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A. J. A. Essers - One of the best experts on this subject based on the ideXlab platform.
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Cyanogen removal from cassava roots during sun-drying.
Food Chemistry, 1996Co-Authors: A. J. A. Essers, R. M. Van Der Grift, A.g.j. VoragenAbstract:Abstract Linamarin levels in sun-drying cassava root pieces showed an exponential decrease, parallel with the moisture loss, and stabilized when moisture levels reached about 15%. Linamarin degradation in thin root segments was significantly slower and less complete than in thick ones. Disinfected longitudinal root halves, oven-dried at 40 °C for 24 h, had significantly higher residual Linamarin levels than the matched ones subjected to humid incubation at 25 °C. Interrupting for one or two days the sun-drying of peeled roots resulted in significantly lower residual levels of Linamarin and of cyanohydrins plus HCN. Linamarin degradation was greater when the interruption was earlier or longer. The rate of dehydration influences Linamarin degradation negatively — mechanisms are discussed. Cyanohydrin removal was completed by prolonged drying. Sun-drying is not very effective in Linamarin removal, and speeding up the drying process, e.g. by reducing the size of the pieces, aggravates this. There is a potential for increasing the effectiveness of cyanogen removal by reducing the initial drying rate, followed by thorough final drying.
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Mechanisms of increased Linamarin degradation during solid-substrate fermentation of cassava
World Journal of Microbiology and Biotechnology, 1995Co-Authors: A. J. A. Essers, M. H. J. Bennik, M. J. R. NoutAbstract:Several fungi and bacteria, isolated from Ugandan domestic fermented cassava, released HCN from Linamarin in defined growth media. In 72 h, a Bacillus sp. decreased the Linamarin to 1% of initial concentrations, Mucor racemosus to 7%, Rhizopus oryzae and R. stolonifer to 30%, but Neurospora sitophila and Geotrichum candidum hardly degraded the Linamarin. Adding pectolytic and cellulolytic enzymes, but not linamarase, to root pieces under aseptic conditions, led to root softening and significantly lower Linamarin contents. Neurospora sitophila showed no linamarase activity, in contrast to M. racemosus and Bacillus sp., both of which were less effective in root softening and decreasing the root Linamarin content. The most important contribution of microorganisms to Linamarin decrease in the solid-substrate fermentation of cassava is their cell-wall-degrading activity, which enhances the contact between endogenous linamarase and Linamarin.
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Removal of cyanogens from cassava roots : studies on domestic sun-drying and solid-substrate fermentation in rural Africa
1995Co-Authors: A. J. A. EssersAbstract:Cassava is an important staple crop, but its potential toxicity has led to some health problems in Africa. The potential toxicity comes from endogenous cyanogenic glucosides, mainly Linamarin, which may degrade by linamarase to cyanohydrins and subsequently to hydrocyanic acid (HCN). A study into a small outbreak of paralysis and poisoning in a cassava-dominated rural area of Mozambique revealed that the walking disability was konzo, a recently identified disease, and suggested that insufficient processing of the bitter cassava roots was a factor in its causation. The usual processing stages to turn roots into flour, sun-drying and heapfermentation, were studied in Uganda and The Netherlands. For evaluation of initial and resulting levels of the cyanogenic compounds, an analytical assay was tested and improved. Mechanisms of cyanogen removal from cassava by sun- drying and heap-fermentation were elucidated, to allow for its optimization. Sun-drying removed cyanogens insufficiently from roots with high initial levels. Dynamics of cyanogen levels are described. Continuing drying below moisture levels of 15% did not diminish Linamarin levels further, but it was useful for further removal of the cyanohydrins formed. The dehydration rate influenced Linamarin breakdown negatively. Reducing the size of the pieces to speed up drying, as done during the konzo outbreak, therefore resulted in higher residual Linamarin levels. Linamarin breakdown can be enhanced by reducing the initial dehydration rate. Microbial contamination may need to be controlled to prevent the formation of microbial toxins. In Uganda and Mozambique certain communities promote fungal growth by heaping and covering the peeled roots. Their aim is to improve the palatability and reduce the toxicity. Cyanogen removal by this solid-substrate fermentation appeared more effective than by sun-drying alone, but several samples of this flour from rural households still had undesirably high levels of cyanogens. Screening of 30 flour samples for mycotoxins was negative, but the formation of mycotoxins cannot be excluded. The humid incubation of cassava extends the time of physiological cell-wall degradation, which allows for linamarase-Linamarin interaction. The microflora had an additional positive effect on cyanogen removal by enhancing the cell-wall degradation. The linamarase activity shown by several microorganisms was of lesser importance. The food grade fungi Neurospora sitophila and Rhizopus oryzae were the most effective in cyanogen removal. Optimization of processing conditions, including the use of starter cultures, is recommended for ensuring safe products.
Marta Izquierdo - One of the best experts on this subject based on the ideXlab platform.
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cyanide bystander effect of the linamarase Linamarin killer suicide gene therapy system
Journal of Gene Medicine, 2002Co-Authors: Maria Luisa Cortés, Monica A. Hughes, Vega Garciaescudero, Marta IzquierdoAbstract:Background The killer-suicide system linamarase/Linamarin (lis/lin) uses the plant gene linamarase (β-glucosidase) to convert the cyanogenic glucoside substrate, Linamarin, into glucose and cyanide. We have studied the bystander effect associated with this new system mediated by the production of the cyanide ion that diffuses freely across membranes. Methods Immunofluorescent staining of cells treated with an anti-linamarase antibody allowed us to localize the enzyme within the cells. Flow cytometry was used to determine the sensitivity of different mixtures of cells, C6lis and C6gfp (green), to Linamarin as a percentage of cell survival. Results We demonstrate here that rat glioblastoma C6 cells carrying the linamarase gene (lis), mixed with naive C6 cells and exposed to Linamarin, induce generalized cell death. Cells expressing lis efficiently export linamarase, whereas Linamarin enters cells poorly by endocytosis; as a result most of the cyanide is produced outside the cells. The study was facilitated by the presence of the green fluorescent protein (gfp) gene in the bystander population. As few as 10% C6lis-positive cells are sufficient to eliminate the entire cell culture in 96 h. Conclusions This bystander mechanism does not preferentially kill toxic metabolite producer cells compared with bystander cells, thus allowing production of sufficient cyanide to cause tumor regression. In this report we confirm the potential of the lis/lin gene therapy system as a powerful tool to eliminate tumors in vivo. Copyright © 2002 John Wiley & Sons, Ltd.
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Cyanide bystander effect of the linamarase/Linamarin killer‐suicide gene therapy system
The journal of gene medicine, 2002Co-Authors: Maria Luisa Cortés, Monica A. Hughes, Vega García-escudero, Marta IzquierdoAbstract:The killer-suicide system linamarase/Linamarin (lis/lin) uses the plant gene linamarase (beta-glucosidase) to convert the cyanogenic glucoside substrate, Linamarin, into glucose and cyanide. We have studied the bystander effect associated with this new system mediated by the production of the cyanide ion that diffuses freely across membranes. Immunofluorescent staining of cells treated with an anti-linamarase antibody allowed us to localize the enzyme within the cells. Flow cytometry was used to determine the sensitivity of different mixtures of cells, C6lis and C6gfp (green), to Linamarin as a percentage of cell survival. We demonstrate here that rat glioblastoma C6 cells carrying the linamarase gene (lis), mixed with naive C6 cells and exposed to Linamarin, induce generalized cell death. Cells expressing lis efficiently export linamarase, whereas Linamarin enters cells poorly by endocytosis; as a result most of the cyanide is produced outside the cells. The study was facilitated by the presence of the green fluorescent protein (gfp) gene in the bystander population. As few as 10% C6lis-positive cells are sufficient to eliminate the entire cell culture in 96 h. This bystander mechanism does not preferentially kill toxic metabolite producer cells compared with bystander cells, thus allowing production of sufficient cyanide to cause tumor regression. In this report we confirm the potential of the lis/lin gene therapy system as a powerful tool to eliminate tumors in vivo. Copyright 2002 John Wiley & Sons, Ltd.
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Successful use of a plant gene in the treatment of cancer in vivo
Gene therapy, 1998Co-Authors: Maria Luisa Cortés, Monica A. Hughes, P. De Felipe, Verónica Martín, Marta IzquierdoAbstract:A new strategy for cancer gene therapy has been developed using a plant gene which encodes the enzyme, linamarase, that hydrolyzes the cyanogenic glucoside substrate, Linamarin, into glucose, acetone and cyanide. Retroviral vectors that carry linamarase as a potential killer-suicide gene cause a marked sensitization to the innocuous substrate, Linamarin, followed by cell death. We show that the system can eradicate very large intracerebral gliomas in vivo helped by a cyanide bystander effect. Animals showing a total regression of the tumor by magnetic resonance imaging (MRI), do not show other appreciable toxic effects.
Anna Stochmal - One of the best experts on this subject based on the ideXlab platform.
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Ultraperformance liquid chromatography tandem mass spectrometry determination of cyanogenic glucosides in Trifolium species.
Journal of agricultural and food chemistry, 2014Co-Authors: Tamar Muzashvili, Barbara Moniuszko-szajwaj, Lukasz Pecio, Wieslaw Oleszek, Anna StochmalAbstract:Cyanogenic glucosides were analyzed by ultra-high-performance liquid chromatography combined with mass spectrometry in 88 Trifolium species grown at the same site. On the basis of the occurrence of cyanogenic glucosides and the Linamarin/lotaustralin ratio species could be grouped into five clusters. Cluster C1 included 37 species, which did not contain cyanogens. Cluster C2 (22 species) included plants containing only lotaustralin. In clusters C3 (14 species), C4 (13 species), and C5 (2 species) both Linamarin and lotaustralin were present but at different ratios. In C3 and C4 the Linamarin/lotaustralin ratio was below 1, whereas in cluster C5 the ratio was much higher. Generally, the total content of cyanogens was below 500 μg/g dry matter. Only in Trifolium repens var. biasoletti and Trifolium montanum extremely high cyanogen concentrations were observed. There was no general rule of occurrence of cyanogens. Samples of the same species from different countries accumulated cyanogens or could be free of these compounds.
Ke-feng Dou - One of the best experts on this subject based on the ideXlab platform.
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the adenovirus mediated linamarase Linamarin suicide system a potential strategy for the treatment of hepatocellular carcinoma
Cancer Letters, 2010Co-Authors: Liang Zhu, Wenjie Song, Desheng Wang, Ke-feng DouAbstract:Suicide gene therapy, also known as gene-directed enzyme prodrug therapy, has the potential to provide a cure for hepatocellular carcinoma (HCC), which is a leading cause of cancer death worldwide. Linamarase from cassava is an enzyme that can hydrolyze the innocuous substrate Linamarin into cyanide, which can block the mitochondrial respiratory chain of cells. To investigate whether linamarase and Linamarin can be used for HCC treatment, a recombinant adenovirus carrying linamarase cDNA was constructed using the ViraPower™ Adenoviral Expression System. In vitro cytotoxicity assays indicated that the system has a dramatic inhibitory effect and a strong bystander effect on HCC cell lines. Application of the recombinant adenovirus with Linamarin in nude mice bearing HCC xenografts also resulted in significant inhibition of the tumors, while the animals did not show any appreciable toxic effects. Our findings provide the first in vitro and in vivo evidence that this system may provide a potential strategy for HCC treatment.
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The adenovirus-mediated linamarase/Linamarin suicide system: a potential strategy for the treatment of hepatocellular carcinoma.
Cancer letters, 2009Co-Authors: Liang Zhu, Wenjie Song, Desheng Wang, Ke-feng DouAbstract:Suicide gene therapy, also known as gene-directed enzyme prodrug therapy, has the potential to provide a cure for hepatocellular carcinoma (HCC), which is a leading cause of cancer death worldwide. Linamarase from cassava is an enzyme that can hydrolyze the innocuous substrate Linamarin into cyanide, which can block the mitochondrial respiratory chain of cells. To investigate whether linamarase and Linamarin can be used for HCC treatment, a recombinant adenovirus carrying linamarase cDNA was constructed using the ViraPower Adenoviral Expression System. In vitro cytotoxicity assays indicated that the system has a dramatic inhibitory effect and a strong bystander effect on HCC cell lines. Application of the recombinant adenovirus with Linamarin in nude mice bearing HCC xenografts also resulted in significant inhibition of the tumors, while the animals did not show any appreciable toxic effects. Our findings provide the first in vitro and in vivo evidence that this system may provide a potential strategy for HCC treatment.
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the killing effect and bystander effect of linamarase Linamarin suicide gene system on human hepatocellular carcinoma cell line hepg2 in vitro
Chinese journal of cellular and molecular immunology, 2008Co-Authors: Ming-hao Chen, Fu-qin Zhang, Ke-feng DouAbstract:AIM: To investigate the killing effect of linamarase/Linamarin (lis/lin) system on hepatocellular carcinoma cell line HepG2 in vitro. METHODS: A cDNA library was built from RNA of cassava by RT-PCR, then the linamarase gene was amplified from it by PCR and cloned into the eukaryotic expression vector plasmid pEGFP-N1, which made up the recombinant plasmid pEGFP-N1-lis. The human HCC cells HepG2 were transfected with the recombinant plasmid mediated by electroporation and screened by G418 to yield the positive clone which was termed HepG2/lis. The expression of lis was confirmed by fluorescent staining, RT-PCR and Western blot. The killing effect and bystander effect of Linamarin with different concentrations on HepG2 was detected by MTT. RESULTS: RT-PCR confirmed the expression of lis gene in HepG2 and Western blot analysis confirmed existence of lis-EGFP fusion protein in HepG2. Linamarin in low concentration had shown notable cytotoxic effect on HepG2/lis. When HepG2/lis cells were mixed with parental HepG2 cells at a ratio of 10:90 and cultivated in 500 mg/L lin medium, significant bystander effect was observed in vitro. CONCLUSION: The linamarase/Linamarin suicide gene system has strong killing effect and bystander effect on HCCs with the concentration of 500 mg/L lin.
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The killing effect and bystander effect of linamarase/Linamarin suicide gene system on human hepatocellular carcinoma cell line HepG2 in vitro
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology, 2008Co-Authors: Ming-hao Chen, Fu-qin Zhang, Ke-feng DouAbstract:AIM: To investigate the killing effect of linamarase/Linamarin (lis/lin) system on hepatocellular carcinoma cell line HepG2 in vitro. METHODS: A cDNA library was built from RNA of cassava by RT-PCR, then the linamarase gene was amplified from it by PCR and cloned into the eukaryotic expression vector plasmid pEGFP-N1, which made up the recombinant plasmid pEGFP-N1-lis. The human HCC cells HepG2 were transfected with the recombinant plasmid mediated by electroporation and screened by G418 to yield the positive clone which was termed HepG2/lis. The expression of lis was confirmed by fluorescent staining, RT-PCR and Western blot. The killing effect and bystander effect of Linamarin with different concentrations on HepG2 was detected by MTT. RESULTS: RT-PCR confirmed the expression of lis gene in HepG2 and Western blot analysis confirmed existence of lis-EGFP fusion protein in HepG2. Linamarin in low concentration had shown notable cytotoxic effect on HepG2/lis. When HepG2/lis cells were mixed with parental HepG2 cells at a ratio of 10:90 and cultivated in 500 mg/L lin medium, significant bystander effect was observed in vitro. CONCLUSION: The linamarase/Linamarin suicide gene system has strong killing effect and bystander effect on HCCs with the concentration of 500 mg/L lin.
Maria Luisa Cortés - One of the best experts on this subject based on the ideXlab platform.
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cyanide bystander effect of the linamarase Linamarin killer suicide gene therapy system
Journal of Gene Medicine, 2002Co-Authors: Maria Luisa Cortés, Monica A. Hughes, Vega Garciaescudero, Marta IzquierdoAbstract:Background The killer-suicide system linamarase/Linamarin (lis/lin) uses the plant gene linamarase (β-glucosidase) to convert the cyanogenic glucoside substrate, Linamarin, into glucose and cyanide. We have studied the bystander effect associated with this new system mediated by the production of the cyanide ion that diffuses freely across membranes. Methods Immunofluorescent staining of cells treated with an anti-linamarase antibody allowed us to localize the enzyme within the cells. Flow cytometry was used to determine the sensitivity of different mixtures of cells, C6lis and C6gfp (green), to Linamarin as a percentage of cell survival. Results We demonstrate here that rat glioblastoma C6 cells carrying the linamarase gene (lis), mixed with naive C6 cells and exposed to Linamarin, induce generalized cell death. Cells expressing lis efficiently export linamarase, whereas Linamarin enters cells poorly by endocytosis; as a result most of the cyanide is produced outside the cells. The study was facilitated by the presence of the green fluorescent protein (gfp) gene in the bystander population. As few as 10% C6lis-positive cells are sufficient to eliminate the entire cell culture in 96 h. Conclusions This bystander mechanism does not preferentially kill toxic metabolite producer cells compared with bystander cells, thus allowing production of sufficient cyanide to cause tumor regression. In this report we confirm the potential of the lis/lin gene therapy system as a powerful tool to eliminate tumors in vivo. Copyright © 2002 John Wiley & Sons, Ltd.
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Cyanide bystander effect of the linamarase/Linamarin killer‐suicide gene therapy system
The journal of gene medicine, 2002Co-Authors: Maria Luisa Cortés, Monica A. Hughes, Vega García-escudero, Marta IzquierdoAbstract:The killer-suicide system linamarase/Linamarin (lis/lin) uses the plant gene linamarase (beta-glucosidase) to convert the cyanogenic glucoside substrate, Linamarin, into glucose and cyanide. We have studied the bystander effect associated with this new system mediated by the production of the cyanide ion that diffuses freely across membranes. Immunofluorescent staining of cells treated with an anti-linamarase antibody allowed us to localize the enzyme within the cells. Flow cytometry was used to determine the sensitivity of different mixtures of cells, C6lis and C6gfp (green), to Linamarin as a percentage of cell survival. We demonstrate here that rat glioblastoma C6 cells carrying the linamarase gene (lis), mixed with naive C6 cells and exposed to Linamarin, induce generalized cell death. Cells expressing lis efficiently export linamarase, whereas Linamarin enters cells poorly by endocytosis; as a result most of the cyanide is produced outside the cells. The study was facilitated by the presence of the green fluorescent protein (gfp) gene in the bystander population. As few as 10% C6lis-positive cells are sufficient to eliminate the entire cell culture in 96 h. This bystander mechanism does not preferentially kill toxic metabolite producer cells compared with bystander cells, thus allowing production of sufficient cyanide to cause tumor regression. In this report we confirm the potential of the lis/lin gene therapy system as a powerful tool to eliminate tumors in vivo. Copyright 2002 John Wiley & Sons, Ltd.
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Successful use of a plant gene in the treatment of cancer in vivo
Gene therapy, 1998Co-Authors: Maria Luisa Cortés, Monica A. Hughes, P. De Felipe, Verónica Martín, Marta IzquierdoAbstract:A new strategy for cancer gene therapy has been developed using a plant gene which encodes the enzyme, linamarase, that hydrolyzes the cyanogenic glucoside substrate, Linamarin, into glucose, acetone and cyanide. Retroviral vectors that carry linamarase as a potential killer-suicide gene cause a marked sensitization to the innocuous substrate, Linamarin, followed by cell death. We show that the system can eradicate very large intracerebral gliomas in vivo helped by a cyanide bystander effect. Animals showing a total regression of the tumor by magnetic resonance imaging (MRI), do not show other appreciable toxic effects.