Evaluation of the estrogenic effects of legume extracts containing phytoestrogens

Seven legume extracts containing phytoestrogens were analyzed for estrogenic activity. Methanol extracts were prepared from soybean (Glycine max L.), green bean (Phaseolus vulgaris L.), alfalfa sprout (Medicago sativa L.), mung bean sprout (Vigna radiata L.), kudzu root (Pueraria lobata L.), and red clover blossom and red clover sprout (Trifolium pratense L.). Extracts of kudzu root and red clover blossom showed significant competitive binding to estrogen receptor beta (ERbeta). Estrogenic activity was determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay. Kudzu root, red clover blossom and sprout, mung bean sprout, and alfalfa sprout extracts displayed increased cell proliferation above levels observed with estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting an ER-related signaling pathway was involved. The ER subtype-selective activities of legume extracts were examined using transiently transfected human embryonic kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist activity toward ERbeta. Using HPLC to collect fractions and MCF-7 cell proliferation, the active components in kudzu root extract were determined to be the isoflavones puerarin, daidzin, genistin, daidzein, and genistein. These results show that several legumes are a source of phytoestrogens with high levels of estrogenic activity.     

Loma salmonae (Protozoa: Microspora) infections in seawater reared coho salmon Oncorhynchus kisutch

Loma salmonae (Putz et al., 1965) infections were observed in five groups of coho salmon, Oncorhynchus kisutch, reared in seawater net-pens in Washington State, U.S.A. in 1984–1986. Ultrastructural characteristics, size of spores, tissues and host infected, and geographical location identified the microsporidium as Loma salmonae. Preserved spores measured 4.4×2.3 (4–5.6×2–2.4) μm and exhibited 14–17 turns of the polar filament. Infections were evident in the gills of some fish before seawater entry, but few parasites were observed and they caused little tissue damage. Infections observed in fish after transfer to seawater were associated with significant pathological changes in the gills. A mixed inflammatory infiltrate was associated with ruptured microsporidian xenomas within the vessels and interstitium of the primary lamellae. Microsporidian spores were dispersed throughout the lesions and were often seen inside phagocytes. The parasite was also observed in the heart, spleen, kidney and pseudobranchs; however, the inflammatory lesions were common only in the heart.

Monthly examination of fish after transfer to seawater showed peak prevalences (33–65%) of gill infections during the summer. Although moribund fish were often infected with other pathogens, the high prevalence of L. salmonae infections and the severity of the lesions it caused, suggested that this parasite significantly contributed to the recurrent summer mortalities observed at this net-pen site.     

Phenotypic differences among three clonal lineages of Phytophthora ramorum

There are three major clonal lineages of Phytophthora ramorum present in North America and Europe named NA1, NA2, and EU1. Twenty‐three isolates representing all three lineages were evaluated for phenotype including (i) aggressiveness on detached Rhododendron leaves and (ii) growth rate at minimum, optimum, and maximum temperatures. Closely related species P. foliorum and P. hibernalis were included in phenotypic tests since these species are encountered in nursery surveys for P. ramorum. Isolates from the NA2 and EU1 lineages were the most aggressive and isolates from the NA1 group were the least aggressive. The NA1 lineage of P. ramorum was the most variable in aggressiveness and growth rate. The variability in the NA1 lineage was due to the presence of non‐wild type (nwt) isolates. There was no significant difference in growth rate among NA1 wild type (wt), NA2, and EU1 lineages at any temperature tested. The difference between wt and nwt P. ramorum isolates is discussed.     

Comparative evaluation of NTA and EDTA for extractive decontamination of Pb-polluted soils

Nitrilotriacetic acid (NTA) and ethylenediaminetetraacetic acid (EDTA) were compared for their ability to solubilize Pb from a highly-contaminated (PbT 21%) soil collected from a battery recycling facility. For chelant concentrations below 0.04 M (representing a 1:1 chelant-to-Pb_T molar ratio), EDTA released 10 to 30% more Pb than NTA. NTA-to-Pb T ratios greater than 1:1 reduced Pb recovery because of readsorption of Pb(NTA)₂ ^4? onto positively-charged oxide soil components at pH < 8.5. For the EDTA system, however, complexation completely bound all coordination sites of Pb and EDTA, leaving no functional groups available for surface adsorption. Thus, Pb recovery progressively increased with higher EDTA concentrations, although the additional Pb release with each EDTA increment became smaller. For pH < 5 and EDTA/Pb of 2:1, Pb recovery exceeded 90%. The addition of 0.5 M NaC1O₄ enhanced Pb recovery by EDTA for pH 5 to 12, but substantially suppressed recovery by NTA for pH < 11. Because Pb release by NTA was diminished by high ionic strength and chelant-to-metal ratios, NTA may be limited as a soil washing reagent. Stronger complexation and consistent Pb desorption behavior by EDTA favors its use.     

Deposition and depletion of five anticoccidials in eggs

Anticoccidials are compounds that are widely used as feed additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a certain time interval for broilers and pullets but not for laying hens. It was shown in the past that carry-over at the feeding mill is found to be the main reason for the presence of residues in eggs. An animal experiment was set up to investigate the effect of carry-over at the feeding mill on the presence of residues of anticoccidials in eggs. For the compounds diclazuril, robenidine, halofuginone and nicarbazin in combination with narasin, two concentration levels were tested: the maximum allowed concentration for broilers (100%) and a concentration corresponding to 5% carry-over during feed preparation. Also dimetridazole was included in the experiment but only at one concentration level. Eggs were sampled during treatment (14 days) and for a period of 30 days after withdrawal of the anticoccidial-containing feed. Residues were determined, and deposition and depletion curves were generated. Analyses were performed by ELISA and LC-MS/MS. For all compounds, substantial residues could be found in the 5% groups, which points out the risk of carry-over at the feeding mill. The distribution of the residues between egg yolk and white was determined by analyzing both fractions.     

Microbial respiration and growth during the decomposition of wheat straw

The effect of N on the disappearance of C from 1.5 g wheat (Triticum aestivum L. var. “Nugaines”) straw decomposing in sand and the response of the biomass to addition of N (adequate to bring the C:N ratio to 48:1) and C (200 mg of glucose-C) were determined. A concept was used that assumed the change in the microbial biomass was proportional to the change in acid hydrolyzable amino acid-N. Microbial respiration (CO₂ evolution and O₂ uptake) and growth were stimulated by the initial addition of N (which brought the original C:N ratio from 150:1 to 48:1), but the addition of the same amount of N to the system at 240 h that had received no N initially resulted in slight if any increase in respiration or microbial growth. The response of the microflora to the 200 mg of glucose-C additions after 240 h indicated the microbial populations were primarily limited by available C rather than available N after only 240 h incubation, even though about 95% of the original straw residue-C plus biomass C remained in the system. Respiratory quotients indicated a qualitative shift over time in the average oxidation state of the substrates being metabolized. It is postulated that the RQ shift resulted, at least in part, from death of the population rather than totally from the availability of the straw substrate.The initial addition of N resulted in 3.8 times the net amino acid production, but only 1.6 times the CO₂-C production over 240 h compared with the control without added N. These results suggest that N availability might result in a change in the growth yield of the microbial population.     

Soil microbial biomass and selected soil enzyme activities: Effect of fertilization and cropping practices

Selected microbiological properties of soils receiving different fertilizer management regimes were studied from adjoining wheat farms in the highly productive Palouse region of eastern Washington. Since 1909, the only N input to the soil of Farm Management System 1 has been through leguminous green manure crops consisting most recently of Austrian winter peas (Pisum sativum ssp. arvense L., Poir), plus native soil fertility for N and all other plant nutrients. The soil of Farm Management System 2 received regular applications of anhydrous ammonia, P and S at recommended rates for the last 30 yr. There were no differences in numbers of soil microorganisms as determined by plate counts; however, soil from management system 1 had significantly higher levels of urease, phosphatase and dehydrogenase at all three samplings and significantly higher soil microbial biomass at the first two samplings. The data indicate that management system 1 soil had a larger and more active soil microflora.