A secretory leukocyte protease inhibitor variant with improved activity against lung infection.

Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI's proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.

Effect of monosodium methanarsonate application on cuticle wax content of cocklebur and cotton plants.

Leaf cuticle waxes were extracted from monosodium methanearsonate (MSMA)-resistant (R) and -susceptible (S) common cocklebur (Xanthium strumarium L.) and cotton (Gossypium hirsutum L.) plants at 0, 3, 5, and 7 days after treatment (DAT) following 1x and 2x MSMA applications. Wax constituents were analyzed by gas chromatography (GC) with flame ionization detection and compared to alkane and alcohol standards of carbon lengths varying from C21 to C30. Differences in waxes were calculated and reported as change per ng mm2-1. Tricosane (C23) was found to increase following MSMA applications. All other alkanes decreased by 7 DAT, with some showing a linear effect over time in the R-cocklebur. Alcohol constituents were also observed to decrease by 7 DAT. Total arsenic in the extracted wax fraction was determined, with greatest quantities detected in the R-cocklebur. Wax changes are not believed to play a role in cotton tolerance, since changes in cuticle concentrations were minimal. Cocklebur resistance to MSMA is not due to cuticle constituents; the wax changes are a secondary effect in response to herbicide application.

Biological activities of Ginkgo extracts.

The biological activity of methanolic the extracts of leaves, roots, leaf-derived callus, root-derived callus, ginkolide A, ginkgolide B, bilobalide and a commercial Ginkgo product (Tanakan) was assessed. Bioassays consisted of the Agrobacterium tumefaciens-induced potato tumor assay and a Kirby-Bauer microbial sensitivity assay with pure strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes. Methanolic extracts of leaves, leaf-derived callus, root-derived callus, bilobalide and Tanakan inhibited tumor formation significantly, but more weakly than the positive control, camptothecin. No activity against E. coli was detected, but extracts from both callus types inhibited the growth of K. pneumonia, P. aeruginosa, S. aureus, S. epidermidis and S. pyogenes. All extracts and reference compounds inhibited the growth of S. pyogenes. Leaf and root tissues contained the highest levels of ginkgolide A, as compared to the callus tissues; leaf tissue contained more of all three marker compounds than the callus tissues.

MSMA resistance studies.

Monosodium methanearsonate (MSMA)-resistant and -susceptible common cocklebur (Xanthium strumarium L.) and cotton (Gossypium hirsutum L.) were treated with MSMA. Plant parameters analyzed were: glutathione synthetase activity, selected amino acid (arginine, glutamic acid, alanine, citrulline, glutamine, and glutathione) content and arsenic content (MSMA, total arsenic, and arsonate). No reduction of arsenic from the parent pentavalent form present in MSMA to the trivalent form was detected. Arginine, glutamic acid, and glutamine content increased in tissue three days after MSMA treatment. Glutathione content decreased during the first three days after treatment; however, five days after treatment the resistant biotype of cocklebur and cotton had elevated glutathione levels (8-20 times greater, respectively). Glutathione Synthetase activity was higher in cotton than in either of the cocklebur biotypes; MSMA did not affect its activity in cotton or either cocklebur biotype. Resistant biotypes have a slightly higher activity than the susceptible biotype. Tolerance of cotton to MSMA may be related to glutathione synthetase activity and possibly to the presence of phytochelatins. Also, increased glutathione levels in the resistant biotype may implicate phytochelatin involvement in the resistance mechanism.

Mycotoxins in root extracts of American and Asian ginseng bind estrogen receptors alpha and beta.

The estrogenic activity of ginseng has been the subject of conflicting reports. Cell proliferation, induction of estrogen-responsive genes, and isolated cases of adverse reactions such as postmenopausal vaginal bleeding and gynecomastia have been reported after ginseng treatment. Other studies report antiproliferative effects with no induction of estrogen-responsive genes. We developed estrogen receptor (ER) alpha and ER alpha competitive binding assays using recombinant receptors and [(3)H]-17 alpha-estradiol to detect phytoestrogens in extracts of Asian ginseng root (Panax ginseng C. A. Meyer) and American ginseng root (Panax quinquefolius L.). Root extracts contained substances that bound both receptor isoforms. These substances had a two to three times greater affinity for ER alpha. Significantly higher binding was found in methanol extracts than in hot water extracts. Subsequent analysis of the extracts revealed significant ER binding attributable to zearalenone, the estrogenic mycotoxin produced by several Fusarium species. The ER showed no binding affinity for Rb1 and Rg1, the major ginsenosides found in P. quinquefolius and P. ginseng, respectively. Thus, ginseng extraction methods, plant species tested, and mycotoxin contaminants may help to explain the disparate literature reports. The prevalence and health significance of fungal contamination in herbal products used for medicinal purposes should be further investigated.

Light and clomazone effects on tobacco (Nicotiana tabacum) callus and leaf discs.

The effects of clomazone on the growth of tobacco (Nicotiana tabacum L. 'NC2326') callus and leaf discs were studied under four light regimes. Callus cultures and leaf discs were grown on Murashige and Skoog medium supplemented with IAA and kinetin. Light regimes were: dark grown callus kept in the dark and also transferred to the light; light grown callus kept in the light and also transferred to the dark. Two-month-old callus (cultured for 2 months from initiation) grew more rapidly than twelve-month-old callus (cultured for 12 months from initiation) under all conditions tested. Callus transferred from light to dark, or from dark to light, increased in fresh weight slower than did the callus maintained totally in light or dark. Clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone) at 140 mg l(-1) or more was lethal to both callus and leaf discs whereas 10 mg l(-1) was stimulatory to growth. Callus tissue responded to clomazone differently depending on the light regime under which it was grown. While clomazone may be affecting the isoprenoid pathway in the callus and leaf disks resulting in growth inhibition, it is possible that other target sites are also being affected and contribute to the reduced growth.

Potato disc tumor induction assay: a multiple mode of drug action assay.

The study reported herein utilized the Agrobacterium tumefaciens-induced potato disc tumor assay. The objective was to verify the detection of antineoplastic activity in the potato disc tumor induction assay, regardless of the mode of antineoplastic drug action. Camptothecin, paclitaxel, podophyllin, vinblastine and vincristine were tested, each with a different mode of action. All drugs tested inhibited tumor induction. The order of activity was: camptothecin = paclitaxel = vinblastine < podophyllin = vincristine. No effect on the viability of the bacterium was detected. The A. tumefaciens-induced potato disc tumor assay was an effective indicator of antitumor activity regardless of the mechanism of drug action. Thus, this assay would be acceptable as a primary general screen for antineoplastic activity of various crude extracts, as well as for purified fractions, regardless of mode of inhibitory action on tumor formation.

Fate of isoxaben in a containerized plant rhizosphere system.

Commercial production of ornamental plants is an important industry in the United States and involves a complex technology that includes the use of herbicides. Isoxaben[N-[3-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2,6-dimethoxybenzamide] is a pre-emergence herbicide used for controlling weeds in many areas including containerized ornamental plants. Degradation was studied in potting mix (80% bark, 20% sand) with three different regimes (sterile, bulk and rhizosphere). The rhizosphere regime contained Switch Grass (Panicum virgatum), and plants were allowed to grow for 14 days before adding isoxaben (10 microg/g potting mix). Isoxaben was degraded to 0.5 microg/g in 60 days giving a half-life of 7 days. Two degradation products were detected: 3-nitrophthalic acid in the rhizosphere and bulk regimes and 4-methoxyphenol in the sterile regime. Microbial population shifts were determined by fatty acid methyl ester profile analysis and were influenced by the introduction of a plant (rhizosphere regime) and by isoxaben addition.

Biological activity of common mullein, a medicinal plant.

Common Mullein (Verbascum thapsus L., Scrophulariaceae) is a medicinal plant that has been used for the treatment of inflammatory diseases, asthma, spasmodic coughs, diarrhea and other pulmonary problems. The objective of this study was to assess the biological activity of Common Mullein extracts and commercial Mullein products using selected bench top bioassays, including antibacterial, antitumor, and two toxicity assays--brine shrimp and radish seed. Extracts were prepared in water, ethanol and methanol. Antibacterial activity (especially the water extract) was observed with Klebsiella pneumonia, Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. Agrobacterium tumefaciens-induced tumors in potato disc tissue were inhibited by all extracts. Toxicity to Brine Shrimp and to radish seed germination and growth was observed at higher concentrations of the extracts.

Atrazine effects on in vitro maturation and in vitro fertilization in the bovine oocyte.

The effect of low levels of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on in vitro oocyte maturation, in vitro capacitation of sperm, or in vitro fertilization of bovine oocytes and on the quality of blastocyst formation was studied. Bovine oocytes collected from abattoir ovaries were matured, fertilized, and developed to the blastocyst stage in vitro. Embryos that reached a morula or blastocyst stage were stained with Hoechst 33258 stain to determine the number of blastomeres per embryo. Three bulls whose fertilization rates were proven consistent among straws were used for this study. Atrazine was tested at concentrations of 0.01, 0.1, 1, and 10 microM in either the maturation medium, sperm capacitation medium, or the fertilization medium. Because atrazine was dissolved in ethanol, an ethanol control was used to determine any possible effects of ethanol on the in vitro process. The addition of atrazine to both the maturation and fertilization media did not result in any significant difference in fertilization rates between the controls and the treatments. In the capacitation medium, a significant difference between the controls and the atrazine levels of 0.1, 1, and 10 microM was noted for one bull. Atrazine did not affect the number of blastomeres per embryo. There was not a significant difference (p>0.05) in the number of blastomeres per embryo between the controls and the different levels of atrazine in each medium. This study indicates that low levels of atrazine do not have an effect on in vitro fertilization rates or the number of blastomeres per embryo produced in vitro.

Microbial degradation of mefenoxam in rhizosphere of Zinnia angustifolia.

The fate of the fungicide mefenoxam was studied in a containerized rhizosphere system. The rhizosphere system used Zinnia angustifolia (Tropic Snow) in a bark/sand potting mix and was compared to bulk potting mix (no plants). Rhizosphere microbial populations were allowed to establish for 3 weeks prior to fungicide addition (20 microg per g mix). Mefenoxam and degradation product concentrations were determined by High HPLC or capillary electrophoresis after extraction. Seventy eight percent of the fungicide originally applied to the rhizosphere was degraded after 21 days compared to 44% in bulk system (no plant). The primary degradation product was the free acid N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine, which accounted for 71% of the applied parent chemical after 30 days. N-(2,6-dimethylphenyl)-acetamide was also detected, but in lesser amounts. Bacterial populations in the rhizosphere increased during the 30-day period, which correlated with an increase in degradation of the parent compound. Pure cultures of Pseudomonas fluorescens and Chrysobacterium indologenes isolated from the rhizosphere system could degrade the applied fungicide (10 microg/ml) almost completely to the free acid within 54 h.

Degradation of isoxaben in soils and an aqueous system.

The degradation of isoxaben [N-[3-(1-ethyl-1-methylpropyl)-5-isoxazolyl]-2,6-dimethoxybenzamide] was studied in soil and in an aqueous system. Soil studies were conducted in Erlenmeyer flasks (treated with 1 microg/g isoxaben) and mineralization studies in Biometer flasks (treated with 1 microg/g unlabeled and 14C-isoxaben) incubated at 23 C. Degradation in the aqueous system was performed in Erlenmeyer flasks under aerobic and anaerobic conditions incubated at 23 degrees C. Incubation mixtures were extracted at selected times and analyzed for isoxaben and degradation products by HPLC with product identification confirmed by GC-MS. After 8 weeks, 78% and 23% of the total isoxaben disappeared in nonsterile and sterile soils, respectively. After 12 weeks, approximately 1% of the labeled isoxaben was recovered as CO2 in the Biometer flask experiments; no volatile products were detected, and 5% and 33% of the total radioactivity was recovered from the nonsterile and sterile soils, respectively. In the aquatic system after 8 weeks, isoxaben had decreased from 1microg/g to 0.1 and 0.004 microg/g under aerobic and anaerobic conditions, respectively. Degradation products detected from the soil studies were 3-nitrophthalic acid and 4-methoxyphenol, and 3-nitrophthalic acid in the aqueous system studies. Microbial activity was considered to be a major factor in the degradation of isoxaben in this study.

Atrazine degradation in a containerized rhizosphere system.

The effect of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on rhizosphere microorganisms and its fate in a containerized rhizosphere system was studied. The rhizosphere system consisted of corn grown in pot containing a defined potting mix of sand and bark with atrazine. Sterilized potting mix and a container without plants served as controls. Atrazine was extracted and analyzed via HPLC. Fluorescent pseudomonad populations increased 100-fold in the rhizposphere during a 60-day incubation period as compared to the nonvegetated control. Atrazine degradation was higher in the rhizosphere system (half-life of 7 days) compared to the nonvegetated control (half-life of greater than 45 days). The major degradation product detected in the rhizosphere system was deisopropylatrazine; other products detected included deethylatrazine, deethylhydroxyatrazine, deisopropylatrazine and hydroxyatrazine. Hydroxyatrazine was detected in the nonvegetated and sterile controls. The containerized rhizosphere system provides an experimental system to study the fate of pesticidal chemicals as well as the effects on microbial populations.

Growth effects, uptake and metabolism of trifluralin in tissue cultures.

Growth effects, uptake and metabolism of trifluralin (alpha, alpha, alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) in carrot (Daucus carota L.) and tobacco (Nicotiana tabaccum L.) callus tissue were determined. Carrot callus was initiated from tap root tissue on Murashige and Skoog medium with kinetin and 2,4-dichlorophenoxy acetic acid (2,4-D). Tobacco callus was initiated from pith tissue on Murashige and Skoog medium with indole-3-acetic acid (IAA) and kinetin.

Aerobic degradation of diuron by aquatic microorganisms.

Degradation of diuron [3-(3,4-dichlorophenyl)-1,1-dimethyl-urea] by microorganisms obtained from pond water and sediment was determined under aerobic conditions. Enrichment procedures were used to isolate cultures capable of degrading the herbicide. Several mixed fungal/bacterial and mixed bacterial cultures were isolated that could degrade diuron. The mixed cultures degraded 67-99% of the added diuron forming from six to seven products which were separated via TLC. The major degradation product detected in most culture extracts was 3,4-dichloroanaline. Other identified products formed were 3-(3,4-dichlorophenyl)-1-methyl-urea and 3-(3,4-dichlorophenyl)urea.

Degradation of selected phenylurea herbicides by anaerobic pond sediment.

Anaerobic degradation of diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea], monuron [3-(4-chlorophenyl)-1,1-dimethylurea], and fenuron [1,1-dimethyl-3-phenylurea] were studied. Herbicide containing media (reduced with cysteine-HCl and under 95% N2:5% CO2 gas phase) were inoculated with pond sediments. Sediment from a diuron-treated pond dehalongenated diuron to 3-(3-chlorophenyl)-1,1-dimethylurea (CPDU) in 17 to 25 days but sterile sediment from the pond did not. Sediments from non-diuron treated ponds were also ineffective. Particles from diuron-treated sediment were essential for dehalogenation and they could not be replaced with other solid surfaces such as clay, sand or cellulose. Diuron was degraded by sediment at 25 and 30 degrees C (maximal rate) but not at 5, 15 and 37 degrees C after 55 days incubation. The product CPDU produced in laboratory cultures was found in sediment of the diuron-treated pond, indicating in situ reductive dechlorination. Sediment-inoculated cultures containing monuron and fenuron showed no degradation after 74 days incubation.

Aerobic and anaerobic degradation of profluralin and trifluralin.

The degradation of profluralin [N-(cyclopropylmethyl)-alpha,alpha,alpha-trifluoro-2,6-dinitro-N-propyl-p-toluidine] and trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) was studied under aerobic and anaerobic soil conditions. Three soils (Goldsboro loamy sand, Cecil loamy sand, Drummer clay loam) were each treated with 1 ppmw herbicide; anaerobic conditions were maintained by flooding. Soil samples were extracted monthly and subjected to TLC analysis. No degradation was detected in sterile controls. Aerobic degradation of both herbicides was greatest in the Cecil loamy sand soil over the entire incubation period. Degradation of profluralin in Cecil soil under aerobic conditions was 86 percent after 4 months with three products detected; 83 percent of the trifluralin was degraded with two products detected. Anaerobic degradation accounted for 72 percent of the profluralin and 78 percent of the trifluralin after 4 months. Degradation of both herbicides increased with incubation time for the first 3 months and decreased slightly thereafter. Generally there was more extensive degradation (percent and in number of products formed) of profluralin than trifluralin under the conditions tested. More degradation products were detected for both herbicides under aerobic conditions than under anaerobic conditions.

Isolation of ioxynil degraders from soil-enrichment cultures.

A soil enrichment technique was used to isolate microorganisms which could degrade ioxynil (3,5-diiodo-4-hydroxybenzonitrile). Many isolates obtained were able to degrade ioxynil to various products. However, only a fungal isolate (Fusarium solani) and a Gram-negative bacterium (Klebsiella ozaenae) released 14CO2 from ring-labeled ioxynil. No appreciable degradation was detected in pure cultures without the addition of exogenous nutrients. Results indicated that the degradation of ioxynil to CO2 proceeded more slowly in pure culture. Ioxynil was degraded in pure culture at a faster rate by F. solani than by K. ozaenae. Analyses of radioactivity distribution in the cultures indicated that a sizable fraction of radioactivity was in the form of polar products. Several degradation products were detected in the ethyl acetate extracts by thin-layer chromatography and subsequent radioautography. Screening of pure cultures of ioxynil degraders revealed that most isolates degraded ioxynil to the same products which were extractable with ethyl acetate.

Degradation of ioxynil and bromoxynil as measured by a modified spectrophotometric method.

A modified spectrophotometric method was developed to estimate ioxynil and bromoxynil residues. The method when compared with a 14C-tracer method was less sensitive but allowed rapid and accurate estimation of the herbicides. A clay loam soil with high organic matter content, which degraded ioxynil completely to CO2, also degraded bromoxynil completely. Bromoxynil degradation proceeded at a faster rate than that of ioxynil. The half-life of degradation was estimated to be 7 days for bromoxynil and 9-10 days for ioxynil. However, soil microorganisms which degraded ioxynil either completely to CO2 or partially did not seem to completely degrade bromoxynil. Degradation products from bromoxynil were detected on thin-layer chromatograms of extracts from pure cultures containing an exogenous carbon source. Complete degradation of bromoxynil and ioxynil in soil could be due to the action of different microorganisms.