A Novel Isourazole Herbicide, Fluthiacet-Methyl, is a Potent Inhibitor of Protoporphyrinogen Oxidase after Isomerization by Glutathione S-Transferase

A novel isourazole herbicide, fluthiacet-methyl (methyl [[2-chloro-4-fluoro-5-[(5,6,7,8-tetrahydro-3-oxo-lH,3H-[l,3,4]thiadiazolo[3,4-a]pyridazin-l-ylidene)amino]phenyrjthio]acetate;experimental code name, KIH-9201) promoted the leakage of electrolytesfrom cotyledons of velvetleaf (Abtilon theophtasti Medic) andcotton (Gossypium hirsutum L.) plants that are sensitive tothis compound. It induced the accumulation of protoporphyrinIX in cotyledons of cotton and inhibited Chl biosynthesis incotyledons of velvetleaf and cotton at low concentrations (I₅₀values, 10–12 nM). Fluthiacet-methyl was converted toits urazole by glutathione S-transferase that had been partiallypurified from velvetleaf. The urazole inhibited protoporphyrinogenoxidase (Protox, EC 1.3.3.4 [EC] ) from some plants, including velvetleaf,at low concentrations (I₅₀ values, 5.1–11 nM), whereasfluthiacet-methyl was not as potent. The effects in vivo (electrolyteleakage and inhibition of Chi biosynthesis) of fluthiacet-methylwere correlated with the inhibition of Protox activity by theurazole and not with the action of fluthiacet-methyl itself.From these results, it is concluded that fluthiacet-methyl inhibitsProtox activity after conversion to the corresponding urazoleby glutathione S-transferase. It is in this way that fluthiacet-methylexerts its effect as a light-dependent peroxidizing herbicide. (Received November 1, 1994; Accepted March 6, 1995)     

Comparative effects of CGA-92194, cyometrinil,and flurazole on selected metabolic processes of isolated soybean leaf cells

The potential adverse phytotoxic effects of the herbicide safeners CGA-92194 {-[1,3-dioxolan-2-yl-methoxy)imino]benzeneacetonitrile}, cyometrinil [-(cyanomethoxy)imino-benzeneacetonitrile] and flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazole-carboxylate] on selected metabolic processes of enzymatically isolated leaf cells of soybean [Glycine max (L.) Merr.] were compared in time- and concentration-course studies. CO₂ fixation, protein synthesis, RNA synthesis, DNA synthesis, and lipid synthesis were assayed by the incorporation of NaH¹⁴CO₃, [¹⁴C]-leucine, [¹⁴C]-uracil, [³H]thymidine, and [¹⁴C]-acetate, respectively, into the isolated cells. CGA-92194 and cyometrinil behaved similarly, and at low concentrations (0.1, 1, and 10 M) they stimulated rather than inhibited the five metabolic processes assayed, following incubation periods of up to 2 h. At the highest concentration of 100 M, both safeners inhibited all metabolic processes of the soybean leaf cells but neither compound exhibited rapid and distinct inhibitions as might be expected in the case of inhibition of a primary target site by a potent inhibitor. At low concentrations and early incubation periods (30 and 60 min), flurazole effects on all metabolic processes were also stimulatory rather than inhibitory. However, the stimulation of CO₂ fixation by 0.1 and 1.0 M was highly significant. At 100 M flurazole was extremely potent on all metabolic processes of soybean leaf cells examined. At the 2-h incubation period, flurazole also inhibited all metabolic processes at concentrations lower than 100 M. The sensitivity of the five metabolic processes to flurazole decreased in the following order: photosynthesis = lipid synthesis >DNA synthesis>protein synthesis>RNA synthesis.Contribution No. 534, Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg VA 24061 USA.     

Evaluation of diuron tolerance and biotransformation by the white-rot fungus Ganoderma lucidum

The white rot basidiomycete Ganoderma lucidum was evaluated for its capability to tolerate and to degrade the herbicide diuron. Diuron at a subtoxic concentration was added at the start of the cultivation in glucose liquid stationary cultures. Under this condition diuron was a laccase inducer. Almost 50% of the initially present diuron was removed after 15 d of cultivation. Two diuron metabolites were found N′-(3,4-dichlorophenyl)-N-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The addition of the cytochrome P450 inhibitors 1-aminobenzotriazole and piperonyl butoxide reduced significantly the capability of the fungus in degrading diuron. The activities of superoxide dismutase and catalase were significantly increased in the mycelial extracts by the presence of diuron. On the other hand, diuron did not cause any significant alteration in the levels of reactive oxygen species. Additionally, laccase could also degrade diuron in vitro and this degradation was increased by the addition of synthetic mediators, 3-ethylbenzthiazoline-6-sulphonic acid and acetylacetone. Significant reduction in the toxicity, as evaluated by the Lactuca sativa bioassay, was observed after G. lucidum treatment. In conclusion, G. lucidum is able to metabolize diuron by intra- and extracellular mechanisms, without the accumulation of toxic products.     

Absorption,translocation, and metabolism of 14C-thifensulfuron in soybean (Glycine max), spurred anoda (Anoda cristata), and velvetleaf (Abutilon theophrasti)

The absorption, translocation, and metabolism of thifensulfuron-methyl {methyl 3-[[[[(4-methoxy)-6-methyl-1,3,5-triazin-2-yl]-amino]-carbonyl] amino]sulfonyl]-2-thiophenecarboxylate} were investigated in tolerant Essex soybean [Glycine max (L.) Merr.], moderately tolerant Vance soybean, and spurred anoda [Anoda cristata (L.) Schlecht.], and susceptible velvetleaf (Abutilon theophrasti Medic.). Radiolabeled (thiophene-2-¹⁴C) thifensulfuron-methyl was absorbed readily by young seedlings of all species following a foliar spray with the herbicide. Spot-applied ¹⁴C-thifensulfuron-methyl was absorbed by the treated leaf of all species. Absorption of thifensulfuron-methyl was limited when excised stems of all species were dipped into the herbicide solution for 2 h. Translocation of absorbed thifensulfuron-methyl to other plant parts was limited in all species, regardless of the method of its application. Root exudation of leaf-applied thifensulfuron-methyl was observed in all species and it was higher in seedlings of spurred anoda and velvetleaf. The two soybean cultivars metabolized 62–70% of absorbed thifensulfuron-methyl at 3 days after treatment with spot-applied ¹⁴C-thifensulfuron. Velvetleaf and tolerant spurred anoda metabolized about 50% of the absorbed herbicide. The major metabolite formed in all species appeared to be deesterified thifensulfuron acid. Differential metabolism seems to be a contributing factor in the selectivity of thifensulfuron-methyl between the two soybean cultivars and velvetleaf. The metabolic basis for the moderate tolerance of spurred anoda to thifensulfuron-methyl is not understood at the present time.Plant Pathology, Physiology, and Weed Science Department, Contribution no. 628.     

Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum

The aim of this work was to establish the pathways of carbohydrate oxidation used in the dark by leaves of Pisum sativum and Triticum aestivum. Segments of young and mature leaves of pea released the carbons of glucose-[¹⁴C] as ¹⁴CO₂ in the order 3,4 > 1 > 2 > 6 whereas in segments of young and mature leaves of wheat the order was 3,4 > 1 > 6 > 2. The detailed labelling of the constituents of mature leaves of wheat by glucose-[1-¹⁴C], -[2-¹⁴C], -[3,4-¹⁴C], and -[6-¹⁴C] was determined and showed that the high yield of CO₂ from C-6 relative to that from C-2 was due to release of C-6 during pentan synthesis. Estimates were made of the maximum catalytic activities of phosphofructokinase and glucose-6-phosphate dehydrogenase in pea and wheat leaves of three ages. The results of all the above investigations strongly indicate that both pea and wheat leaves in the dark oxidize carbohydrate via glycolysis and the pentose phosphate pathway with the latter accounting for no more than a third of the total. No evidence was obtained of any major change in the relative activities of the two pathways during the development of either type of leaf.     

Effects of sethoxydim on the metabolism of isolated leaf cells of soybean [Glycine max (L.) Merr.]

The effects of the herbicide sethoxydim {2-[1-ethoxyimino)-butyl]-5-[2-(ethylthio)-propyl]-3-hydroxy-2-cyclohexen-1-one} on selected metabolic processes of enzymatically isolated leaf cells from soybeans [Glycine max (L.) Merr., cv. Essex] were studied. Photosynthesis, protein, ribonucleic acid (RNA), and lipid synthesis were measured by the incorporation of NaH¹⁴CO₃, [¹⁴C]leucine, [¹⁴C]uracil, and [¹⁴C]acetic acid into the isolated soybean cells, respectively. Time-course and concentration studies included incubation times of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 M of sethoxydim. Lipid synthesis was the most sensitive and first metabolic process inhibited by the lowest concentration of sethoxydim. Photosynthesis was not affected significantly by sethoxydim and did not appear to be a target site involved in its herbicidal action. RNA and protein syntheses were inhibited significantly but only by the high concentrations of sethoxydim. It is suggested that sethoxydim exhibits its phytotoxic action by altering or modifying the lipid composition of plant membranes.Abbreviations MES [2-(N-morpholino)-ethanesulfonic acid] - HEPES [N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid]     

Transition of Lipid Synthesis from Chloroplasts to a Cytoplasmic System during Hardening in Chlorella ellipsoidea

Chlorella ellipsoidea Gerneck (IAM C-27) was synchronously grown and cells at an intermediate stage in the ripening phase of the cell cycle were hardened at 3 C for 48 hours. At various times of hardening, the cells were pulse-labeled for 4 minutes with [¹⁴C]NaHCO₃ in the light or with [¹⁴C]glucose in the dark, and the incorporation rate of ¹⁴C into total lipids was determined. A high incorporation rate of [¹⁴C]NaHCO₃ at zero time of hardening decreased after 6 hours. In the next 15 hours, a distinct increase was noted. This increase occurred prior to the development of frost hardiness. Cycloheximide completely inhibited both the increase and the development, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea remarkably lowered the high incorporation rate at zero time. The incorporation rate of [¹⁴C]glucose increased along with hardiness in the dark. These results suggest that the major site of lipid synthesis shifts from chloroplasts to a cytoplasmic system during hardening of Chlorella.     

Ribohomopolymer formation as a source of error in the radioactive precursor incorporation studies of nuclear DNA-dependent RNA synthesis

Isolated rat liver nuclei contain ribohomopolymer polymerases with relative activities in the following order: Poly (A) (100%) > Poly (C) (62%) > Poly (U) (34%) > Poly (G) (13%). Because these enzymes share the same substrates with the nuclear DNA-dependent RNA polymerases in nuclei, labelled precursor is therefore concurrently incorporated into both RNA and ribohomopolymer. Thus, experiments designed to study DNA-dependent RNA synthesis are subjected to error. It is estimated when [¹⁴C]ATP is used as the labelled precursor, the error is as high as 35%; [¹⁴C]CTP, 20%; [¹⁴C]UTP or [¹⁴C]GTP, 10%.     

Effects of Light and Inhibitors on Glutamate Metabolism in Leaf Discs of Vicia faba L: Sources of ATP for Glutamine Synthesis and Photoregulation of Tricarboxylic Acid Cycle Metabolism

Metabolism of [¹⁴C]glutamate was studied in leaf discs of Vicia faba L. in light and in darkness. In white light glutamine was the main labeled product. In the dark label was principally in compounds closely associated with tricarboxylic acid cycle metabolism, predominantly aspartate. Entry of label from glutamate into tricarboxylic acid metabolism appeared to be at least partially by decarboxylation of glutamate to γ-amino butyric acid, followed by conversion to succinate. 3-(3,4-dichlorophenyl)-1, 1-Dimethylurea inhibited light-enhanced synthesis of glutamine and caused reversion toward the dark pattern of metabolism. Methionine sulfoximine severely inhibited glutamine synthesis and caused accumulation of labeled malate.     

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.     

Effects of Photosystem II Herbicides on the Photosynthetic Membranes of the Cyanobacterium Aphanocapsa 6308

The effects of the photosystem II herbicides diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on the photosynthetic membranes of a cyanobacterium, Aphanocapsa 6308, were compared to the effects on a higher plant, Spinacia oleracea. The inhibition of photosystem II electron transport by these herbicides was investigated by measuring the photoreduction of the dye 2,6-dichlorophenol-indophenol spectrophotometrically using isolated membranes. The concentration of herbicide that caused 50% inhibition of electron transport (I₅₀ value) in Aphanocapsa membranes for diuron was 6.8 × 10⁻⁹ molar and the I₅₀ value for atrazine was 8.8 × 10⁻⁸ molar. ¹⁴C-labeled diuron and atrazine were used to investigate herbicide binding with calculated binding constants (K) being 8.2 × 10⁻⁸ molar for atrazine and 1.7 × 10⁻⁷ molar for diuron. Competitive binding studies carried out on Aphanocapsa membranes using radiolabeled [¹⁴C]atrazine and unlabeled diuron revealed that diuron competed with atrazine for the herbicide-binding site. Experiments involving the photoaffinity label [¹⁴C]azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-2-triazine) and autoradiography of polyacrylamide gels indicated that the herbicide atrazine binds to a 32-kilodalton protein in Aphanocapsa 6308 cell extracts.     

Herbicide and plastoquinone-binding proteins of Photosystem II reaction center complexes from the thermophilic cyanobacterium,Synechococcus sp.

Photoaffinity labeling of Synechococcus Photosystem (PS) II preparations with radioactive azido-derivatives of three herbicides and of plastoquinone was carried out to identify herbicide and plastoquinone-binding proteins. [¹⁴C]Azido-atrazine and [¹⁴C]azido-monuron specifically labeled the 28 kDa polypeptide of the PS II reaction center complex, which is sensitive to 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). No specific labeling of this polypeptide with azido-atrazine was found in CP2-b (PS II reaction center lacking the 40 kDa subunit) which is insensitive to DCMU. [³H]Azido-dinoseb reacted with the 28 kDa polypeptide and the 47 kDa chlorophyll-carrying protein. The labeling with [³H]azido-plastoquinone resulted in the incorporation of the radioactivity exclusively into the 47 kDa polypeptide. It is concluded that the 28 kDa polypeptide is the herbicide-binding protein of the cyanobacterium and that the 47 kDa polypeptide has a binding site for plastoquinone and for phenol-type herbicides.     

Ethylene formation in Pisum sativum and Vicia faba protoplasts

Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-1-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-¹⁴C]methionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Evidence for two different herbicide-binding proteins at the reducing side of Photosystem II

The binding behaviour at the thylakoid membrane of the radioactively labelled phenolic inhibitors 2-iodo-4-nitro-6-[2′,3′-³H]isobutylphenol and 3,5-diiodo-4-hydroxy[U-¹⁴C]benzonitrile (ioxynil) has been studied. As judged from displacement experiments with other herbicides, phenolic herbicides and herbicides as represented best by 3-(3,4-dichlorophenyl)-1,1-dimethylurea have different binding sites at the reducing side of Photosystem II. The binding parameters of phenolic herbicides are not, or only slightly, affected by trypsin treatment of chloroplasts. In chloroplasts, besides free pigments, lipids, and the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex, a protein of molecular weight 41 000 is radioactively labelled by the photoaffinity label 4-nitro-2-azido-6-[2′,3′-³H]isobutylphenol. The amount of radioactivity bound to the 41 kDa protein is diminished if chloroplasts are incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prior to addition of the photoaffinity label, but not if the 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol is used instead. These two compounds are characteristic representatives of inhibitiors acting at the reducing or the oxidizing site of plastoquinone, respectively. Based on these results, a model for two different herbicide-binding proteins at the reducing side of Photosystem II is presented.     

Activity of the pentose phosphate pathway during lignification

Summary We did this work to see if there is a correlation between lignin synthesis and the activity of the pentose phosphate pathway. Excision of the third internode of the stem of Coleus blumei Benth. followed by incubation on sucrose and indoleacetic acid led to extensive formation of tracheids. During this lignification we determined the activities of glucose-6-phosphate dehydrogenase and fructose-1,6-diphosphate aldolase, and the extent to which [1-¹⁴C]-,[3,4-¹⁴C]-, and [6-¹⁴C]glucose labelled CO₂ and the major cellular components. The results indicate that the pentose phosphate pathway was active during lignification, and that the activity of this pathway relative to glycolysis increased at the onset of lignification. Explants of storage tissue of Helianthus tuberosus L. were cultured under conditions which caused extensive lignification. ¹⁴CO₂ production from [1-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose indicated activity of the pentose phosphate pathway during tracheid formation. We suggest that lignification is accompanied by appreciable activity of the pentose phosphate pathway and that this could provide the reducing power for lignin synthesis.Abbreviations NADP nicotinamide-adenine dinucleotide phosphate - IAA indoleacetic acid     

Rapid mineralization of the phenylurea herbicide diuron by Variovorax sp. strain SRS16 in pure culture and within a two-member consortium

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations, and consequently, it is frequently detected as a major water contaminant in areas where there is extensive use. We constructed a linuron [N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea]- and diuron-mineralizing two-member consortium by combining the cooperative degradation capacities of the diuron-degrading organism Arthrobacter globiformis strain D47 and the linuron-mineralizing organism Variovorax sp. strain SRS16. Neither of the strains mineralized diuron alone in a mineral medium, but combined, the two strains mineralized 31 to 62% of the added [ring-U-(14)C]diuron to (14)CO(2), depending on the initial diuron concentration and the cultivation conditions. The constructed consortium was used to initiate the degradation and mineralization of diuron in soil without natural attenuation potential. This approach led to the unexpected finding that Variovorax sp. strain SRS16 was able to mineralize diuron in a pure culture when it was supplemented with appropriate growth substrates, making this strain the first known bacterium capable of mineralizing diuron and representatives of both the N,N-dimethyl- and N-methoxy-N-methyl-substituted phenylurea herbicides. The ability of the coculture to mineralize microgram-per-liter levels of diuron was compared to the ability of strain SRS16 alone, which revealed the greater extent of mineralization by the two-member consortium (31 to 33% of the added [ring-U-(14)C]diuron was mineralized to (14)CO(2) when 15.5 to 38.9 mug liter(-1) diuron was used). These results suggest that the consortium consisting of strains SRS16 and D47 could be a promising candidate for remediation of soil and water contaminated with diuron and linuron and their shared metabolite 3,4-dichloroaniline.     

The effect of glucose on the control of carbohydrate metabolism in ripening bananas

The effect of exogenous glucose on the major fluxes of carbohydrate metabolism in cores of climacteric fruit of banana (Musa cavendishii Lamb ex Paxton) was determined with the intention of using the effects in the application of top-down metabolic control analysis. Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21 °C. Cores were removed from climacteric fruit and incubated in 100 or 200 mM glucose for 4 or 6 h. The rates of starch breakdown, sucrose and fructose accumulation and CO₂ production were measured. The steady-state contents of hexose monophosphates, adenylates and pyruvate were determined. In addition, the detailed distribution of label was determined after supply of the following: [U-¹⁴C]-, [1-¹⁴C]-, [3,4¹⁴C]and [6-¹⁴C]glucose, and [U-¹⁴C]glycerol. The data were used to estimate the major fluxes of carbohydrate metabolism. Supply of exogenous glucose led to increases in the size of the hexose-monophosphate pools. There was a small stimulation of the rate of sugar synthesis and a major increase in the rate of starch synthesis. Starch breakdown was inhibited. Respiration responded to the demand for ATP by sugar synthesis. The effect of glucose on fluxes and metabolite pools is discussed in relation to our understanding of the control and regulation of carbohydrate metabolism in ripening fruit.Abbreviations Glc6P glucose-6-phosphate - Glc1P glucose-1-phosphate - Fru6P fructose-6-phosphate - AEC adenylate energy charge We thank Geest Foods Group, Great Dunmow, Essex, UK for giving us the bananas. SAH thanks the managers of the Broodbank Fund for a fellowship.     

Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I₅₀ values for growth, chlorophyll (Chl), β-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [¹⁴C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.     

Effects of 1-Aminocyclopropane-1-carboxylic Acid Production on the Changes in the Polyamine Levels in Hiproly Barley Callus after Auxin Withdrawal

Contents of polyamines and 1-aminocyclopropane-1-carboxylic acid (ACC) in Hiproly barley callus were examined under different culture conditions. After auxin withdrawal, the contents of free polyamines changed conversely to the contents of ACC. In the absence of auxin, incorporation of l-[3,4–¹⁴C]methionine into polyamines and the activity of S-adenosylmethionine decarboxylase (SAMDCase) in the callus increased, then remained stable, but incorporation of l-[3,4- ¹⁴C]methionine into ACC, precursor of ethylene and ACC synthase activity once declined and increased again.

Aminooxyacetic acid (AOA) affected the increase in the levels of polyamines in the callus. 1- Aminoisobutyric acid (AIB) had a slight effect on the polyamine production. The incorporation of l-[3,4–¹⁴C]methionine into ACC and ACC synthase activity were inhibited by AOA, but not by « 4 AIB. AOA stimulated the activity of SAMDCase, and also enhanced the incorporation of l-[3,4- ¹⁴C]methionine into polyamines in the callus. Methylglyoxal-bis(guanylhydrazone) (MGBG) greatly enhanced the ACC production. The rate of incorporation of l-[3,4–¹⁴C]methionine into ACC and ACC synthase activity in the callus were significantly enhanced by MGBG. MGBG strongly inhibited SAMDCase activity and the incorporation of l-[3,4–¹⁴C]methionine into polyamines. Moreover, the synthesis of polyamines was inhibited by MGBG.

These results suggested that in Hiproly barley callus ACC production has an important effect on changes in the polyamine levels, and that polyamine and ethylene biosynthetic pathways are regulated by competition against each other.     

Species differences and mechanism of action of A<Subscript>3</Subscript> adenosine receptor allosteric modulators

Activity of the A₃ adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A₃AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[³⁵S]thio]triphosphate ([³⁵S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A₃ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A₃ARs but provided only weak activity at mouse A₃ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A₃AR agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N ⁶-(4-amino-3-[¹²⁵I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([¹²⁵I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A₃AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [¹²⁵I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A₃AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.