT [μm] = 49.50 + 1.28 x, r ² = 0.16**), specific leaf mass (W_s [g (DM) m⁻²] = 6.73 + 1.31 x, r ² = 0.43**), and leaf succulence (L_S [g(FM)] m⁻² = 94.2 + 1.58 x, r ² = 0.19**). However, a negative relationship was found between SPAD values and either specific leaf area [SLA (m² kg⁻¹) = 35.1 − 0.37 x, r ² = 0.38**] or the leaf water content (LWC [%]= 80.0 − 0.42 x, r ² = 0.58**). Leaf Chl contents predicted by the general calibration equation significantly differed (p<0.01) from those estimated by species-specific calibration equations. We conclude that to improve the accuracy of the SPAD-502 leaf thickness and LWC should be taken into account when calibration equations are to be obtained to convert SPAD values into absolute Chl content.     

Inherent G418-resistance in hybridization of industrial yeasts

Eight strains of sake yeast exhibited inherent-resistance to 100 μg/ml of Geneticin (G418). Fourteen wine yeasts and 1 shochu yeast (Saccharomyces cerevisiae) and 1 miso yeast (Zygosaccharomyces rouxii) were inherent G418-sensitive. The petites converted from inherent G418-resistants by treatment with ethidium bromide retained G418-resistance (ϱ⁻ G418R), and thus were hybridized by electrofusion with the wine yeast W3 (ϱ⁺ G418S, wild type). A lag phase of 12–18 h was required prior to administration of the drug in glycerol medium when selecting G418-resistant hybridization products. Colonies were formed in the regeneration medium at a frequency of about 1 × 10⁻⁵ per used protoplasts. No growth of any parental strain (10⁶/_~10⁷ protoplasts) separately subjected to electrofusion and regeneration was observed. The hybridization products were G418-resistant “grande” strains (ϱ⁻ G418R) in which the genetic traits of parental strains had been complemented. Uninucleate cells (DAPI staining) of the hybridization products showed CHEF electrophoretic karyotypes similar to that of wine yeast, but possessed a single chromosome (approx. 320 kb) presumably from sake yeast.     

The <Emphasis Type="Italic">pydA</Emphasis>–<Emphasis Type="Italic">pydB</Emphasis> fusion gene produces an active dioxygenase–hydrolase that degrades 3-hydroxy-4-pyridone,an intermediate of mimosine metabolism

The objective of this research was to construct a pydA–pydB hybrid gene that encodes a functional dioxygenase–hydrolase (PydA–PydB) fusion protein for degradation of 3-hydroxy-4-pyridone (HP). HP is an intermediate in both synthesis and degradation of mimosine, a toxic amino acid produced by the tree legume Leucaena leucocephala. Computer-generated models of the fusion proteins suggested that joining of PydA and PydB with 0, 3, or 7 glycine residues as a linker should produce a functional PydA–PydB fusion protein. Accordingly, three hybrid genes, G0, G3, and G7, were constructed in which pydA and pydB were connected with 0, 9, and 21 nucleotides, respectively, encoding the glycine residues of the linker region. When these hybrid genes were expressed in Rhizobium and Escherichia coli, only one of them, G3, produced a functional PydA–PydB fusion protein, having both the dioxygenase and hydrolase activities. The G3 hybrid gene could complement both pydA and pydB mutants of Rhizobium, and E. coli lysate containing the overexpressed G3 protein was able to degrade HP. This hybrid gene may be useful for developing mimosine-free L. leucocephala plants in the future.     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Acetylcholine Synthesis and CO2 Production from Variously Labeled Glucose in Rat Brain Slices and Synaptosomes

Abstract: The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-¹⁴C]glucose, and ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O₂ or antimycin) or stimulated (2,4- dinitrophenol [DNP] or 35 mm -K⁺) ¹⁴CO₂ production from [2-¹⁴C]- or [3,4-¹⁴C]glucose. Incorporation of [U-¹⁴C]glucose into ACh was reduced under low O₂ and by antimycin or DNP (by 51-93%) and stimulated by 35 mm -K⁺ (by 30-60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-¹⁴C]- and [2-¹⁴C]glucose were linearly related (r= 0.741 and 0.579, respectively). The difference in the rate of ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low 0₂ and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K⁺ stimulation in slices) compared with control values under 100% O₂. ACh synthesis and efflux were more closely related (r= 0.860) than ACh synthesis and ¹⁴CO₂ production from variously labeled glucoses.