The Intra-cellular Localization of the Herbicide Diphenamid in Corn Root .Tip

The intra-cellalar localization of the herbicide N,N-dimethyl–2,2-diphenylacetamide (diphenamid) was examined in 10 mm root tips of corn seedlings (Zea mays L. cv. Shawnee). Experiments are described which culminate in the observation that a significant, measurable effect on root growth occurs within 12 hours after treatment with diphenamid. Corn seedlings were then treated with 14-C-diphenamid for 12 hours and the 10 mm root tips excised and homogenized. By use of differential centrifugation and gel filtration, the root homogenate was separated into fractions; as follows; cell wall, mitochondria, microsomal, protein and eluate. The highest concentration of the herbicide was found in the root tips. Furthermore, most of the radioactivity was found in the eluate. Results are discussed as to localization of diphenamid in the microsomal and protein fraction and possible mode of action.     

Metabolism of diclofop-methyl (methyl-2-[4-(2',4'-dichlorophenoxy)phenoxy] propanoate) in cell suspensions of diploid wheat (Triticum monococcum)

The metabolism of the herbicide, diclofop-methyl (methyl-2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate, in cell suspensions of resistant diploid wheat (Triticum monococcum L.) was determined 1, 8, and 24 h after treatment with ¹⁴C-diclofop-methyl. The ¹⁴C-labeled products were identified by thin layer chromatographic comparisons to appropriate standards. Eight hours after treatment with 5 μM diclofop-methyl in 0.8% acetone (neither of which were toxic to the cell suspensions) 87.2% (84.0% methanol soluble, 3.2% methanol insoluble) of the total ¹⁴C recovered (90.4%) was in the cells and 12.8% was in the medium. Major metabolites found in methanol extracts of the cells were diclofop (2-[4-(2′,4′-dichlorophenoxy)phenoxylpropionic acid), diclofop hydroxylated at an undetermined position on the 2,4-dichlorophenyl ring (ring-OH diclofop), and conjugates of ring-OH diclofop. Acid hydrolysis of the conjugated metabolite(s) yielded ring-OH diclofop and diclofop. Twenty-four hours after treatment 70–75% of the total ¹⁴C recovered was present as conjugated metabolites. With the exception of ring-OH diclofop, all metabolites present in the cells were also recovered from the medium. A metabolite found in low concentrations in the medium that yielded diclofop upon hydrolysis was identified as an ester conjugate. Toxic concentrations of diclofop-methyl (10 and 20μM) had no effect on the metabolism of the herbicide, although the rate of uptake was slower than for cells treated with 5 μM herbicide. The products of diclofop-methyl metabolism in cell suspensions of T. monococcum were compared to previous data from T. aestivum intact plant metabolism of diclofop-methyl.     

Application of X-ray microanalysis to cell suspensions of oil palm (Elaeis guineensis Jacq.)

X-ray microanalysis has been used to determine the elemental composition of oil-palm (Elaeis guineesis) cell suspensions without the use of cryoprotectants. Results based on individual cells were gathered over a typical growth cycle of 14 d. During the log phase (5–7 d) there is an increase in the number of cells containing high concentrations of both K (400 mmol kg^-1 dry weight) and P (400 mmol kg^-1 dry weight). Morphologically these cells had thin cell walls and were frequently joined to other cells (two to five cells per clump).     

Preparative isolation and purification of four flavonoids from the petals of nelumbo nucifera by high‐speed counter‐current chromatography

Introduction – Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective – To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high‐speed counter‐current chromatography (HSCCC). Methodology – Following an initial clean‐up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC‐PAD, ESI‐MS, ¹H‐NMR and ¹³C‐NMR. Results – The separation was performed using a two‐phase solvent system composed of ethyl acetate–methanol–water–acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow‐rate of 1.0 mL/min in the head‐to‐tail elution mode. Ultimately, 5.0 mg syringetin‐3‐O‐β‐d‐glucoside, 6.5 mg quercetin‐3‐O‐β‐d‐glucoside, 12.8 mg isorhamnetin‐3‐O‐β‐d‐glucoside and 32.5 mg kaempferol‐3‐O‐β‐d‐glucoside were obtained from 125 mg crude sample. Conclusion – The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright © 2009 John Wiley & Sons, Ltd.     

Formation of edulinine and furoquinoline alkaloids from quinoline derivatives by cell suspension cultures of Ruta graveolens

The formation of furoquinoline alkaloids and of edulinine, elaborated by cell suspension cultures of Ruta graveolens, was found to occur by way of 4-hydroxy-2-quinolone. Other substrates transformed to furoquinolines included 4-hydroxy- and 4-methoxy-3-(3-methyl-2-butenyl)-2-quinolone, known earlier as natural precursors in studies with whole plants. Involvement of dictamnine as a natural precursor of 8-methoxydictamnine (γ-fagarine) and skimmianine was proved using ¹⁴C-labelled compounds. Edulinine in the cell suspensions was formed from such substrates as 4-hydroxy-N-methyl-2-quinolone, 4-hydroxy-3-(3-methyl-2-butenyl)-N-methyl-2-quinolone and its 4- methyl ether; this is probably the natural biosynthetic sequence. Changes in alkaloid yields were noted upon prolonged subculturing.     

The biosynthesis of cyanogenic glucosides in Linum usitatissimum (linen flax) in Vitro

Microsomal preparations from dark-grown Linum usitatissimum (linen flax) seedlings synthesize acetone cyanohydrin, the precursor of the cyanogenic glucoside linamarin, from valine in the presence of NADPH. N-Hydroxyvaline and isobutyraldoxime, which are predicted intermediates in the pathway, are also converted into products. These microsomal preparations also convert isoleucine into 2-butanone cyanohydrin the precursor of lotaustralin. The biosynthetic activity is located exclusively in the developing cotyledons.     

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

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.     

Photosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv. Xanthi) Cell Suspension Cultures

Photoheterotrophic growth of cell suspensions of Nicotiana tabacum L. (cv. Xanthi) in organic culture medium enriched in sucrose (30 g per liter) showed a classical sigmoid growth curve. The cells developed functional chloroplast structures during the exponential growth phase, when their chlorophyll content increased steadily. A limited drop (30%) in the chlorophyll amount and structural changes of the plastids (starch accumulation) were observed during the lag phase. The measurements of photosynthetic capacities (O₂ evolution and CO₂ fixation) during the growth cycle revealed changes in the photosynthetic ratio (O₂/CO₂), which was near 1 during the lag and stationary phases and near 2 during exponential growth. During exponential growth there was also a rapid NO₃^? uptake. Analysis of label distribution among the products of ¹⁴CO₂ fixation showed that both CO₂ assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic pathway) were operative with an important increase of the capacity of the latter during the exponential growth phase. Maximum rate of oxygen evolution, either endogenous or with p-benzoquinone as Hill reagent, as well as the increased CO₂ Fixation capacity via the non-autotrophic pathway during the exponential phase were concomitant with a high cyanide inhibited O₂ uptake.     

Acylation of steryl glucosides by phospholipids. Solubilization and properties of the acyl transferase.

Particulate enzyme preparations of cotton fibers catalyze the acylation of exogenous steryl glucoside to form acylated steryl glucoside. The acyl transferase involved in this reaction was solubilized by treatment of the membrane fractions with Triton X-100 and was partially purified by chromatography on DEAE-cellulose and gel filtration. This solubilized enzyme had an absolute requirement for Triton X-100 and phospholipid in order to catalyze the acylation of the steryl glucoside. The best phospholipid substrate was phosphatidylethanolamine but egg and soybean phosphatidylcholine were also active. The phospholipid was shown to function as an acyl donor by demonstrating that [¹⁴C]fatty acid from ¹⁴C-labeled phospholipid could be transferred to steryl-[³H]glucoside to form [¹⁴C,³H]acylated steryl glucoside. Saponification of this compound yielded [¹⁴C]fatty acid and steryl-[⁷H]glucoside.     

Cyanogenic and non-cyanogenic pyridone glucosides from Acalypha indica (Euphorbiaceae)

Seven cyanopyridone derivatives and one corresponding seco compound have been isolated from a methanolic extract of the inflorescences and leaves of Acalypha indica L. (Euphorbiaceae). The absolute configuration of the main cyanogenic glucoside acalyphin, (−)-(5R,6S)-5-cyano-5-β-d-glucopyranosyloxy-6-hydroxy-4-methoxy-1-methyl-2(5,6-dihydro)-pyridone, was deduced from an X-ray crystallographic study. In addition, the 6R-epimer of acalyphin, epiacalyphin, and the corresponding pair of N-demethyl derivatives were isolated. The corresponding amide of acalyphin and a 1′,2′-glucosyl-fused epiacalyphin amide were isolated from air-dried material. Structural elucidation was performed by means of ¹H and ¹³C NMR-spectra, chiroptical methods such as CD-spectroscopy and optical rotation. Two further corresponding derivatives, an aromatized compound and an open-chain structure, were isolated from the aqueous phase.     

Reduction of Hg(II) to Hg(0) by Biogenic Magnetite from two Magnetotactic Bacteria

Understanding the biogeochemical cycle of the highly toxic element mercury (Hg) is necessary to predict its fate and transport. In this study, we determined that biogenic magnetite isolated from Magnetospirillum gryphiswaldense MSR-1 and Magnetospirillum magnetotacticum MS-1 was capable of reducing inorganic mercury [Hg(II)] to elemental mercury [Hg(0)]. These two magnetotactic bacteria (MTB) lacked mercuric resistance operons in the genomes. However, they revealed high resistance to Hg(II) under atmospheric conditions and an even higher resistance under microaerobic conditions (1% O₂ and 99% N₂). Neither strain reduced Hg(II) to Hg(0) under atmospheric conditions. However, a slow rate (0.05–0.21 µM·d^?1) of Hg(II) loss occurred from late log phase to stationary phase in two MTBs' culture media under microaerobic conditions. Increased Hg(II) entered both cells under microaerobic conditions relative to atmospheric conditions. The majority of Hg(II) was still blocked by the cell membrane. Hg(II) reduction was more effective when biogenic magnetite was extracted out, with or without the magnetosome membrane envelope. When magnetosome membrane was present, 8.55–13.53% of 250 nM Hg(II) was reduced to Hg(0) by 250 mg/L biogenic magnetite suspension within 2 hours. This ratio increased to 55.07–64.70% while magnetosome membrane was removed. We concluded that two MTBs contributed to the reduction of Hg(II) to Hg(0) at a slow rate in vivo. Such reduction was more favorable to occur when biogenic magnetite is released from dead cells. It proposed a new biotic pathway for the formation of Hg(0) in aquatic systems.     

Kinetics of N-(Delta-Isopentenyl)Adenosine Degradation in Tobacco Cells: EVIDENCE OF A REGULATORY MECHANISM UNDER THE CONTROL OF CYTOKININS

Uptake and degradation of the cytokinin, N⁶-(Δ²-isopentenyl) adenosine, were studied in tobacco cells grown as cell suspensions. Degradation occurs by cleavage of the isopentenyl chain which gives adenylic products. Rate of N⁶(Δ²-isopentenyl)[8-¹⁴C]adenosine degradation increases several-fold after a 3- to 4-hour delay when cells have been exposed to a cytokinin. Consequently, only rates of N⁶-(Δ²-isopentenyl)adenosine degradation measured during the first 3 hours of incubation with [8-¹⁴C]-N-⁶(Δ²-isopentenyl)adenosine are representative of the intrinsic in vivo cytokinin degradative activity of tobacco cells. Within these limits, it appears that cytokinin degradative activity is high in cytokinin-autonomous tobacco cells, as indicated by the half life of the supplied N⁶(Δ² isopentenyl adenosine (about 3 hours) when it is supplied at the physiological concentration of 0.2 micromolar. This cytokinin degradative activity appears to be under the control of cytokinins themselves because N⁶-(Δ²-isopentenyl)adenosine degradative activity is increased several-fold following a 3- to 4-hour delay after these cells have been exposed to a cytokinin.     

Simultaneous measurement of nicotinamide and its catabolites,nicotinamide N-oxide,N1-methyl-2-pyridone-5-carboxamide,and N1-methyl-4-pyridone-3-carboxamide,in mice urine

Nicotinamide N-oxide is a major nicotinamide catabolite in mice but not in humans and rats. A high-performance liquid chromatographic method for the simultaneous measurement of nicotinamide, nicotinamide N-oxide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone-3-carboxamide in mice urine was developed by modifying the mobile phase of a reported method for measurement of nicotinamide N-oxide.     

Aerobic Mineralization of Trichloroethylene, Vinyl Chloride, and Aromatic Compounds by Rhodococcus Species

Two Rhodococcus strains which were isolated from a trichloroethylene (TCE)-degrading bacterial mixture and Rhodococcus rhodochrous ATCC 21197 mineralized vinyl chloride (VC) and TCE. Greater than 99.9% of a 1-mg/liter concentration of VC was degraded by cell suspensions. [1,2-¹⁴C]VC was degraded by cell suspensions, with the production of greater than 66% ¹⁴CO₂ and 20% ¹⁴C-aqueous phase products and incorporation of 10% of the ¹⁴C into the biomass. Cultures that utilized propane as a substrate were able to mineralize greater than 28% of [1,2-¹⁴C]TCE to ¹⁴CO₂, with approximately 40% appearing in ¹⁴C-aqueous phase products and another 10% of ¹⁴C incorporated into the biomass. VC degradation was oxygen dependent and occurred at a pH range of 5 to 10 and temperatures of 4 to 35°C. Cell suspensions degraded up to 5 mg of TCE per liter and up to 40 mg of VC per liter. Propane competitively inhibited TCE degradation. Resting cell suspensions also degraded other chlorinated aliphatic hydrocarbons, such as chloroform, 1,1-dichloroethylene, and 1,1,1-trichloroethane. The isolates degraded a mixture of aromatic and chlorinated aliphatic solvents and utilized benzene, toluene, sodium benzoate, naphthalene, biphenyl, and n-alkanes ranging in size from propane to hexadecane as carbon and energy sources. The environmental isolates appeared more catabolically versatile than R. rhodochrous ATCC 21197. The data report that environmental isolates of Rhodococcus species and R. rhodochrous ATCC 21197 have the potential to degrade TCE and VC in addition to a variety of aromatic and chlorinated aliphatic compounds either individually or in mixtures.     

Synthesis of (+)-(R)- and (−)-(S)-5-hydroxy-2-methyl-2-dipropylaminotetralin: Effects on rat hippocampal output of 5-HT, 5-HIAA,and DOPAC as determined by in vivo microdialysis

Racemic 5-methoxy-2-methyl-2-dipropylaminotetralin ( 3 ) has been prepared by a short synthetic route, in which the N,N-dipropyliminium perchlorate of 5-methoxy-2-tetralone ( 4 ) is a key intermediate. Racemic 3 was resolved by crystallization of the corresponding diastereomeric di-p-toluoyltartrates. The enantiomeric excess (%ee) of the phenolic derivatives of (+)-(R)- and (?)-(S)-3 [(+)-(R)- and (?)-(S)-2] was determined by ¹HNMR spectroscopic analysis of the corresponding diastereomeric (?)-(R)-1,1′-binaphthyl-2,2′-diylphosphoric acid salts utilizing ¹³C satellites. X-ray crystallography established the absolute configuration of (?)-(S)-2 · HCl. The enantiomers of 2 were tested for hippocampal output of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and dihydroxyphenylacetic acid in rats by use of in vivo microdialysis. The (?)-(S)-enantiomer appeared to affect 5-HT-turnover, whereas (+)-(R)- 2 was inactive. Results obtained provide support for the previously reported hypothesis that the inactivity of (?)-(S)- 2 at central DA receptors is caused by the steric bulk of the C(2)-methyl group. This makes it possible to define a “DA D2 receptor essential volume.” © 1993 Wiley-Liss, Inc.     

Effects of Peroxidation Products of Linoleic Acid on Tryptophan–nicotinamide Metabolism in Rats

The flavin and pyridine nucleotide coenzymes are involved in the detoxication of autoxidation products of lipids. In tryptophan-nicotinamide metabolism, kynurenine 3-hydroxylase and N¹-methylnicotinamide (MNA) oxidase contain FAD as a coenzyme. So, the effects of dietary autoxidation products of linoleic acid on the metabolism of tryptophan-nicotinamide were investigated using rats. The administration of linoleic acid hydroperoxides or secondary products reduced the urinary excretion of xanthurenic acid, nicotinamide and its metabolites such as MNA, N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) as compared with the group administered saline or linoleic acid. Among the enzyme activities involved in the tryptophan-nicotinamide metabolism, the activity of NAD⁺ synthetase was decreased by the administration of linoleic acid hydroperoxides or secondary products. The activities of tryptophan oxygenase and 4-Py-forming MNA oxidase were also decreased by the administration of secondary products. These results indicate that the conversion of tryptophan to nicotinamide would be lower in the groups administered the hydroperoxides and secondary products than in saline and linoleic acid groups.     

Biotransformation of [ring-U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-phase system

The biotransformation of [¹⁴C]4-n-nonylphenol (5 mg l^–1; 10 mg l^–1) by Agrostemma githago cell suspensions was studied using a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v). The highly lipophilic 4-n-nonylphenol was applied via n-hexadecane phase. After 7 d of incubation, more than 85% of applied 4-n-nonylphenol was absorbed by the cells, and 40% was transformed to 10 side-chain monohydroxylated metabolites (two with additional double bond at side-chain). The primary metabolites were analyzed by GC-EIMS. In the cells, the monohydroxylated products and residual 4-n-nonylphenol were present as glycosides. The method proved to be suitable for the production of primary metabolites of 4-n-nonylphenol on a larger scale for identification purposes and for metabolic profiling of the compound.     

Fate of radioactivity in Atlantic Salmon (Salmo salar) following intragastric administration of (methyl-14C)-trichlorfon

The antiparasitic drug Neguvon® (Bayer), with the active component trichlorfon (0,0-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate), is extensively used in fish farms in Norway. The fate of (methyl-¹⁴C)-trichlorfon was tested in Atlantic salmon (Salmo salar) by liquid scintillation counting at day 1, 4, 7, 14, and 30 post administration, and by autoradiography on selected organs 24 h after administration. The remaining radioactivity was found to be high compared to earlier measurements of the trichlor)fon content made by gas chromatography (Brandal 1977). The grains in autoradiographic preparations of muscle were unevenly distributed both in the muscle as a whole and within muscle fibers. In liver the grains were evenly distributed, but were absent from fat vacuoles. The study indicate that the radioactive residues in salmon muscle do not represent trichlorfon or its derivative dichlorvos (0,0-dimethyl-0-(2,2-dichlorovinyl)-phosphate), but rather hydrophilic metabo)lites of these compounds.     

Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod ⁺fix^- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod⁺ fix^- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.