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Cytogenetic damage in Allium cepa root meristems induced by Dursban 4 and Antracol WP 7 pesticides
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
added to the dsDNA before hybridization. The color of the solution in test reaction was compared with the set of standards and the presence of dsDNA was determined. The setup is able to detect picogram level of dsDNA and colorimetric method is found tenfold more sensitive then visualized under UV trans-illuminator. The advantages of this method are (1) Isothermal amplification does not require thermal cycler. (2) For the visualization of the amplified fragment restriction digestion and gel electrophoresis is not required. (3) Less instrumentation hence carried out on site. (4) Cost effective and operated with formal training. (5) Rapid detection of pathogen within 60 min. (6) The pathogenic load can be determined visually. The array is being developed to detect a range of pathogens. In future this technology will also be utilized to detect genetically modified seeds and bioterrorism. doi:10.1016/j.nbt.2009.06.915
5.0.12 Cytogenetic damage in Allium cepa root meristems induced by Dursban 4 and Antracol WP 7 pesticides A. Ergene 1,∗ , S. Tan 1 , F. Arslanoglu 1 , E. Yalcin 2 1
2
Yilmaz 1 , S.
Topcu 1 , A.
Kaya 1 , I.
Kirikkale University, Kirikkale, Turkey Giresun University, Turkey
Nowadays cytotoxic effects of 900 different chemical and 60,000 composition structured pesticides were determined particularly. The examination of the effects of pesticides on plants is very important and useful due to the major nutrient property of plants for humans. The Allium test, used since the late 1930s and standardized in 1985 is a very sensitive and reliable plant assay method in environmental monitoring. It is based on the assessment of the toxic and genotoxic potential of chemicals such as pesticides in species of the genus Allium by measuring the mean root growth and by recording mitotic activity (mitotic index), mitotic abnormalities and chromosomal aberrations in meristematic root tip cells. In our experiment Dursban 4 (insecticide) and Antracol WP 7 (fungicide) were added to tap water in amounts appropriate to achieve concentrations of 0.3%, 0.6% and 0.9% to study their cytotoxic effect on the root tip cells of shallot Allium cepa. All tested solutions in concentrations of 0.9% caused significant inhibition of shallot root growth. The most common aberrations observed in all treatments were alterations in anaphase cells (bridges, chromosome laggards and multipolarity), changes in telophase cells. Other aberrations, such as chromosome breaks and micronuclei, were also observed. The results are a cytological demonstration that Dursban 4 and Antracol WP 7 induce in A. cepa root meristems chromosomal abnormalities and formation of micronucleus. doi:10.1016/j.nbt.2009.06.916
5.0.13 Direct delivery of bacterial type III secretion system effector proteins to the eukaryotic cell as a tool to study bacterial pathogenesis A. Jalili 1,2,∗ , J. Atherton 1 , R. Delahay 1 1
2
University of Nottingham, Nottingham, United Kingdom Mashad University of Medical Sciences, Mashad, Islamic Republic of Iran
Pathogenic bacteria that employ type III secretion systems inject a repertoire of up to 40 effector proteins directly into the host eukaryotic cells to which they attach. Evidence suggests that the translocated bacterial effectors can act in a synergetic or antagonistic manner to subvert host cellular processes. Such activity promotes colonisation of the parent bacterium and ultimately defines the outcome of the bacterial infection. To fully understand effector protein function it is necessary to study the activity of individual proteins both in isolation and in the context of the entire effector repertoire. Approaches have traditionally employed transfection techniques to assess individual effectors in the absence of the effector repertoire and more commonly, examination of the phenotypic consequences of deleting up to two individual effectors from the repertoire by the generation of effector mutants strains. However, both approaches have limitations which reduce their experimental application; transfection is cumbersome, optimisation is needed with each cell line, and long incubation times are required, whereas, deletion of multiple effector genes in different genomic locations is impractical. As a solution to the characterisation of effector proteins both alone and in the context of the entire effector repertoire, this study aims to exploit and develop an emerging technology, based on carrier peptide Pep-1, for delivering individual and defined subsets of recombinant bacterial effectors to the host eukaryotic cell in in vitro models of infection. To assess the efficacy of the approach, two effector proteins, Tir and TccP from the diarrhoeagenic pathogens, enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC, respectively) were selected for study. EPEC/EHEC Tir is a membrane-localised effector crucial for the subversion of host cellular signalling and remodelling of the actin cytoskeleton. We show that Pep-1 can efficiently deliver recombinant Tir to the host cell in far greater quantity than Tir alone. However, although Pep-1-delivered recombinant Tir is membrane localised, it is only partially activated by host kinase modification and therefore only minimally functional. This observation suggests a probable limitation for Pep-1 mediated delivery of membrane associated effectors. Translocated EHEC TccP however, is predominantly cytoplasmic and functions to physically link EHEC Tir with host signalling complexes. Preliminary data suggest that Pep-1 delivered TccP retains native functionality. Experiments are in progress to further define TccP activity and to assess tandem and sequential delivery of recombinant effector subsets. doi:10.1016/j.nbt.2009.06.917
S372
www.elsevier.com/locate/nbt
ABSTRACTS
added to the dsDNA before hybridization. The color of the solution in test reaction was compared with the set of standards and the presence of dsDNA was determined. The setup is able to detect picogram level of dsDNA and colorimetric method is found tenfold more sensitive then visualized under UV trans-illuminator. The advantages of this method are (1) Isothermal amplification does not require thermal cycler. (2) For the visualization of the amplified fragment restriction digestion and gel electrophoresis is not required. (3) Less instrumentation hence carried out on site. (4) Cost effective and operated with formal training. (5) Rapid detection of pathogen within 60 min. (6) The pathogenic load can be determined visually. The array is being developed to detect a range of pathogens. In future this technology will also be utilized to detect genetically modified seeds and bioterrorism. doi:10.1016/j.nbt.2009.06.915
5.0.12 Cytogenetic damage in Allium cepa root meristems induced by Dursban 4 and Antracol WP 7 pesticides A. Ergene 1,∗ , S. Tan 1 , F. Arslanoglu 1 , E. Yalcin 2 1
2
Yilmaz 1 , S.
Topcu 1 , A.
Kaya 1 , I.
Kirikkale University, Kirikkale, Turkey Giresun University, Turkey
Nowadays cytotoxic effects of 900 different chemical and 60,000 composition structured pesticides were determined particularly. The examination of the effects of pesticides on plants is very important and useful due to the major nutrient property of plants for humans. The Allium test, used since the late 1930s and standardized in 1985 is a very sensitive and reliable plant assay method in environmental monitoring. It is based on the assessment of the toxic and genotoxic potential of chemicals such as pesticides in species of the genus Allium by measuring the mean root growth and by recording mitotic activity (mitotic index), mitotic abnormalities and chromosomal aberrations in meristematic root tip cells. In our experiment Dursban 4 (insecticide) and Antracol WP 7 (fungicide) were added to tap water in amounts appropriate to achieve concentrations of 0.3%, 0.6% and 0.9% to study their cytotoxic effect on the root tip cells of shallot Allium cepa. All tested solutions in concentrations of 0.9% caused significant inhibition of shallot root growth. The most common aberrations observed in all treatments were alterations in anaphase cells (bridges, chromosome laggards and multipolarity), changes in telophase cells. Other aberrations, such as chromosome breaks and micronuclei, were also observed. The results are a cytological demonstration that Dursban 4 and Antracol WP 7 induce in A. cepa root meristems chromosomal abnormalities and formation of micronucleus. doi:10.1016/j.nbt.2009.06.916
5.0.13 Direct delivery of bacterial type III secretion system effector proteins to the eukaryotic cell as a tool to study bacterial pathogenesis A. Jalili 1,2,∗ , J. Atherton 1 , R. Delahay 1 1
2
University of Nottingham, Nottingham, United Kingdom Mashad University of Medical Sciences, Mashad, Islamic Republic of Iran
Pathogenic bacteria that employ type III secretion systems inject a repertoire of up to 40 effector proteins directly into the host eukaryotic cells to which they attach. Evidence suggests that the translocated bacterial effectors can act in a synergetic or antagonistic manner to subvert host cellular processes. Such activity promotes colonisation of the parent bacterium and ultimately defines the outcome of the bacterial infection. To fully understand effector protein function it is necessary to study the activity of individual proteins both in isolation and in the context of the entire effector repertoire. Approaches have traditionally employed transfection techniques to assess individual effectors in the absence of the effector repertoire and more commonly, examination of the phenotypic consequences of deleting up to two individual effectors from the repertoire by the generation of effector mutants strains. However, both approaches have limitations which reduce their experimental application; transfection is cumbersome, optimisation is needed with each cell line, and long incubation times are required, whereas, deletion of multiple effector genes in different genomic locations is impractical. As a solution to the characterisation of effector proteins both alone and in the context of the entire effector repertoire, this study aims to exploit and develop an emerging technology, based on carrier peptide Pep-1, for delivering individual and defined subsets of recombinant bacterial effectors to the host eukaryotic cell in in vitro models of infection. To assess the efficacy of the approach, two effector proteins, Tir and TccP from the diarrhoeagenic pathogens, enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC, respectively) were selected for study. EPEC/EHEC Tir is a membrane-localised effector crucial for the subversion of host cellular signalling and remodelling of the actin cytoskeleton. We show that Pep-1 can efficiently deliver recombinant Tir to the host cell in far greater quantity than Tir alone. However, although Pep-1-delivered recombinant Tir is membrane localised, it is only partially activated by host kinase modification and therefore only minimally functional. This observation suggests a probable limitation for Pep-1 mediated delivery of membrane associated effectors. Translocated EHEC TccP however, is predominantly cytoplasmic and functions to physically link EHEC Tir with host signalling complexes. Preliminary data suggest that Pep-1 delivered TccP retains native functionality. Experiments are in progress to further define TccP activity and to assess tandem and sequential delivery of recombinant effector subsets. doi:10.1016/j.nbt.2009.06.917
S372
www.elsevier.com/locate/nbt