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Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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.

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.