Transformation of metolachlor in soil inoculated with a Streptomyces sp.

Microbial detoxication of pesticides may offer a promising alternative to existing physical-chemical treatment methods. We investigated a strain of Streptomyces sp. which can transform metolachlor in a liquid medium for its ability to decontaminate herbicide-treated soil. A cell suspension of Streptomyces sp. was added to a silt loam soil (Hagerstown, pH 6.1) which was amended with 10 g of metolachlor containing 5 nCi ring-UL-¹⁴C metolachlor per gram of soil, and the mixture was incubated at 28°C. Inoculation of the sterile soil resulted in the rapid transformation of metolachlor. Analyses of one-week-old samples indicated that approximately 70% of the added radioactivity was recovered in the ethyl acetate and water fractions as products from the inoculated reaction mixture, whereas in the uninoculated control less than 8% of the ¹⁴C was found as products and about 80% was recovered in the form of unchanged metolachlor. In native soil, however, the rate of metolachlor disappearance was not enhanced by Streptomyces inoculation. In inoculated sterile soil the yields of products were affected by inoculum size, inoculation temperature and substrate concentration, but these variables had no effect on product formation in the inoculated native soil. Addition of Na₂CO₃ (200 g/g soil) into native soil significantly promoted growth of Streptomyces due to the higher pH (7.8) and also stimulated transformation of metolachlor by 30%. Our results suggest that proliferation of the inoculated organisms under favorable conditions is essential for their function as metolachlor degraders in native soil.     

In vitro hormonal stimulation of [14C]acetate incorporation by Heliothis armigera pheromone glands

Isolated pheromone gland preparations of Heliothis armigera incorporateradioactivity from [¹⁴C]sodium acetate at a linear rate for 24 h when incubated in a physiological saline. This incorporation is stimulated when methanolic or partially purified brain-complex extracts are present in the incubation medium and the stimulation is dose-dependent. The radioactive products extracted from the pheromone glands and media by hexane, revealed incorporation of radioactivity into a product exhibiting the same mobility as (Z)-11 hexadecenal, the main component of the pheromone of H. armigera, as analyzed by thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). In addition, gas chromatographic (GC) analysis of the hexane soluble products after incubation in the absence of [¹⁴C]sodium acetate revealed a significant stimulation of the concentration of (Z)-11 hexadecenal by brain-complex extracts.     

Cytokinin Biosynthesis in Mutants of the Moss Physcomitrella patens

Three cytokinin-over-producing mutants of the moss, Physcomitrella patens, have been shown to convert [8-¹⁴C]adenine to N⁶-[¹⁴C](Δ²-isopentenyl)adenine, the presence of which was confirmed by thin layer chromatography, high performance liquid chromatography, and recrystallization to constant specific radioactivity. The labeled cytokinin was detected in the culture medium within 6 hours and the tissue itself appears to contain both labeled N⁶-(Δ²-isopentenyl)adenine and N⁶-(Δ²-isopentenyl)adenosine monophosphate.     

Coronatine biosynthesis: Incorporation of l-[U-14C]isoleucine and l-[U-14C] threonine into the 1-amido-1-carboxy-2-ethylcyclopropyl moiety

Addition of either l-[U-¹⁴C]threonine or l-[U-¹⁴C]isoleucine to 2.7-day-old shaking liquid cultures of Pseudomonas syringae pv. atropurpurea resulted in incorporation of radioactivity into coronatine, but not into N- coronafacoylvaline, another phytotoxin excreted by P.s. atropurpurea. In contrast, addition ofl-[U-¹⁴C]valine did not lead to incorporation of radioactivity into coronatine, but instead into coronafacoylvaline. Acid hydrolysis of the purified [¹⁴C] coronatine obtained after incorporation of either [¹⁴C]isoleucine or [¹⁴C]threonine demonstrated that > 94% of the radioactivity was present in the 1-amido-1-carboxy-2-ethylcyclopropyl moiety of coronatine, and < 6 % was in the coronafacoyl moiety. These findings are used to propose a biosynthetic pathway for coronatine.     

Absorption and excretion of tablet disintegrating β-cyclodextrin polymer in rats

Absorption and excretion of a new tablet disintegrating agent, a crosslinked β-cyclodextrin polymer was investigated following per os administration in the rat. The polymer, which is insoluble but swells in water, was prepared from β-cyclodextrin by reacting with [2-¹⁴C]epichlorohydrin in an alkaline medium. Radioactivity of blood, urine, faeces, exhaled carbon dioxide and the gastrointestinal tract was determined by a liquid scintillation method. No radioactivity could be detected in the blood up to 24 h after the administration of the polymer. Radioactivity of urine and exhaled carbon dioxide together did not exceed 0·11% of the total administered radioactivity, 98% of which was found in the large intestine and the faeces. Therefore, it is assumed that β-cyclodextrin polymer could not be absorbed from the gastrointestinal tract.     

Incorporation of [14C]-2 deoxy-D-glucose into the lipids of normal cells as compared to virus-transformed cells

[¹⁴C]-2 deoxy-D-glucose is incorporated into the glycolipids of both normal and transformed cells. The chromatographic pattems of [¹⁴C]-2 deoxy-D-glucose labeled lipids differ markedly in oncornavirus and herpes simplex virus-transformed cells as compared to normal and virus-infected but not transformed cells. Deoxyglucoselabeled lipids with intermediate chromatographic mobility were enriched in normal and virus-infected but not transformed cells. Studies with a murine sarcoma virus-infected cell line which is temperature-sensitive for transformation indicated that the altered chromatographic pattern of [¹⁴C]-2 deoxy-D-glucose labeled lipids was related to the expression of the transformed phenotype.     

Uptake of benzyladenine by excised watermelon cotyledons

The uptake of 8-[¹⁴C]N⁶-benzyladenine (BA) was studied in excised watermelon (Citrullus vulgaris Schrad.) cotyledons 24 hours after the start of imbibition. The passive nature of this uptake is suggested by the following evidence: (a) no sign of saturation on increasing external concentration of BA; (b) no decrease in uptake under conditions that inhibit ATP synthesis; (c) no change in amount of radioactivity absorbed when cotyledons are frozen and thawed before the uptake test. About two-thirds of the radioactivity taken up is released after 12 hours of washing. If the washing is performed at 2 C very little radioactivity is released.     

A radiotracer method for determining ingestion of Bacillus sphaericus by mosquito larvae

Bacillus sphaericus was labeled with carbon-14 by culturing in a medium containing U-¹⁴C-l-amino acid mixture or d-[U-¹⁴C]glucose, the former giving consistently higher labeling. Less than 5% of the label was lost during the 2-hr experimental period. Groups of mosquito larvae were exposed to labeled bacteria for variable times, washed to remove external radioactivity, and then were either analyzed by combustion and liquid scintillation counting for internal ¹⁴C or held in fresh water for mortality counts. Culex quinquefasciatus larvae rapidly ingested bacteria during the first 30 min followed by a reduced rate of accumulation. the rate and quantity of bacteria ingested was greater with later instars. The method is applicable for the study of environmental and physiological factors influencing the ingestion and susceptibility of different species of mosquito larvae to bacterial pathogens.     

Movement and Fate of p-Nitrophenyl-alpha,alpha,alpha-Trifluoro-2-Nitro-p Tolyl Ether-1'-C in Peanut Seedlings

Autoradiographs and liquid scintillation counts of peanut (Arachis hypogaea L., var. Starr) seedlings indicate that p-nitrophenyl-α,α,α-trifluoro-2-nitro-p-tolyl ether-1′-¹⁴C (C-6989) is rapidly absorbed from nutrient solution but only 6.5% of the radioactivity absorbed is translocated to the shoot after 144 hr, with the acropetal movement being confined to the stem and petiole.     

Microbial growth on C1 compounds: Uptake of [14C]formaldehyde and [14C]formate by methane-grown Pseudomonas methanica and determination of the hexose labelling pattern after brief incubation with [14C]methanol

1. A study has been made of the incorporation of carbon from [¹⁴C]formaldehyde and [¹⁴C]formate by cultures of Pseudomonas methanica growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compounds for periods of up to 1min., has been analysed by chromatography and radioautography. 3. Radioactivity was fixed from [¹⁴C]formaldehyde mainly into the phosphates of the sugars, glucose, fructose, sedoheptulose and allulose. 4. Very little radioactivity was fixed from [¹⁴C]formate; after 1min. the only products identified were serine and malate. 5. The distribution of radioactivity within the carbon skeleton of glucose, obtained from short-term incubations with [¹⁴C]methanol of Pseudomonas methanica growing on methane, has been investigated. At the earliest time of sampling over 70% of the radioactivity was located in C-1; as the time increased the radioactivity spread throughout the molecule. 6. The results have been interpreted in terms of a variant of the pentose phosphate cycle, involving the condensation of formaldehyde with C-1 of ribose 5-phosphate to give allulose phosphate.     

The metabolism of [3−3H]oleanolic acid-3-O-mono-[14C]glucoside in isolated cells from Calendula officinalis leaves

The radioactive precursor, [3?³H]oleanolic acid-3-O-mono-[¹⁴C]glucoside was administrated to isolated cells obtained from the leaves of Calendula officinalis. The radioactivity of the precursor was incorporated into fractions containing free oleanolic acid, individual glucosides, glucuronide F and other glucuronides. The ratio of ³H: ¹⁴C radioactivity in these fractions indicated that glucosides were formed in a process involving direct glycosylation of the precursor, whereas the glucuronides were formed from oleanolic acid released by hydrolysis of the precursor. Dynamics curves showed that glucoside II formed by direct glycosylation of the precursor was intensively transformed to other derivatives.     

Identification of metolachlor mineralizing bacteria in aerobic and anaerobic soils using DNA-stable isotope probing

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of the popular soil-applied pre-emergent herbicide, metolachlor is unknown. To address this knowledge gap, we utilized DNA-based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor or ¹³C-metolachlor. Mineralization of metolachlor was confirmed as noted from the evolution of ¹⁴CO₂ from ¹⁴C-metolachlor-treated microcosms and clearly demonstrated the efficient utilization of the herbicide as a carbon source. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts indicated that fragment 307 bp from aerobic soil and 212 bp from anaerobic soil were detected only in the herbicide-treated (both unlabeled metolachlor and ¹³C-metolachlor) soils when compared to the untreated control microcosms. T-RFLP profiles from the ultracentrifugation fractions illustrated that these individual fragments experienced an increase in relative abundance at a higher buoyant density (BD) in the labeled fractions when compared to the unlabeled herbicide amendment fractions. The shift in BD of individual T-RFLP fragments in the density-resolved fractions suggested the incorporation of ¹³C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the ¹³C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic mineralization and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soil.     

Preparation of 14C-Labeled Sterigmatocystin in Liquid Media

¹⁴C-labeled sterigmatocystin was prepared from surface cultures of Aspergillus versicolor A-18074 maintained in liquid media by multiple additions of [1-¹⁴C]acetate to the cultures. The highest yield of 7.75 mg/10 ml was found with a sucrose-asparagine-ammonium medium in which more than 3% of the radioactivity of the added [1-¹⁴C]acetate was recovered in the purified [ring-¹⁴C] sterigmatocystin. The method offers an easy way to prepare ¹⁴C-labeled sterigmatocystin for studies of this mycotoxin.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to more unsaturated acyclic, monocyclic, and dicyclic carotenes by a cell-free preparation of red tomato fruits

This paper reports the conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C|phytofluene and the conversion of the latter compound to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from the plastids of red tomato fruits. Each of these radioactive compounds was also converted to labeled neurosporene, lycopenc, α-carotene, and β-carotene by the same enzyme system. The incorporation of each substrate into more unsaturated carotenes was carried out under nitrogen at pH 7.5–8.2 (borate buffer), at 25 °C in the dark.Proof of the formation of the above carotenes from each of the three radioactive substrates was demonstrated by cochromatography with authentic nonradioactive carotenes on an alumina chromatographic column. A close correspondence between radioactivity and light absorbance for each carotene was observed. Confirmation of these conversions was achieved by cochromatography with authentic samples on thinlayer plates. Final proof for the formation of the acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed.Maximum conversion of cis- and trans-phytofluenes to more unsaturated carotenes by the red tomato fruit enzyme system appears to be dependent upon the presence of NADP⁺, FAD, and Tween 80. The formation of the carotenes is also increased in the presence of Mg²⁺ or Mn²⁺ ions.     

Kinetin incorporated into tobacco callus ribosomal RNA and transfer RNA preparations

Kinetin, N⁶-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) callus RNA isolated from rapidly growing tissue cultured in the presence of N⁶-furfuryladenine-8-¹⁴C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N⁶-furfuryladenosine (fr⁶A) in the rRNA and tRNA preparations from the tobacco callus. The rRNA contained over 90% of these fr⁶ A moieties. The extent of kinetin incorporation was four times greater than that observed for N⁶-benzyladenine. The radiochemical purity of the recovered fr⁶ A was confirmed by three successive chromatographic purifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 eluted with water). A cytokinin-active ribonucleoside with elution volumes corresponding to fr⁶ A was isolated from the tobacco callus rRNA preparation. This compound was analyzed by gas-liquid chromatography and rigorously characterized as N⁶-furfuryladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative.     

Enzymatic degradation of heparin-related mucopolysaccharides from the surface of endothelial cell cultures

When cultures of endothelial cells prelabeled with H₂^{3 5}SO₄ are exposed to a purified preparation from induced Flavobacterium heparinum containing heparinase and heparitinase activities, radioactivity accumulates in the supernatant medium. After further treatment in vitro with crude enzyme this material migrates, in part, as glucosamine (N, O-disulfated glucosamine), a breakdown product characteristic of heparin and heparin-related mucopolysaccharides. After exposure of the cultures to the purified enzyme, the amount of acid-insoluble ^{3 5}S radioactivity that can be removed with EDTA is decreased compared to that that can be removed from control cultures. Since the amount of radioactivity that is released as breakdown products is much higher than the amount of radioactivity that is secreted into the supernatant medium as intact (non-dialysable) mucopolysaccharide chains in control plates, the action of the enzyme appears to be on the cell itself. The data presented support previous studies suggesting that chains of heparitin sulfate that are accessible to the action of the enzyme are present at the surface of endothelial cells.     

Catabolic Fate of Streptomyces viridosporus T7A-Produced, Acid-Precipitable Polymeric Lignin upon Incubation with Ligninolytic Streptomyces Species and Phanerochaete chrysosporium

Degradation of ground and hot-water-extracted corn stover (Zea mays) lignocellulose by Streptomyces viridosporus T7A generates a water-soluble lignin degradation intermediate termed acid-precipitable polymeric lignin (APPL). The further catabolism of T7A-APPL by S. viridosporus T7A, S. badius 252, and S. setonii 75Vi2 was followed for 3 weeks in aerated shake flask cultures at 37°C in a yeast extract-glucose medium containing 0.05% (wt/vol) T7A-APPL. APPL catabolism by Phanerochaete chrysosporium was followed in stationary cultures in a low-nitrogen medium containing 1% (wt/vol) glucose and 0.05% (wt/vol) T7A-APPL. Metabolism of the APPL was followed by turbidometric assay (600 nm) and by direct measurement of APPL recoverable from the medium. Accumulation and disappearance of soluble low-molecular-weight products of APPL catabolism were followed by gas-liquid chromatography and by high-pressure liquid chromatography, utilizing a diode array detector. Identified and quantified compounds present in culture media included p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, protocatechuic acid, vanillic acid, and vanillin. The further catabolism of these APPL-derived aromatic compounds varied with the culture examined, and only S. setonii and P. chrysosporium completely degraded all of them. Some new intermediates of APPL metabolism also appeared in culture media, but the patterns were culture specific. Additional evidence from high-pressure liquid chromatography analyses indicated that one strain, S. badius, converted a water-soluble fraction evident by high-pressure liquid chromatography (7 to 10 min retention time range) into new products appearing at shorter retention times. Mineralization of a [¹⁴C-lignin]APPL was also followed. The percent ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-APPL, ¹⁴C-labeled water-soluble products, and cell mass-associated radioactivity, were determined for each microorganism after 1 and 3 weeks of incubation in bubbler tube cultures at 37°C. P. chrysosporium evolved the most ¹⁴CO₂ (10%), and S. viridosporus gave the greatest decrease in recoverable ¹⁴C-APPL (23%). The results show that S. badius was not able to significantly degrade the APPL, while the other microorganisms demonstrated various APPL-degrading abilities. The significance of these findings relative to the fate of APPLs in nature was discussed.     

The Metabolism of the Herbicide Diphenamid (N-N-dimethyl-2,2-diphenyl-acetamide) in Cell Suspensions of Soybean (Glycine max)

The fate of the herbicide diphenamid was determined in cell suspensions of soybean [Glycine max (L.) Merr. ‘Wilkin’] at different stages of cell growth: early log phase (3 to 7 d), log phase (7 to 14 d), and stationary phase (14 to 18 d). [Carbonyl-¹⁴C]-diphenamid was added to the suspensions as an acetone solution. Neither diphenamid (2 to 3 μM) nor acetone (0.5% v/v) was phytotoxic. The ¹⁴C-labeled products were identified tentatively by thin layer chromatographic comparison with reference compounds. The major metabolic products formed were N-hydroxymethyl-N-methyl-2,2-diphenylacetamide, N-methyl-2,2-diphenylacetamide, 2,2-diphenylacetamide, and two polar metabolites (0.9 to 25% of the applied ¹⁴C activity) that appeared to be glucose conjugates; one an acidic glucoside. All metabolites were found in both the cell extract and the culture medium, except for the acidic glucoside, which was recovered in small amounts only from the cell extracts. These products were the same as those recovered from intact plants. Similar results were obtained from cell suspensions of different ages. The rate of metabolism by log phase cells was slightly less than the rate for either young or old cells. The results indicated that soybean cell suspensions can be used to obtain reliable information on the fate of agricultural chemicals in soybeans.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.