Potential of 2,4-Dihydroxybenzoic Acid as an Oviposition Stimulant for Mass-Reared Ladybird Beetles.

The discovery of inexpensive, readily available bioflavonoids, and their degradation products that boost the reproductive potential of mass-reared predators is the overarching goal of this research. We tested the hypothesis that 2,4-dihydroxybenzoic acid (DHBA), an inexpensive degradation product of morin (a flavonol bioflavonoid), stimulates oviposition by the ladybird beetle Coleomegilla maculata (DeGeer). We also tested the hypothesis that C. maculata females must touch or taste DHBA to stimulate oviposition. We setup bioassays in communal cages (housing 10 females) and solitary cages (housing 1 female). In communal cages, nearly all egg clutches were found in or near the chemical dish with DHBA only. Provisioning cages with a tissue substrate reduced oviposition in the chemical dish. Regardless of oviposition site, egg number per clutch did not increase in communal cages or solitary cages with DHBA only. Affixing DHBA to the base of the chemical dish, then covering it with a nylon screen, reduced oviposition. This study suggests that females must touch or taste DHBA to stimulate oviposition. The physiological mechanism involved in oviposition stimulation requires further study. DHBA could potentially serve as a weak oviposition stimulant for predatory ladybird beetles in some mass-rearing systems.

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran.

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C6H8O3 and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.

Bioactive crude plant seed extracts from the NCAUR oilseed repository.

Over four-hundred crude extracts from 202 plant species distributed among 131 plant families were evaluated for their bioactivity against brine shrimp (Artemia salina). Activity was determined for both the organic (CH2Cl2:MeOH) and aqueous extracts against A. salina in a 96 well-plate assay. Of the greater than four-hundred extracts tested, 21 organic and 6 aqueous extracts demonstrated potent cytotoxic activity (LC50 = < 100 microg/ml). Three of these organic extracts (Crateva religiosa, Diospyros dichrophylla, and Olax subscorpioidea) were chosen for chemical investigations due to their high activity and a lack of prior investigations. Chemical analysis of these extracts resulted in the isolation of oleanolic acid (1) and 4-epi-hederagenin (2) from C. religiosa, isodiospyrin (3) from D. dichrophylla, and santalbic acid (4) from O. subscorpioidea.

Estrogenic and antiproliferative properties of soy sapogenols in human breast cancer cells in vitro.

Two soy sapogenols, soyasapogenol A (SA) and soyasapogenol B (SB) were tested for their estrogenic activities in estrogen responsive MCF-7 or estrogen-insensitive MDA-MB-231 (MDA) human breast cancer cells. SB and SA had differential actives on cell proliferation with 10 microM SB being growth inhibitory to MDA cells with no significant effect at any concentration on MCF-7 cells. SA also inhibited MDA cell proliferation at 10 micro, but at this same dose stimulated a 2.5-fold increase in MCF-7 proliferation. SA (0.1-10 microM) induced pS2 mRNA levels and the induction was blocked by co-treatment of cells with the anti-estrogen ICI 182,780. SA also induced the formation of an ER-ERE DNA complex measured by electrophoretic mobility shift assay. In summary, these results show that soyasapogenol A is estrogenic, whereas soyasapogenol B is growth inhibitory.

Isolating antigenotoxic components and cancer cell growth suppressors from agricultural by-products.

Commercial processing wastes or by-products of crops were found to be sources of antimutagens and human tumor cell growth suppressors. We developed a microplate method to measure genomic DNA damage in Chinese hamster ovary cells with a modified single cell gel electrophoresis (SCGE) assay. This allowed us to measure the repression of 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage by very small amounts of complex mixtures, fractions or individual chemicals isolated from agricultural by-products. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, repressed induced genomic DNA damage in mammalian cells. PCC was separated into a series of chemically defined fractions and two fractions (PCC70 and PCC100) repressed mutagen-induced damage. Of the isoflavones isolated from soybean fraction PCC70, daidzein expressed antigenotoxic activity, however, genistin and genistein enhanced DNA damage. An antigenotoxic response also was observed with a fraction isolated from corn distillate solids (CDS40). We developed a microplate assay to measure the suppression of the growth rate of human cancer cells in which the cytostatic/cytotoxic status at each concentration of the test sample was quantitatively determined. Genistein, genistin, daidzein and daidzin isolated from soybean fraction PCC70 expressed a wide range of growth suppression of HT-29 human colon cancer cells. The biological assays were integrated with, and directed, the separation and analytical chemistry component of this project. Compounds were purified from biologically active fractions and the structure of individual chemicals was determined with analytical HPLC and LC-mass spectroscopy (LC-MS). This research may lead to the isolation of novel chemoprotectants from agronomic commercial processing products and by-products.

Evaluation of conventional resistance to European corn borer (Lepidoptera: Crambidae) and western corn rootworm (Coleoptera: Chrysomelidae) in experimental maize lines developed from a backcross breeding program.

Plant resistance is a promising control method for the two most damaging insect pests of maize, Zea mays L.: the European corn borer, Ostrinia nubilalis (Hübner), and the western corn rootworm Diabrotica virgifera virgifera LeConte. Fifteen experimental lines of maize, derived from a backcross breeding program designed to introgress resistance to European corn borer from Peruvian maize into two U.S. Corn Belt adapted inbred lines, were evaluated for resistance to European corn borer and western corn rootwonrm. The experimental lines were in the second generation of backcrossing. All experimental lines were resistant to leaf blade feeding by European corn borer. These lines had low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, a chemical commonly associated with leaf blade feeding resistance, indicating that this was not the mechanism of resistance to leaf blade feeding in these lines. Eleven experimental lines were resistant to leaf sheath and collar feeding by European corn borer. Useful sources of European corn borer ovipositional nonpreference and root feeding resistance to western corn rootworm were not identified. Some of the lines evaluated in this study may provide useful sources of resistance to both leaf blade and leaf sheath and collar feeding by European corn borer.

Characterization and antimutagenic activity of soybean saponins.

An extract was prepared from a commercial soybean-processing by-product (soybean molasses) and was fractionated into purified chemical components. In previous work, this extract (phytochemical concentrate, PCC) repressed induced genomic DNA damage, whole cell clastogenicity and point mutation in cultured mammalian cells. In the current study, a chemical fraction was isolated from PCC using preparative high-performance liquid chromatography (HPLC). This fraction, PCC100, repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage in Chinese hamster ovary (CHO) cells as measured by single cell gel electrophoresis (alkaline Comet assay). Using liquid chromatography-electrospray ionization-mass spectroscopy and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, PCC100 was shown to consist of a mixture of group B soyasaponins and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) soyasaponins. These include soyasaponins I, II, III, IV, V, Be, betag, betaa, gammag and gammaa. Purified soyasapogenol B aglycone prepared from fraction PCC100 demonstrated significant antigenotoxic activity against 2AAAF. To our knowledge, these data demonstrate for the first time the antimutagenic activity of soybean saponins in mammalian cells.

Antimutagenic activity of chemical fractions isolated from a commercial soybean processing by-product.

Commercial products of agronomic crop plants may become a reliable and inexpensive source of phytonutrients, such as antimutagenic food supplements. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, was able to repress induced genomic DNA damage, whole cell clastogenicity, and point mutation in mammalian cells. In this paper we separated PCC into a series of chemically defined fractions and determined their ability to repress induced mutagenic damage in Chinese hamster lung cells, Chinese hamster ovary cells and human lymphocytes. Fraction PCC70 (PCC 70% methanol eluate) contained the flavonoids from PCC and daidzin and genistin repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage measured with single cell gel electrophoresis. Genistein, however, enhanced the genotoxic impact of 2AAAF. Fraction PCC100 (PCC 100% methanol eluate) had the greatest level of antigenotoxic activity against 2AAAF in CHO cells and repressed the genotoxic capacity of the dietary carcinogen 2-amino-3-methylimidazo-(4,5-f)quinoline (IQ) in human lymphocytes. These data indicate that commercial soybean products and by-products may be a source of chemoprotective food additives.

Acylated flavonoids in callus cultures of Citrus aurantifolia.

Two new acylated flavonol glycosides were isolated along with kaempferol 3-O-beta-rutinoside from 10-year-old callus cultures of Mexican lime. The structures of these new compounds are kaempferol 3-O-beta-D-glucopyranoside-6"-(3-hydroxy-3-methyl glutarate) and kaempferol 3-O-beta-D-glucopyranoside-6"-(3-hydroxy-3-methyl glutarate)-7-O-beta-D-glucopyranoside.

Ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase in Prochloron.

Cell-free extracts of Prochloron didemni were assayed for ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) and phosphoribulokinase (EC 2.7.1.19), two key enzymes in the reductive pentose-phosphate cycle. In an RuBP-dependent reaction, the production of two molecules of 3-phosphoglycerate per molecule of CO2 fixed was shown. Phosphoribulokinase activity was demonstrated by the production of ADP from ribulose 5-phosphate (Ru5P) and ATP and by measurement of ATP-, Ru5P-dependent (14)CO2 fixation in the presence of excess spinach RuBP carboxylase. When Prochloron RuBP carboxylase was purified from cell-free extracts by isopycnic centrifugation in reoriented linear 0.2 to 0.8 M sucrose gradients, the enzyme sedimented to a position which corresponded to that for the 520,000-dalton spinach enzyme. After polyacrylamide gel electrophoresis (PAGE) of Prochloron enzyme, a major band of enzyme activity corresponded to that for the spinach enzyme. Considerably more additional carboxylase activity was found in a less mobile species than was the case for spinach RuBP carboxylase. Sodium dodecyl sulfate-PAGE of the Prochloron enzyme indicates that it is composed of both large (molecular weight, MW=57,500) and small (MW=18,800) subunits.