Is the Cry1Ab protein from Bacillus thuringiensis (Bt) taken up by plants from soils previously planted with Bt corn and by carrot from hydroponic culture?

The uptake of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) by various crops from soils on which Bt corn had previously grown was determined. In 2005, the Cry1Ab protein was detected by Western blot in tissues (leaves plus stems) of basil, carrot, kale, lettuce, okra, parsnip, radish, snap bean, and soybean but not in tissues of beet and spinach and was estimated by enzyme-linked immunosorbent assay (ELISA) to be 0.05 +/- 0.003 ng g(-1) of fresh plant tissue in basil, 0.02 +/- 0.014 ng g(-1) in okra, and 0.34 +/- 0.176 ng g(-1) in snap bean. However, the protein was not detected by ELISA in carrot, kale, lettuce, parsnip, radish, and soybean or in the soils by Western blot. In 2006, the Cry1Ab protein was detected by Western blot in tissues of basil, carrot, kale, radish, snap bean, and soybean from soils on which Bt corn had been grown the previous year and was estimated by ELISA to be 0.02 +/- 0.014 ng g(-1) of fresh plant tissue in basil, 0.19 +/- 0.060 ng g(-1) in carrot, 0.05 +/- 0.018 ng g(-1) in kale, 0.04 +/- 0.022 ng g(-1) in radish, 0.53 +/- 0.170 ng g(-1) in snap bean, and 0.15 +/- 0.071 ng g(-1) in soybean. The Cry1Ab protein was also detected by Western blot in tissues of basil, carrot, kale, radish, and snap bean but not of soybean grown in soil on which Bt corn had not been grown since 2002; the concentration was estimated by ELISA to be 0.03 +/- 0.021 ng g(-1) in basil, 0.02 +/- 0.008 ng g(-1) in carrot, 0.04 +/- 0.017 ng g(-1) in kale, 0.02 +/- 0.012 ng g(-1) in radish, 0.05 +/- 0.004 ng g(-1) in snap bean, and 0.09 +/- 0.015 ng g(-1) in soybean. The protein was detected by Western blot in 2006 in most soils on which Bt corn had or had not been grown since 2002. The Cry1Ab protein was detected by Western blot in leaves plus stems and in roots of carrot after 56 days of growth in sterile hydroponic culture to which purified Cry1Ab protein had been added and was estimated by ELISA to be 0.08 +/- 0.021 and 0.60 +/- 0.148 ng g(-1) of fresh leaves plus stems and roots, respectively. No Cry1Ab protein was detected in the tissues of carrot grown in hydroponic culture to which no Cry1Ab protein had been added. Because of the different results obtained with different commercial Western blot (i.e., from Envirologix and Agdia) and ELISA kits (i.e., from Envirologix, Agdia, and Abraxis), it is not clear whether the presence of the Cry1Ab protein in the tissues of some plants under field condition and in carrot in sterile hydroponic culture was the result of the uptake of the protein by the plants or of the accuracy and sensitivity of the different commercial kits used. More detailed studies with additional techniques are obviously needed to confirm the uptake of Cry proteins from soil by plants subsequently planted after a Bt crop.

Microbial populations and enzyme activities in soil in situ under transgenic corn expressing cry proteins from Bacillus thuringiensis.

Transgenic Bt crops produce insecticidal Cry proteins that are released to soil in plant residues, root exudates, and pollen and that may affect soil microorganisms. As a continuation of studies in the laboratory and a plant-growth room, a field study was conducted at the Rosemount Experiment Station of the University of Minnesota. Three Bt corn varieties that express the Cry1Ab protein, which is toxic to the European corn borer (Ostrinia nubilalis Hübner), and one Bt corn variety that expresses the Cry3Bb1 protein, which is toxic to the corn rootworm complex (Diabrotica spp.), and their near-isogenic non-Bt varieties were evaluated for their effects on microbial diversity by classical dilution plating and molecular (polymerase chain reaction-denaturing gradient gel electrophoresis) techniques and for the activities of some enzymes (arylsulfatases, acid and alkaline phosphatases, dehydrogenases, and proteases) involved in the degradation of plant biomass. After 4 consecutive years of corn cultivation (2003-2006), there were, in general, no consistent statistically significant differences in the numbers of different groups of microorganisms, the activities of the enzymes, and the pH between soils planted with Bt and non-Bt corn. Numbers and types of microorganisms and enzyme activities differed with season and with the varieties of corn, but these differences were not related to the presence of the Cry proteins in soil. The Cry1Ab protein of Bt corn (events Bt11 and MON810) was detected in most soils during the 4 yr, whereas the Cry3Bb1 protein was not detected in soils of Bt corn (event MON863) expressing the cry3Bb1 gene.

Release of the recombinant proteins, human serum albumin, beta-glucuronidase, glycoprotein B from human cytomegalovirus, and green fluorescent protein, in root exudates from transgenic tobacco and their effects on microbes and enzymatic activities in soil.

We determined the release in root exudates of human serum albumin (HSA), beta-glucuronidase (GUS), glycoprotein B (gB) from human cytomegalovirus, and green fluorescent protein (GFP) from genetically modified transgenic tobacco expressing the genes for these proteins in hydroponic culture and non-sterile soil. GUS, gB, and GFP were expressed in the plant but were not released in root exudates, whereas HSA was both expressed in the plant and released in root exudates, as shown by a 66.5-kDa band on SDS-PAGE and Western blot and confirmed by ELISA. Root exudates from GUS and gB plants showed no bands that could be attributed to these proteins on SDS-PAGE, and root exudates from GFP plants showed no fluorescence. The concentration of HSA in root exudates was estimated to be 0.021 ng ml(-1), whereas that in the plant biomass was estimated to be 0.087 ng ml(-1). The concentration of HSA in soil was estimated to be 0.049 ng g(-1). No significant differences in the number of microorganisms and the activity of selected enzymes were observed between rhizosphere soil of non-modified and HSA tobacco.

Effect of lidocaine on production of migration inhibitory factor and on macrophage motility: in vitro exposure of guinea pig lymphocytes and macrophages.

Exposure of lymphocytes from guinea pigs sensitized to keyhole limpet hemocyanin and of macrophages from nonsensitized animals to noncytotoxic doses of lidocaine (10(-4) to 10(-6) M) resulted in the inhibition of the production of macrophage migration inhibitory factor and of macrophage motility. The inhibition of both processes was related to the concentration of lidocaine in the medium. The effects of lidocaine, a membrane-stabilizing drug, were apparently related to its ability to interact with the cell surface and cause changes in the surface ionic configuration of the cells, as determined by cell electrophoresis. The drug conferred permanent changes in the surface of lymphocytes at all concentrations tested, but the changes in the surface of macrophages induced in the presence of 10(-5) and 10(-6) M of the drug were reversible. The presence of noncytotoxic doses of lidocaine in the cellular environment resulted in significant changes in cellular functions that appeared to be related to the ability of the drug to interact with cell membranes in a manner determined by the specific surface properties of the cell.

Developing standards for environmental toxicants: the need to consider abiotic environmental factors and microbe-mediated ecologic processes.

This article suggests and discusses two novel aspects for the formulation of standards for environmental toxicants. First, uniform national standards for each pollutant will be underprotective for some ecosystems and overprotective for others, inasmuch as the toxicity of a pollutant to the indigenous biota is dependent on the physicochemical properties of the recipient environment. As the number of chemicals that need regulation is immense and as microbes appear to respond similarly to pollutant-abiotic factor interactions as do plants and animals, it is suggested that microbial assays be used initially to identify those abiotic factors that most influence the toxicity of specific pollutants. Thereafter, additional studies using plants and animals can focus on these pollutant-abiotic factor interactions, and more meaningful standards can then be formulated more rapidly and inexpensively. Second, it is suggested that the response to pollutants of microbe-mediated ecologic processes be used to quantitate the sensitivity of different ecosystems to various toxicants. Such a quantification, expressed in terms of an "ecological dose 50%" (EcD50), could be easily incorporated into the methodologies currently used to set water quality criteria and would also be applicable to setting criteria for terrestrial ecosystems.

Larvicidal Cry proteins from Bacillus thuringiensis are released in root exudates of transgenic B. thuringiensis corn, potato, and rice but not of B. thuringiensis canola, cotton, and tobacco.

Larvicidal proteins encoded by cry genes from Bacillus thuringiensis were released in root exudates from transgenic B. thuringiensis corn, rice, and potato but not from B. thuringiensis canola, cotton, and tobacco. Nonsterile soil and sterile hydroponic solution in which B. thuringiensis corn, rice, or potato had been grown were immunologically positive for the presence of the Cry proteins; from B. thuringiensis corn and rice, the soil and solution were toxic to the larva of the tobacco hornworm (Manduca sexta), and from potato, to the larva of the Colorado potato beetle (Leptinotarsa decemlineata), representative lepidoptera and coleoptera, respectively. No toxin was detected immunologically or by larvicidal assay in soil or hydroponic solution in which B. thuringiensis canola, cotton, or tobacco, as well as all near-isogenic non-B. thuringiensis plant counterparts or no plants, had been grown. All plant species had the cauliflower mosaic virus (CaMV) 35S promoter, except rice, which had the ubiquitin promoter from maize. The reasons for the differences between species in the exudation from roots of the toxins are not known. The released toxins persisted in soil as the result of their binding on surface-active particles (e.g. clay minerals, humic substances), which reduced their biodegradation. The release of the toxins in root exudates could enhance the control of target insect pests, constitute a hazard to nontarget organisms, and/or increase the selection of toxin-resistant target insects.

Differentiation by electrophoresis and enzymoserology of proteinases, produced by strains of Staphylococcus epidermidis biotypes 1 and 4.

Electrophoretic and serologic characterization of extracellular proteinases from 111 isolates (80 biotype 1 and 31 biotype 4) of Staphylococcus epidermidis (coagulase negative) of human origin were conducted. Seventy-seven (60 of biotype 1, 17 of biotype 4) of the 111 proteinase-positive isolated of S. epidermidis were classified by serological differentiation of their proteolytic enzymes with known specific antisera. Thirty-two (18 of biotype 1, 14 of biotype 4 (isolates of S. epidermidis produced proteolytic enzymes that did not react with any of the known group antisera and were classified as NR (non-reacting). No classification of the enzymes of two other isolates was possible. The NR proteinase enzymes from the 32 cultures that could not be classified by serological differentiation with the known specific antisera fitted into one of three electrophoretic mobility patterns: NR1, NR2 or NR3. Seventy-eight isolates of S. epidermidis biotype 1 were placed into four proteinase sero-groups; 49 in F, 11 in B, 11 in NR2, and 7 in NR3. Thirty-one isolates of S. epidermidis biotype 4 were placed into one of two groups: 17 in C and 14 in NR. All enzymes tested migrated towards the anode at pH 8.6, and the order of electrophoretic mobility was NR3 greater than F greater than NR2 greater than NR1 greater than B greater than C.

Differentiation of strains of Staphylococcus epidermidis biotypes 1 and 4 by aminopeptidase profiles.

Seven different strains of Staphylococcus epidermidis and one strain of S. aureus were catagorized by their aminopeptidase activity. Eight different profiles were obtained: five profiles for S. epidermidis biotype 4 (each representing a different known serotype), two profiles for S. epidermidis biotype 1 (one for each strain used) and one for the strain of S. aureus used. Despite some similarities, distinct patterns of the hydrolysis of 22 different amino acid-beta-naphthylamides were evident for each individual strain. The evaluation of aminopeptidase profiles provides another means for distinguishing between strains of the same species and can, therefore, be used advantageously and in combination with serotyping as an epidemiological tool.

Staphylococcus epidermidis biotype 4: epidemiological conclusions from five different typing methods.

There is presently no accepted method for marking individual strains of Staphylococcus epidermidis. Consequently, five parameters for distinguishing such strains were examined and compared for their epidemiological efficacy: biotyping, serotyping, proteinase grouping, aminopeptidase profiles and antibiograms. Both biotyping and proteinase grouping were of limited use in identifying a particular strain, although they were helpful in initially categorizing such strains. Antibiograms were least useful because of similarities in susceptibility patterns among isolates. Serotyping and amino-peptidase profiles provided the best means of identifying an individual strain for epidemiological use. The applicability of these typing methods was demonstrated during a one year epidemiological study at a chronic disease hospital.

Dibromochloropropane (DBCP): a review.

A highly persistent, lipophilic, brominated organochlorine which is effectively used against nematodes, dibromochloropropane (DBCP) has been produced for agriculture since 1955. In 1975, production of DBCP in the United States reached 25 million lbs. However, investigations with laboratory animals, some of which were published in the early 1960s, have shown that DBCP decreases sperm mobility and spermatogenesis, disturbs the estrous cycle, reduces phagocytosis by white blood cells, and induces malignant tumors. Later studies with procaryotic and eucaryotic cells, including human sperm, have demonstrated DBCP to be mutagenic and to effect the genome adversely. In 1977 many of the employees at the Occidental Chemical plant in Lathrop, California, who had handled DBCP, were found to be either azoospermic or oligospermic. Subsequent surveys of employees handling DBCP at other chemical plants confirmed these findings. In 1977 on edible crops and in 1979 DBCP per se was detected in well waters. As a result of these studies, the Occupational Safety and Health Administration (OSHA) and the Environmental Protection Agency (EPA) in 1977 promulgated regulations restricting the use and handling of DBCP. In 1979, the EPA banned almost all agricultural uses of DBCP.

Supplementation with selenium and human immune cell functions. I. Effect on lymphocyte proliferation and interleukin 2 receptor expression.

Selenium (Se) is an essential nutritional factor that was shown by us to alter the expression of the high affinity interleukin 2 receptor (Il2-R) and its subunits, cell proliferation, and clonal expansion of cytotoxic T-lymphocytes in mice. This study shows that dietary supplementation of Se-replete humans with 200 micrograms/d of sodium selenite for 8 wk, or in vitro supplementation with 1 x 10(-7) M Se (as sodium selenite), result in a significant augmentation of the ability of peripheral blood lymphocytes to respond to stimulation with 1 microgram/mL of phytohemagglutinin or alloantigen (mixed lymphocyte reaction) and to express high affinity Il2-R on their surface. There was a clear correlation between supplementation with Se and enhanced 3H-thymidine incorporation into nuclear DNA, preceded by enhanced expression of high affinity Il2-R. Supplementation with Se can apparently modulate T-lymphocyte mediated immune responses in humans that depend on signals generated by the interaction of interleukin 2 with Il2-R.

Supplementation with selenium and human immune cell functions. II. Effect on cytotoxic lymphocytes and natural killer cells.

This study examined the effect of dietary (200 micrograms/d for 8 wk) supplementation with selenium (as sodium selenite) on the ability of human peripheral blood lymphocytes to respond to stimulation with alloantigen, develop into cytotoxic lymphocytes, and to destroy tumor cells, and on the activity of natural killer cells. The participants in the study were randomized for age, sex, weight, height, and nutritional habits and given selenite or placebo tablets; all participants had a selenium replete status as indicated by their plasma Se levels prior to supplementation. The data indicated that the supplementation regimen resulted in 118% increase in cytotoxic lymphocyte-mediated tumor cytotoxicity and 82.3% increase in natural killer cell activity as compared to baseline values. This apparently was related to the ability of the nutrient to enhance the expression of receptors for the growth regulatory lymphokine interleukin-2, and consequently, the rate of cell proliferation and differentiation into cytotoxic cells. The supplementation regimen did not produce significant changes in the plasma Se levels of the participants. The results indicated that the immunoenhancing effects of selenium in humans require supplementation above the replete levels produced by normal dietary intake.

Regulation of cellular immune responses by selenium.

Selenium (Se) is an essential nutritional factor that affects the development and expression of cell-mediated immune responses directed toward malignant cells. These studies have shown that dietary (2 ppm for 8 wk) or in in vitro (1 x 10(-7)M) supplementation with Se (as sodium selenite) results in a significant enhancement of the proliferative responses of spleen lymphocytes from C57Bl/6J mice in response to stimulation with mitogen or antigen. Se deficiency (0.02 ppm for 8 wk) had the opposite effect. The alterations in the ability of the cells to proliferate, which occurred in the absence of changes in the endogenous levels of interleukin-2 (Il2) or interleukin 1, were apparently related to the ability of Se to alter the kinetics of expression of high-affinity Il2 receptors on the surface of activated lymphocytes. This resulted in an enhanced or delayed clonal expansion of the cells, and in an increased or decreased frequency of cytotoxic cells within a given cell population. The changes in tumor cytotoxicity were paralleled by changes in the amounts of lymphotoxin produced by the activated cells. Dietary Se modulations had a comparable effect on macrophage-mediated tumor cytodestruction. The results also suggested that Se exerts its effect 8-24 h after stimulation, and that it most likely affects processes in the cytoplasmic and/or nuclear compartments of activated lymphocytes.

Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells.

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1 x 10(-7)M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1 x 10(-7)M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear 3H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of II-2 through its ability to enhance the expression of intermediate affinity II-2R on these cells.

Acceleration by montmorillonite of nitrification in soil.

A soil naturally containing montmorillonite (M) was amended with 10% M and sequentially perfused with glyeme, with fresh glyeme being added every 16--17d after nitrification of the previously added glycine-nitrogen had reached a plateau. In some systems, the old perfusates were replaced each time with a fresh glycine solution; in others, the initial perfusate was not replaced but only adjusted each time to the original 200 ml volume and a comparable glycine concentration (140 micrograms NH2-N/ml). The incorporation of M enhanced the rates of heterotrophic degradation of glycine and subsequent autotrophic nitrification, but these stimulatory effects decreased with each successive perfusion. The reasons for these decreases are not known, but they did not appear to be related to inorganic nutrition, as perfusion with a mixed cation solution after five perfusion cycles did not significantly enhance nitrification in either the check or M-amended soils during three subsequent perfusions with glycine. The enhancement of nitrification by M appeared to be a result, in part, of the greater buffering capacity of the M-amended soil, as indicated by lesser reductions in the pH of perfusates from the M-amended soil, by titration curves of the soils, and by the greater and longer stimulation of nitrification in the check soil amended with 1% CaCO3, which had a greater buffering capacity than did M. The stimulation by CaCO3 may also have been partially the result of providing CO2 for the autotrophic nitrifyers. Significant concentrations of nitrite accumulated only in perfusates from soil amended with CaCO3. Air-drying and remoistening the soils enhanced nitrification of subsequently added glycine, especially in the check soil. The importance of pH-mediation, of the production of inhibitors, and/or of feed-back inhibition was indicated by the lower rate and extent of nitrification in systems wherein the perfusates were not replaced between successive additions of glycine. Although the results of these studies confirmed previous observations that M enhances the rate of nitrification in soil, the mechanisms responsible for this stimulation are still not known.

Effect of montmorillonite and kaolinite on nitrification in soil.

A soil not naturally containing montmorillonite (M) was amended with approximately 5, 10 or 20% M or kaolinite (K), maintained in a greenhouse under periodic cultivating and alternate wetting and drying of more than two years, and then used in perfusion studies. The incorporation of M enhanced the rate of both heterotrophic degradation of glycine and subsequent autotrophic nitrification in direct relation to the amounts of M added. In soil amended with K, neither degradation nor nitrification was stimulated. The addition of M shortened the lag phase before nitrification was initiated, increased the pH of both the soil and the perfusates, and increased the rate, but not the extent, of oxidation of ammonium to nitrite and nitrate. The addition of CaCO2 or MgCO3, but not of CaSO4, also enhanced the rate of nitrification. The effects observed may have resulted from the influence of M on the pH, buffering capacity, and other soil conditions necessary for maximum activity of nitrifying microorganisms.