[Show abstract][Hide abstract] ABSTRACT: Cranberry juice (CJ) and grape juice (GJ) from Vaccinium macrocarpon and Vitis labrusca, respectively, and purified proanthocyanidins (PACs) from these species are recognized to possess antiviral activity. The
effects of CJ and GJ on tight junction (TJ) structure and function among rotavirus-infected monkey kidney epithelial cells
(MA-104) in monolayer cultures were evaluated. Antiviral activity by cranberry PACs of rotavirus in cell-free suspension was
investigated by a rotavirus antigen [i.e., viral capsid protein 6 (VP6)] capture enzyme-linked immunosorbent assay (ELISA)
and by transmission electron microscopy (TEM). MA-104 monolayers were treated with CJ, GJ, or cranberry juice cocktail (CJC)
drink before inoculation with rotavirus. TJ function and structural integrity were measured by changes in transepithelial
electrical resistance (TEER) and by reduction of signal intensity of the TJ α-claudin 1 by immunofluorescence. The inhibitory
activity of CJ and GJ on viral RNA synthesis, as a function of viral concentration, was determined by reverse transcription
polymerase chain reaction (rtPCR). After 4days, virus-infected monolayers pretreated with GJ (Concord and Niagara GJs) had
TEER readings similar to uninfected controls. CJ and CJC also had a significant protective effect (P<0.05) on TJ function, but to a lesser extent than GJ. Disorganization of TJ integrity commenced at 24- to 36-h post-viral
inoculation, but this effect was reduced by pretreatment with CJ or GP of monolayer cultures. TEM showed aggregation of rotavirus
by cranberry PACs. The destruction of rotavirus capsid proteins VP6, in cell-free suspension was inversely related to the
concentration of cranberry PACs (C-PAC). Loss of rotavirus RNA by CJ or GJ was inversely related to viral infectivity titers.
CJ, GJ, or PAC-associated antiviral activity has been linked to modifications in cellular physiologic events and to physical
factors (e.g., PAC-mediated viral aggregation) that probably compromise viral infectivity. Multiple cell physiological and
physical events must be considered when determining the mechanisms associated with the antiviral (i.e., rotavirus) activity
of CJ, GJ, and PACs.
KeywordsRotavirus infectivity–Proanthocyanidins–Cell function/structure–Tight junctions–Electron microscopy
[Show abstract][Hide abstract] ABSTRACT: Rhizopus stolonifer and Cunninghamella blakesleeana trained after five passages to tolerate elevated concentrations of copper simultaneously acquired a tolerance to elevated levels of cadmium, cobalt, nickel, and lead. The acquired tolerance to copper was not stable in the absence of the metal, as serial transfers to a copper-free medium progressively reduced the level of tolerance, and after the sixth passage on a copper-free medium, the tolerance was equivalent to that of the nontrained parentals ("untraining"). However, the untrained fungi regained the same tolerance to copper as the original copper-trained fungi after only three passages on copper-amended medium ("retraining"). The tolerance to copper was apparently the result of physiological adaptation rather than of the selection of resistant cells or the induction of mutation. The metal may have activated genes that coded for biochemical processes that conferred tolerance to copper as well as to other heavy metals. Furthermore, the tolerance to copper of the mycelia was transferred to the sporangiospores. The copper-trained fungi did not appear to produce extracellular metabolites that complexed with and, hence, excluded and detoxified the copper. However, the mycelia of the trained fungi removed approximately twice as much copper from solution than those of the nontrained parentals, suggesting that the tolerance to copper resulted from the binding of the metal to the cell wall or from an intracellular detoxification mechanism.
Canadian Journal of Microbiology 02/2011; 31(5):485-492. DOI:10.1139/m85-090 · 1.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Globulins, albumins, and basic proteins were extracted from seeds of red kidney bean (Phaseolus vulgaris), and their distribution was in a ratio of about 3:2:1, respectively. The globulin fraction constituted a major portion of the reserve proteins and was hydrolyzed rapidly during germination. More than 90% of the basic proteins, extractable with 0.05 N acetic acid, disappeared 12 days after germination. Although the decrease in total albumin was not as marked as with the other two fractions, a number of components of this fraction disappeared during the early stages of germination, but several new components were detected about 8 days after germination. The apparent synthesis of new globulin components during germination was also observed, but no synthesis of basic protein could be detected.
Canadian Journal of Botany 01/2011; 48(7):1347-1350. DOI:10.1139/b70-203 · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The globulins and albumins in seeds of nine species of conifers were distinguished by disc electrophoresis. On the basis of their higher stain intensity, the slower moving proteins, located in the upper half of the separation gels, appeared to be dominant. Only one globulin and one albumin band were homologous in all species examined, although several globulin and albumin bands appeared to be common to many species. The degree of similarity in protein patterns between species classified within the same genus and the degree of difference between genera were not as great as those reported for seeds of some angiosperms.
Canadian Journal of Botany 01/2011; 48(11):1911-1912. DOI:10.1139/b70-280 · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Isozyme patterns of L-leucine-amino-peptidase (LAP), esterase, and peroxidase in extracts from conifer seeds (eight species of Pinus, Abies concolor Hoopes, and Pseudotsuga taxifolia Britt.) indicated that certain specific isozyme patterns might be useful in systematic studies of conifers. The isozyme patterns of LAP from these seeds showed an apparent phylogenetic relationship. Eight different LAP bands were detected, and all eight species of Pinus displayed the same three LAP bands. The two slowest moving LAP bands were found to be common to all 10 species, whereas the mobility of the other LAP bands varied among species. Isozyme patterns of esterase from seeds of Pinus, Abies, and Pseudotsuga varied from species to species, and no species displayed the same number of bands. The number of esterase bands detected varied from 5 to 16, and no phylogenetic relationships were apparent. The banding patterns of peroxidase from these conifer seeds, which ranged from three to nine bands, also appeared to be species specific, and no common trends or homologies were apparent.
Canadian Journal of Botany 01/2011; 51(11):2201-2205. DOI:10.1139/b73-282 · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The possible transfer of genes from Bacillus thuringiensis subsp. kurstaki (Btk) to indigenous Bacillus spp. was investigated in soil samples from stands of cork oak in Orotelli (Sardinia, Italy) collected 5 years after spraying of the stands with a commercial insecticidal preparation (FORAY 48B) of Btk. Two colonies with a morphology different from that of Btk were isolated and identified as Bacillus mycoides by morphological and physiological characteristics and by 16S rDNA analysis. Amplification by the polymerase chain reaction (PCR) of the DNA of the two isolated B. mycoides colonies with primers used for the identification of the Btk cry genes showed the presence of a fragment of 238 bp of the cry1Ab9 gene that had a similarity of 100% with the sequence of the cry1Ab9 gene present in GenBank, indicating that the isolates of B. mycoides acquired part of the sequence of this gene from Btk. No cells of Btk or B. mycoides carrying the 238-bp fragment of the cry1Ab9 gene were isolated from samples of unsprayed control soil. However, the isolates of B. mycoides were not able to express the partial Cry1Ab protein. Hybridization with probes for IS231 and the cry1Ab9 gene suggested that the inverted repeated sequence, IS231, was probably involved in the transfer of the 238-bp fragment from Btk to B. mycoides. These results indicate that transfer of genes between introduced Btk and indigenous Bacillus spp. can occur in soil under field conditions.
[Show abstract][Hide abstract] ABSTRACT: Earthworms, which play a key role in biogeochemical processes in soil ecosystems, could be negatively affected by the cultivation of transgenic Bt crops. Studies to date have found few effects of Bt maize on earthworm species. If adverse effects occur, they are likely to be chronic or sub-lethal and expressed over large spatial and temporal scales. Our objective in the present study was to investigate potential effects on earthworm populations in soil cultivated with Bt maize in a large multiple-year field study. We surveyed the earthworm populations in 0.16-ha experimental field plots of two varieties of Cry1Ab Bt maize, one variety of Cry3Bb1 Bt maize, and three non-transgenic control varieties cultivated for four years. Four earthworm species were found in our sample: Aporrectodea caliginosa, Aporrectodea trapezoides, Aporrectodea tuberculata (collectively, the A. caliginosa species complex), and Lumbricus terrestris. We found no significant differences in the biomass of juveniles and adults for all four species between Bt and non-Bt maize varieties. From this and previous studies, we conclude that the effects of Cry1Ab and Cry3Bb1 Bt maize on the A. caliginosa species complex and L. terrestris are small. Nonetheless, general conclusions about the effects of Bt maize on earthworm populations are not warranted due to the small number of species tested. In future laboratory studies, earthworm species should be selected according to their association with a Bt crop and the impact of that species to valued soil ecosystem processes.
[Show abstract][Hide abstract] ABSTRACT: Bacillus thuringiensis (Bt) is a useful alternative or supplement to synthetic chemical pesticides in agriculture, forest management, and control of mosquitoes and some other biting insects. When modified Bt cry genes are inserted into a plant species (e.g., corn, cotton, potato, canola, rice), the plant expresses active larvicidal proteins in its tissues. The toxins continue to be synthesized during growth of the plants, making the plant toxic to various insect pests throughout their life or as biomass incorporated into soil. If production exceeds consumption, inactivation, and degradation, the toxins could accumulate to concentrations that may enhance the control of target pests or constitute a hazard to nontarget organisms, such as the soil microbiota, beneficial insects (e.g., pollinators, predators and parasites of insect pests), and other animal classes. The accumulation and persistence of the toxins could also result in the selection and enrichment of toxin-resistant target insects. Persistence is enhanced when the toxins are bound on surface-active particles in the environment (e.g., clays and humic substances) and, thereby, rendered more resistant to biodegradation while retaining toxic activity. Moreover, major problem we face today is of "Molecular pharming" that utilizes transgenic plants and animals for production of pharmaceuticals and chemicals for their use in human beings and industries respectively. Their release to the environment, especially to soil and potentially to waters of the pharmaceutical and industrial products of transgenic plant and animal "pharms" could pose a hazard to the environment. In contrast to the products of most transgenic plants currently available commercially (e.g., the insecticidal proteins from subspecies of Bt) that primarily target insects and other pests. These "pharms" are being genetically engineered to express products for use primarily in human beings. Consequently, these products constitute a class of compounds that is seldom found in natural habitats and that primarily target "higher level" eukaryotes. Hence, they are xenobiotics with respect to the environment, and their persistence in and effects on the environment have not been adequately studied and sober risk assessments on a case-by-case basis must be made before major releases of such transgenic organisms.
[Show abstract][Hide abstract] ABSTRACT: 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.
Bulletin of Environmental Contamination and Toxicology 06/2009; 83(1):48-58. DOI:10.1007/s00128-009-9760-2 · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Cry3Bb1 protein, insecticidal to the corn rootworm complex (Diabrotica spp.), of Bacillus thuringiensis (Bt) subsp. kumamotoensis was released in root exudates of transgenic Bt corn (event MON863) in sterile hydroponic culture (7.5 +/- 1.12 ng/ml after 28 days of growth) and in nonsterile soil throughout growth of the plants (2.2 +/- 0.62 ng/g after 63 days of growth). Kitchawan soil, which contains predominantly kaolinite (K) but not montmorillonite (M), was amended to 3 or 6% (vol./vol.) with K (3K and 6K soils) or M (3M and 6M soils) and with 1, 3, 5, or 10% (wt./wt.) of ground biomass of Bt corn expressing the Cry3Bb1 protein and incubated at 25 +/- 2 degrees C at the -33-kPa water tension for 60 days. Soils were analyzed for the presence of the protein every 7 to 10 days with a western blot assay (ImmunoStrip) and verified by ELISA. Persistence of the protein varied with the type and amount of clay mineral and the pH of the soils and increased as the concentration of K was increased but decreased as the concentration of M was increased. Persistence decreased when the pH of the K-amended soils was increased from ca. 5 to ca. 7 with CaCO(3): the protein was not detected after 14 and 21 days in the pH-adjusted 3K and 6K soils, respectively, whereas it was detected after 40 days in the 3K and 6K soils not adjusted to pH 7. The protein was detected for only 21 days in the 3M soil and for 14 days in the 6M soil, which were not adjusted in pH. These results indicate that the Cry3Bb1 protein does not persist or accumulate in soil and is degraded rapidly.
Transgenic Research 09/2008; 17(4):609-20. DOI:10.1007/s11248-007-9133-8 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human serum albumin (HSA), β-glucuronidase (GUS), and the Cry3Bb1 protein from Bacillus thuringiensis subsp. kumamotoensis are expressed by genetically-modified plants. Commercial samples of these proteins adsorbed and bound rapidly on the clay minerals, kaolinite (K) and montmorillonite (M). Adsorption increased as the concentration of protein increased and then reached a plateau. The greatest amount of adsorption and binding occurred with the Cry3Bb1 protein, of which there was no desorption: 6.7 ±0.21 μg adsorbed and bound μg− 1 of M; 2.1 ± 0.39 μg adsorbed and bound μg− 1 of K. With GUS, 2.2 ± 0.29 μg adsorbed and 1.7 ±0.21 μg bound μg− 1 of M; 1.5 ± 0.28 μg adsorbed and 1.0 ± 0.03 μg bound μg− 1 of K. HSA was adsorbed and bound the least: 1.2 ±0.04 μg adsorbed and 0.8 ± 0.05 μg bound μg− 1 of M; 0.4 ± 0.05 μg adsorbed and 0.4 ± 0.03 μg bound μg− 1 of K. However, X-ray diffraction analyses indicated that only HSA intercalated M, and none of the proteins intercalated K, a nonswelling clay. When bound, the proteins were not utilized for growth by mixed cultures of soil microorganisms, whereas the cultures readily utilized the free (i.e., not adsorbed or bound) proteins as sources of carbon and energy. The enzymatic activity of GUS was significantly enhanced when bound on the clay minerals. These results indicated that recombinant proteins expressed by transgenic plants could persist and function in soil after release in root exudates and from decaying plant residues as the result of the protection provided against biodegradation by binding on clay minerals.
[Show abstract][Hide abstract] ABSTRACT: Recent applications of biotechnology, especially genetic engineering, have revolutionized crop improvement and increased the availability of valuable new traits. A current example is the use of the insecticidal Cry proteins from the bacterium, Bacillus thuringiensis (Bt), to improve crops, known as Bt crops, by reducing injury from various crop pests. The adoption of genetically modified (GM) crops has increased dramatically in the last 11 years. However, the introduction of GM plants into agricultural ecosystems has raised a number of questions, including the ecological impact of these plants on soil ecosystems. Crop residues are the primary source of carbon in soil, and root exudates govern which organisms reside in the rhizosphere. Therefore, any change to the quality of crop residues and rhizosphere inputs could modify the dynamics of the composition and activity of organisms in soil. Insect-resistant Bt crops have the potential to change the microbial dynamics, biodiversity, and essential ecosystem functions in soil, because they usually produce insecticidal Cry proteins through all parts of the plant. It is crucial that risk assessment studies on the commercial use of Bt crops consider the impacts on organisms in soil. In general, few or no toxic effects of Cry proteins on woodlice, collembolans, mites, earthworms, nematodes, protozoa, and the activity of various enzymes in soil have been reported. Although some effects, ranging from no effect to minor and significant effects, of Bt plants on microbial communities in soil have been reported, using both culturing and molecular techniques, they were mostly the result of differences in geography, temperature, plant variety, and soil type and, in general, were transient and not related to the presence of the Cry proteins. The respiration (i.e., CO2 evolution) of soils cultivated with Bt maize or amended with biomass of Bt maize and other Bt crops was generally lower than from soils cultivated with or amended with biomass of the respective non-Bt isolines, which may have been a result of differences in chemical composition (e.g., the content of starch, soluble N, proteins, carbohydrates, lignin) between Bt plants and their near-isogenic counterparts. Laboratory and field studies have shown differences in the persistence of the Cry proteins in soil, which appear to be the result primarily of differences in microbial activity, which, in turn, is dependent on soil type (e.g., pH, clay mineral composition, other physicochemical characteristics), season (e.g., temperature, water tension), crop species (e.g., chemical composition, C:N ratio, plant part), crop management practices (e.g., till vs. no-till), and other environmental factors that vary with location and climate zones. This review discusses the available data on the effects of Cry proteins on below-ground organisms, the fate of these proteins in soil, the techniques and indicators that are available to study these aspects, and future directions.
[Show abstract][Hide abstract] ABSTRACT: 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 (Ostrinianubilalis Hubner), and one Btcorn 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Studies were performed to investigate the effect of several cranberry and grape juice extracts on the inhibition of reovirus infectivity following cell culture inoculation. Infectivity testing was performed utilizing cranberry juice extracts NutriCran-100 and NutriCran-90. At 5% extract concentrations, titers were reduced by ca. 50%. Cranberry cocktail juice caused an infectivity loss of ca. 10%. We ascribe these data to higher concentrations of proanthocyanidins (PACs) in the cranberry extracts. Further testing was performed utilizing purified high and low molecular weight cranberry PAC fractions (CB HMW and CB LMW, respectively), a cranberry flavonol glycoside (CB EToAc), cranberry anthocyanins (CB CA), and a grape PAC extract. Reovirus titers were reduced to undetectable levels at PAC concentrations < or =0.2%. CB CA had no effect on the inhibition of infectivity titers. Loss of infectivity titers was in the order: GP PAC>CB HMW>CB LMW>CB EToAc. Probe homogenization of CB HMW enhanced the extract to efficacy levels equal to that of grape PAC. Reovirus dsRNA segments were undetectable 96-h postcranberry cocktail juice pretreatment of MA-104 cell cultures. This study indicates an inhibition of reovirus infectivity titers by cranberry or grape juices or their purified PAC extracts. Viral inhibition probably occurs at the host cell surface.
[Show abstract][Hide abstract] ABSTRACT: Studies were undertaken to investigate the antiviral effects of comestible juices, especially cranberry juice, on non-related viral species. After exposure of bacteriophage T2 to a commercially available cranberry (Vaccinium macrocarpon) juice cocktail (CJ), virus infectivity titer was no longer detectible. After a 60-min exposure to orange (OJ) and grapefruit juices (GJ), phage infectivity was reduced to 25-35% of control, respectively. Similar data were observed for the bacteriophage T4. CJ inactivation of phage T4 was rapid, dose-dependent, and occurred at either 4 or 23 degrees C. Neither pH nor differences in sugar/carbohydrate levels among the juices may be ascribed to the recognized antiviral effects. Further studies were performed to identify the occurrence of antiviral activity by CJ to a mammalian enteric virus. The treatment of the simian rotavirus SA-11 with a 20% CJ suspension was sufficient to inhibit hemagglutination. Under scanning and transmission electron microscopy, CJ was observed to inhibit the adsorption of phage T4 to its bacterial host cells and prevented the replication of rotavirus in its monkey kidney (MA-104) host cells, respectively. The data suggest, for the first time, a non-specific antiviral effect towards unrelated viral species (viz., bacteriophages T2 and T4 and the simian rotavirus SA-11) by a commercially available cranberry fruit juice drink.