Impact on environment, ecosystem, diversity and health from culturing and using GMOs as feed and food.

Modern agriculture provides the potential for sustainable feeding of the world's increasing population. Up to the present moment, genetically modified (GM) products have enabled increased yields and reduced pesticide usage. Nevertheless, GM products are controversial amongst policy makers, scientists and the consumers, regarding their possible environmental, ecological, and health risks. Scientific-and-political debates can even influence legislation and prospective risk assessment procedure. Currently, the scientifically-assessed direct hazardous impacts of GM food and feed on fauna and flora are conflicting; indeed, a review of literature available data provides some evidence of GM environmental and health risks. Although the consequences of gene flow and risks to biodiversity are debatable. Risks to the environment and ecosystems can exist, such as the evolution of weed herbicide resistance during GM cultivation. A matter of high importance is to provide precise knowledge and adequate current information to regulatory agencies, governments, policy makers, researchers, and commercial GMO-releasing companies to enable them to thoroughly investigate the possible risks.

Development of immunochromatography strip-test using nanocolloidal gold-antibody probe for the rapid detection of aflatoxin B1 in grain and feed samples.

An immunochromatography (ICG) strip test using a nanocolloidal gold-antibody probe was developed and optimized for the rapid detection of aflatoxin B1 (AFB1). A monoclonal antibody specific to AFB1 was produced from the cloned hybridoma cell (AF78), coupled with nanocolloidal gold, and distributed on the conjugate pad of the ICG strip test. The visual detection limit of the ICG strip test was 0.5 ng/ml, and this method showed a cross-reaction to aflatoxin B2, G1, and G2. In total, 172 grain and feed samples were collected and analyzed by both the ICG strip test and HPLC. The results of the ICG strip test showed a good agreement with those obtained by HPLC. These results indicated that the ICG strip test has a potential use as a rapid and cost-effective screening tool for the determination of AFB1 in real samples and could be applied to the preliminary screening of mycotoxin in food and agricultural products, generating results within 15 min without complicated steps.

Comparative proteomic analysis associated with term placental insufficiency in cloned pig.

Somatic cell-derived nuclear transfer (scNT) is a method of animal cloning in which the oocyte reprograms a somatic cell nucleus to divide and execute developmental programs. Despite many successes in this field, cloning by scNT remains very inefficient. Unlike other cloned animals, pigs derived by scNT have placentas with severe villous hypoplasia. To obtain a better understanding of the protein networks involved in this phenomenon, we assessed global protein expression profiles in term placentas from scNT-derived and control animals. Proteomic analysis of term placentas from scNT-derived animals identified 43 proteins that were differentially expressed compared to control animals. Among them, 14-3-3 proteins and Annexin V, which are closely involved in the apoptotic signaling pathway, were significantly down- and up-regulated, respectively. Western blot analysis and immunohistochemistry indicated that down-regulation of 14-3-3 proteins in scNT-derived placentas induced apoptosis of cytotrophoblast cells via mitochondria-mediated apoptosis. Taken together, our results suggest that placental insufficiency in scNT-derived placentas may be due to apoptosis, induced in part by the down-regulation of 14-3-3 proteins and up-regulation of Annexin V. They also indicate that proteomic maps represent an important tool for future studies of placental insufficiency and pathology.

Immunochromatography using colloidal gold-antibody probe for the detection of atrazine in water samples.

An immunochromatography (ICG) strip test for rapid detection of atrazine in water samples was developed. A monoclonal antibody (MAb) specific to atrazine was produced from the cloned hybridoma cell (AT-1-M3) and used to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and ICG strip. MAb conjugated to colloidal gold, and that was applied to the conjugate pad of the ICG strip. The visual detection limit for the ICG strip was 3 ng/mL. This test required only 10 min to get results and one step of sample to perform the assay. The results of water samples spiked with 5, 10, 20, and 50 ng/mL of atrazine by ICG strip were in good agreement with those obtained by DC-ELISA. The ICG strip was sufficiently sensitive and accurate to be useful for rapid screening of atrazine in various water samples.

Dynamic control of oligosaccharide modification in the mammary gland: linking recombinant human erythropoietin functional analysis of transgenic mouse milk-derived hEPO.

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin alpha and beta are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and alpha1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin alpha derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin alpha. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use.

Significant IgG-immunoreactivity of the spermatogonia of the germ cell-depleted testis after busulfan treatment.

Busulfan kills spermatogonia with the exception of a few that are attached to the basal membrane of the seminiferous epithelium. In mice, these remaining spermatogonia reacted strongly to a goat anti-mouse IgG antibody. Spermatogonia in untreated testes rarely showed the same reactivity. Testicular IgG levels are normally minimal but increase markedly, 4 weeks after busulfan treatment before peaking at week 6. Laser scanning cytometry analysis of control and busulfan-treated testicular cells showed busulfan treatment increased the frequency of cells that were positive for not only IgG (from 0.67+/-0.29 to 16.5+/-3.8%) but also for alpha6-integrin, beta1-integrin, GFR(-1 and/or Ret. Thus, an enrichment in putative male stem cells correlates with appearance of IgG expression. Confocal microscopy revealed busulfan-treated cells contained both IgG and GFRalpha-1, and that the initial surface IgG became intracellular in the weeks following busulfan treatment. The basement membranes of the seminiferous tubules were compromised by busulfan treatment as the mRNA expression profiles of various adhesion molecules in the basement membranes were altered and electron microscopy revealed severe damage. Serum IgG levels increased in a manner corresponding with the increase in testicular IgG levels. Thus, it appears that in the busulfan-treated testis, small breaches of the blood-testis barrier leak IgG that is then taken up by a significant number of spermatogonia. When the busulfan-resistant germ cells were transferred into recipient germ cell-depleted testes, they settled and repopulated the recipient testes. Thus, the IgG-bearing cells observed after busulfan treatment may be putative spermatogonial stem cells.

Detection of aflatoxin-producing molds in Korean fermented foods and grains by multiplex PCR.

An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of the organophosphorus pesticide parathion-methyl.

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the detection of parathion-methyl (PM) was developed and optimized. Fluorescein-labeled PM derivatives (tracers) with different structures were synthesized and purified by thin-layer chromatography. The influence of immunogen and tracer structures on the assay characteristics was investigated. PM concentration determinable by the FPIA ranged from 25 to 10000 ppb. The detection limit was 15 ppb. Methanol extracts of vegetable, fruit, and soil samples were diluted 1/10 for the analysis. Recovery in spiked samples averaged between 85 and 110%. The method developed is characterized by high specificity and reproducibility (CV ranged from 1.5 to 9.1% for interassay and from 1.8 to 14.1% for intra-assay). The FPIA method can be applied to the screening of food and environmental samples for PM residues without complicated cleanup.

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.