Insulin and Erythropoietin Production

Insulin and Erythropoietin Production Insulin is a protein (polypeptide) discovered in 1921 by Banting with the pancreas being the site of its production. It is made up of 51 amino acids, divided into 2 chains; A and B, bonded by disulfide linkages. Chain A is made up of 21 amino acids with an intra-disulphide linkage, while chain B is made up of 30 amino acids (4). Why Insulin? Insulin is important in glucose metabolism, and is being used for the treatment of Diabetes mellitus; a metabolic disorder of glucose in the body. Initially, Insulin from animals was used to treat this disorder however nowadays synthesized human Insulin is being used, this is because; it is fast absorbed by the body, it has less allergic reactions, it contains less impurities, and it produces good results (3). Recombinant process of producing Insulin Synthetic Insulin was first produced in 1983 through genetic Engineering, which involve extraction of the human DNA (1), once extracted, the gene for Insulin is isolated, and enzymes are used to cut it. The gene is then cut using enzymes and put into the plasmid of a vector, where in most cases E. coli plasmid is used. Since Insulin contains two chains, two pieces of DNA are extracted, and the genes for the two chains are linked to ÃŽ ² galactosidase enzyme of the bacteria. The plasmids formed are then inserted into a host cell E. coli and sealed using another enzyme called ligase. And the host on replicating produces the enzymes each containing one of the two chains each. Production is followed by extracting and purifying the chains which are mixed in a reaction to reconstitute the disulphide bridges (1). ESCHERICHIA COLI AS RECOMBINANT INSULIN HOST Entero-bacillus, gram-negative E. coli is about 1 2ÃŽ ¼m, it can survive in the presence/absence of oxygen, and it also grows in an optimum pH and temperature of 7.0 and 37oC respectively. It utilizes glucose as its major carbon source and can also use other carbon sources like pyruvate, glycerol, acetate, and other sugars. K-12 and B strains are mostly used in the laboratory (20) Reasons for choosing E. coli Genetic Engineering technologies were developed using E. coli as a role organism, and so, the genetics of E. coli are well known among other microorganisms, as such its the most used organism for the production of different proteins (14). Moreover E. coli has a well known safety and production abilities, stable plasmid, controllable promoter, cheaper and easily cultured (6), E. coli also has fast growth rate, its easy to handle, and has well known fermentation skills and the ability to produce high protein content (14). That is why most of the proteins licensed recently by FDA and EMEA, were produced in E. coli (5). With these, and the fact that Insulin is a simple polypeptide (protein) which does not require glycosylation for its bioactivity and stability, E. coli carrying the plasmids for production of insulin will be used as the host for the production of Insulin Strain and plasmids: BL21 strain containing the pMYW-A and pMYW-B plasmids and temperature repressor ÃŽ »-c1857, will be used for insulin production (21). Growth strategy The various growth strategies that will be used to grow E. coli in order to make it happy and produce the desired product (11) include: Medium: E. coli needs nutrients like carbon, nitrogen and others; thus a carbon source; glycerol will be provided since its cheaper and more soluble than glucose (12), a source of nitrogen in the form of ammonium sulphate will also be provided. However such nutrients in large quantities can inhibit the growth of E. coli, as such a defined medium that contain optimum concentrations 20gl-1 glycerol and 2gl-1 ammonium sulphate will be used (11). The medium will also consist of the following; 3gl-1 KH2PO4, 1gl-1 MgSO4.7H2O, 0.8gl-1 citrate, and 6gl-1 K2HPO4 (23). Some trace elements will also be added to the medium. (23) Process and culture-strategies: E. coli will be grown submerged in a sterile controlled stirred tank reactor, and fed-batch will be used as the growth strategy so as to avoid accumulation of acetate which can be inhibits its growth, and reduce the production of the insulin (18). The growth strategy will be divided into two; initially batch mode will be used to initiate growth, after which the fed-batch exponential feeding will be used to produce the insulin (21). After adapting the medium and feeding method, oxygen transfer rates (OTRs) had to be increased through a suitable bioreactor design and over-head pressure (16). Large scale reactors usually reach high ORTs using air and normal aeration pressure, and so the oxygen partial pressure (pO2) will be increased by adding pure oxygen to the air-stream entering the reactor, thus increasing its oxygen transfer rates (16) DO will be maintained at 40% of air saturation and aeration rate at 1vvm. Foaming arising due to large number of cells and high aeration-rates will be solved by use of impellers for stirring simultaneously at 300rpm and the use of antifoam (ucolub N115) (16, 21). The process temperature and pH will be maintained at 30oC and 6.8 respectively so as to avoid partial proteolysis of the insulin protein. Bioreactor Design: Bioreactor vessel is usually cylindrical and made up of stainless steel. It is composed of impeller for stirring, Air sparger is placed at the bottom of the vessel for introduction of air, it has some inlets for introduction of acid/alkali for pH control and also for introduction of antifoams, nutrients and inoculum; It is also has pH, DO and temperature probes for sensing (22), Microbial activity during fermentation usually produces heat, so the bioreactor design must allow for removal of heat, and this can be achieved by cooling with jackets and coils (16) Bioreactors must also be designed in a way that it can withstand high temperature and pressure and to allow cleaning-up and sterilizing (22). Growth analysis Temperature, pH, DO, foam, partial oxygen and carbon dioxide pressures, will be analysed on-line, other parameters like biomass, will be analysed by using optical density (OD600) and dry cell weight (offline). Cell viability will be analysed by using flow cytometry, the concentrations of substrates and metabolites by enzymatic methods while insulin will be analysed using electrophoresis methods like SDS-PAGE, and ELISA, while its purity will be determined by HPLC (8). Limitations/Problems There are several problems that may arise during processing and can limit the use of this organism for Insulin production, these are; Poor secretion because of the structure of its membrane (and tough cell wall), small amount of foldases, chaperones and increased concentrations of proteases, leading to low productivity (7). Solutions to this problem include all measures taken to increase quality of secretion and production such as: Use of secretion systems like the system of ÃŽ ±-haemolysin (7) co-expression after co-cloning of foldases and chaperones (13) Improving the rates of gene-expression and using proteases deficient mutants like BL21 (18). use of E. coli mutants that are deficient of cell-wall (12) Limited post translational-modifications; including disulfide-linkage formation, which is important for the insulin stability and biological activity (9). Solutions to this problem include; Production of insulin with altered amino acid sequences through genetic engineering (9) Using E. coli mutants to enhance the formation of disulfide linkages e.g. Origami (15) iii. Exporting proteins into the periplasm which has disulphide bonding mechanisms (19). Codon biases; due to large quantities of exact transfer-RNAs found in E. coli, the codons in the human-genes are often different from those that are found in this organism. This results in inefficient expression of some of these rare codons by the organism resulting in an unexpected protein synthesis termination or wrong incorporation of the amino acids (12). This problem can be solved by replacing codons that are rare in the desired gene by codons that are often found in the E. coli and by co-expressing the rare transfer-RNAs (15). Acetate is usually formed as a by-product, and is inhibitory to growth of the cells (20). Solution is by using a fed-batch feeding method and by limiting DO level (11). Another problem is that large proteins are often obtained in an insoluble form (5); forming aggregates called inclusion bodies; IBs (20). This can be solved by adjustment of temperature, increasing the strength of the promoter, adjusting the number of plasmids, concentrations of the inducer, and the composition of the media (9). Erythropoietin EPO EPO is a glycoprotein that is produced in the renal cortex of the kidney (10, 11). It has also being shown to be present in the brain, spleen, liver and the lungs (7, 17). It is made up of 165 amino acids of about 18kDa (25), with a number of carbohydrates linked to the polypeptide through O and N glycosidic-bonds giving the glycoprotein a total weight of 34kDa.Two disulphide linkages hold the molecule together (15) and the carbohydrates are responsible for the stability of the glycoprotein in-vivo,and increasing its half-life in the body (24). Why EPO? EPO functions to regulate the amount of red blood cells (RBC) in the blood by controlling the proliferation and differentiation of its immature cells to mature cells (1, 2, 22,). It is also involved in the growth and formation of blood vessels, and healing of wounds (6), it functions in the brain is not clear, but studies showed the glycoprotein to have some protective effects (18). Because of these functions EPO has being used in the treatment of anaemia caused by kidney failure and other causes (25). Recombinant production of EPO Despite its importance, EPO in body is found in very small amounts and mostly in the urine (4), as such there is the requirement to produce EPO in large amounts, this leads to the work of isolating the glycoprotein from the urine (12, 21), and was used to identify its amino acid sequences, and synthesis of its DNA (9, 12), furthermore the human erythropoietin genes were cloned by Lin et al. (17), and consequently recombinant human EPO (rhuEPO) was produced in 1985 using CHO cells (14, 16). Chinese -Hamster- Ovary (CHO-Cells) as rhuEPO host: These are epithelial cells derived from the ovary of Chinese hamster (a mammal). They grow well in culture and looks like cobble stones. The cells usually attach to a surface available but can be grown in suspension (20). CHO cells are grown best at 37oC and at pH 7.4; they are cultured in a suitable complex medium which can support their growth for many generations (20). CHO cell lines are now available from cell culture collections like the American type culture collection; ATCC. Moreover human EPO expression plasmids are now also commercially available, and are usually used for production of EPO using the CHO cells (27). Reasons for choosing CHO-cells Karthik et al. (13) showed that CHO-cells are being used extensively in the industries for the production of many proteins, because they have demonstrated, to possess some qualities like: They can modify biological products post-translationally; Proteins produce in CHO-cells have high glycosylation quality making them compatible and stable (13) Safety of the product; Studies in 1989 have shown that most viruses do not multiply in CHO-cells (13) Ability to adapt easily and be grown in suspension (13). Products can now be purified to contain less contaminant (13). CHO cells have being used for a long time; as such much data has being accumulated for regulatory reasons (13). They are easy to manipulate genetically (13). The isolation of cells deficient in Dihydrofolate-reductase enzymes leads to stable clones selection and genes amplification to increase production (13). With all these, and the fact that EPO is a glycoprotein that requires glycosylation for its stability and activity, recombinant CHO cells are chosen to produce EPO. Cell lines and plasmids: Cell lines which have the capability of glycosylating proteins (Pro-5), harboring the pGEX-HET-puro expression plasmid, will be used to produce the recombinant human erythropoietin (27). Growth strategy Medium: Complex culture medium will be provided with; Glucose as a source of carbon and energy, Amino acids as source of nitrogen, Salts will be included to make the solution isotonic Vitamins and hormones will be added as co-factors Serum is usually added to the culture medium to enhance the growth of the cell (20), but has the following disadvantages: It chemicals are not defined and can cause cell growth inconsistency between batches (20) It is very expensive (20) The serum may contain proteins which can be difficult to separate and purify from the proteins secreted by the cells during downstream processing (20) It increases foaming and can be a source of contamination by viruses. (20) Therefore a serum-free (SF) media (16) will be used for the growth of the E. coli. Process and culture-strategies: The cells will be grown adherent on micro-carriers in a sterile controlled packed bed reactor, and perfusion method of production where some amounts of the medium is removed and replaced by fresh one and the cells are grown slowly will be used (28); because it was found to improve the glycosylation of the proteins more than fed-batch where there is fast growth of cells, (8). Before, many processes were run in a simple batch method, but nowadays, Perfusion or fed-batch methods are mostly employed and higher products are now realized (22). The production will be carried out in two stages; the growth stage and the production stage. Normally stirring will be kept at 100 to 150 rpm, foaming will be avoided by adding Pluronic F68 (16).Temperature will be maintained at 37oC initially during growth and then reduced to 33oC during production, as was shown to increase the overall protein production, while maintaining the quality of the glycoprotein (3, 26). pH w ill be kept at 7.1 initially and then reduced to 6.8 (8, 26), by passing CO2 gas to the culture or by addition of concentrated sodium-bicarbonate solution in low quantities, because CO2 is also toxic to the cells and can also affect the production of EPO (20). In order to avoid the depletion of oxygen, the oxygen transfer rates (OTRs) will be increased above its utilization rate, with a constant supply of pure oxygen and air, while DO will be maintained at 20-50% of air saturation (20). Bioreactor Design: Since the cells are big and fragile, the design of the bioreactor has to be considered. Mammalian cell culture bioreactors are designed with bottoms that are round and are usually made up of glass/stainless steel (20). The impellers are usually marine or pitched blade types fitted at the end of mechanical drives shafts so that both vertical and horizontal mixing are allowed at low stirring-rates (20). Temperature is controlled through coiled pipes or open ended fermenter jacket (20). pH, DO and temperature probes are used for sensing and have both air inlet and outlet for respiration. Growth Analysis Temperature, pH and DO will be monitored on-line, because cells are immobilized, biomass formed cannot be measured directly therefore it will be monitored by measuring rate of glucose consumed daily and the rate of lactate produced (28) Cell viability by flow cytometry, Glucose, glutamine, and lactate concentrations will be analysed using multi-parameter Bio-analytical system (26); while ammonia formed as waste product of amino acid metabolism, will be analysed by colorimetric assay and by the use of detection-kit (26). EPO formed will be analysed using HPLC to determine its purity and its quality by Isoelectric focusing, SDS, and Bradford assay (26). The activity of EPO will be analysed by bioassay and by the use of protein assay-kit (27) Limitations/Problems. There are many limitations associated with CHO cells culture processes and they include; They are fragile and highly sensitive to shear stress caused by agitation and bubble because the cells are large and have only cell membrane (20). This is usually solved using a suitable bioreactor-design and use of Pluronic F68 (20). They need a complex medium including serum which can cause problems in the downstream processing and is expensive (20). Solution to this is by using serum- free media (24, 25). Low yield of proteins have been produced from these cells, the productivity using the microbes being higher than the use of these cells. They also have slow growth rates (13). The problem of low productivity and slow growth rates can be solved through selecting cell lines that are better and optimizing cultural-strategies. Ammonia and lactate are generated during growth and can inhibit growth and also affect glycosylation (8). Solution is by optimizing the strategies of feeding and by monitoring (8). Glycosylation differences may arise from the EPO produced in the CHO-cells and the human EPO as seen in the way the two are sialylated terminally, as a result that the CHO-cells are not able to express an enzyme called alpha-2,6, sialyltransferase (27). Solution is by the use of CHO-cells harboring alpha-2, 6, sialyltransferase-cDNA expression-cassettes (27). REFERENCES: 1. Alcamo, I., DNA Technology; the Awesome-Skill. Farming-dale. New York: Academic Press. (2001). 2. Banting Grolier Electronic publishing www.littletree.com.au/dna.htm accessed on 30/12/2010 3. Carbs information, www.carb-information.com/insulin-synthetic.htm accessed on 30/12/ 2010. 4. Charce, R.E., and Frank, B.H., Research, Production and Safety of Biosynthetic Human Insulin. (1993). www.littletree.com.au/dna.htm accessed on 30/12/2010. 5. Ferrer-Miralles N. Domingo-Espà ­n, J. Corchero, J.L. Và ¡zquez, E. and Villaverde, A. Microb. fact. for recombinant pharmaceuticals, Microbial factories , 8:17, 2009. 6. Fox, S. Improved processes and new capacity for pipeline to commercial production. Biopharmaceutical contract manufacturing, Volume 1 (report). High Tech Business Decisions: San Jose, CA. 2005 7. Genschev, I., Dietrich, G., Goebel, W.,The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol. 10: 39-45. 2002 8. Hewitt C.J., Nebe-von Caron G., Axelsson B., McFarlane C.M, Nienow A.W Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multi-parameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration. Biotech. Bioeng. 70: 381-390. 2000 9. Hite P.F, Barnes A.M.J.P.E. Exhuberance over Exubera. Clinical Diabetes 24: 110-114. 2006. 10. Jana, S., Deb, J.K. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotech. 67: 289-29. 2005. 11. Joseph S., and Raphael F., growing E. coli to high- cell density-A historical perspective on method development Biotech. Advances 23: 345-357 2005. 12. Korz D.J, Rinas U., Hellmuth K, Sanders E.A, Deckwer W.D. Simple fed-batch technique for high cell density cultivation of E. coli. J Biotechnology, 39: 56-65. 1995. 13. Kujau, M.J., Hoischen, C., Riesenberg, D., Gumpert, J. Expression and secretion of functional mini-antibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and E. coli: a comparison of the synthesis capacities of L-form strains with E. coli producer strain. Appl. Microbiol. Biotech. 49: 51-58. 1998. 14. Lund, P.A. Microbial molecular chaperones. Advanc. Microbiol. Physiol. 44: 93-140. 2001 15. Makrides S.C. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60: 512-5388. 1996. 16. Meyer, H.P. Brass, J. Jungo, C. Klein, J. Wenger, J. and Mommer, R. an emerging Star for Therapeutic and Catalytic Protein Production. Bioprocess International. 2008. 17. Nacelle, G. J. V. and Coppel, R. L. Reshaping Life; Key Issues in Genetic Engineering, Novo-Nordisk Promotional Brochure. Melbourne: Melbourne University Press. 1989. 18. Schmidt, F.R. Recombinant expression systems in pharmaceutical industry. Appl. Microbiol. Biotech. 65:363-37. 2004. 19. Wacker M., Linton D., Hitchen P.G., Nita-Lazar M., Haslam, S.M., North, S.J., Panico M., Morris H.R., Dell A., Wren, B.W., Aeb, M. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298:1790-1793. 2002. 20. Demain, L. A., and Vaishnav, P. Production of recombinant proteins by microbes and higher organisms. Biotech.Advan. 27: 297-306. 2009. 21. Schmidt, M., Raman Babu, K., Khanna, N., Marten, S., Rinas, U., Temperature- induced production of recombinant human insulin in high cell density culture of recombinant Escherichia Coli. Journal of Biotech. 68:71-83. 1999. 22. Ratledge, C. and Kristiansen, B. Basic biotechnology. Cambridge: Cambridge university press. 2001. 23. Tabandeh, F., Shojaosadati, S.A., Zomorodipour, A., Khodabandeh, M., Sanati, M.H., Yakhchali, B. Heat induced production of human growth hormone by high cell density cultivation of recombinant E. coli. Biotech. Letters. 26: 245-250. 2004.

Pros and Cons of Genetic Engineering in Plants Essay

Less tillage needed, especially with crops containing herbicide tolerance transgenes, therefore conserves fertility through minimizing soil damage through compression. |GE agriculture claims low tillage weed control: this can be achieved by ending the practice of monoculture and instead introducing proper crop rotations designed specifically to combat the weeds of the particular locality. Monoculture creates a weed paradise. | All countries face problems caused by alien species accidentally or deliberately introduced into a new environment (e.g. prickly pear in Australia). The main factor permitting this is international travel, but nobody has suggested that this should be banned. The problem of alien species is manageable, as would be the problem of genetic pollution caused by spread of seeds or pollen. As regards pollen contamination from GM varieties and the call for compensation for growers of non-GM or organic varieties whose crops are contaminated, if one is to be fair one migh t reasonably expect growers of non-GM and organic varieties to compensate growers of GM crops if they are contaminated with non-GM or organic pollen. |Genetic pollution from transgenes spreads into other organisms through pollen, seeds and microbial processes. It is fundamentally different from other forms of pollution because once the genes are out, they can’t be recalled. The best example of pollen contamination is provided by the canola seed, which was multiplied in Canada. It was officially confirmed in May 2000 that this seed was contaminated with unapproved GM canola seed and accidentally shipped to UK and other countries. By then it had been planted in Europe and large acreages of the young crop had to be destroyed. According to Advanta, the contamination occurred because of cross-pollination in Canada, where the seed was produced. The nearest source of GM contamination was 4 kilometers away.| Organic farming has long accepted accidental contamination from herbicide sp rays from neighboring farms. If there is concern about GMOs, DNA tests can be carried out.|Risks destroying organic farming, which rules out the use of GM organisms. Who will compensate organic farmers for the extra surveillance and analysis, which will be needed to ensure that the organic food chains remain free of GMOs?| The Starlink debacle is indeed a lesson that the GM food producers will learn from. Identity Preservation Systems are being put in place, verified by DNA analysis, to ensure that GM and non-GM supplies are kept separate.|The massive contamination in 2000 of the USA corn (maize) crop and human food chain by Starlink, a variety that is not approved for human consumption, shows that genetic pollution from transgenic crops to non-transgenic crops and food is inevitable. Starlink maize produces the Cry9C protein, which may be a human allergen. Two other major contaminations of ordinary seed (maize and canola seed) with GM seed have already occurred leading to emergency recalls of the product.| Reduces labor costs. |Sustainable organic agriculture creates much needed jobs in depressed rural economies.| Environmentally relatively benign herbicides are used and less of them. Opposing GM crops forces farmers to use herbicide resistant varieties which have not been made by GM such as those resistant to sulphonylurea herbicides which more readily give rise to herbicide resista nt weeds.|Promotes â€Å"agribusiness†, therefore more herbicide use. Herbicides are responsible for much illness in farm workers and contaminate drinking water.| Enhances biodiversity by allowing weeds to continue growing for longer thus providing nutrition for animals. After weed kill a mulch forms which hosts a thriving population of insects, arthropods etc.|The total herbicides used with herbicide tolerant crops kill all weeds thus reducing biodiversity in the field.| No insecticidal sprays needed on crops that have insecticidal Bacillus thuringiensis (Bt)-toxin genes engineered into them. Plants with Bt or other insecticidal genes are likely to give rise to lower levels of mycotoxins in the final food product. Less insect damage means less opportunity for fungi to infect the plant and bring toxic substances.|As with weed control, control of insect damage is achievable with properly designed crop rotation and other forms of good husbandry such as intercropping. Healthy pl ants not imbalanced by chemical fertilizers build up their own defenses against insect attack. | GM plants are carefully tested for environmental and ecological impact, including their effects on earthworms and beneficial insects. Bt crops target only insects, which attack the crop. Future insect resistance genes will be engineered to express in leaves and stem rather than in pollen and seed. There is already evidence that the Bt gene is expressed less in Bt corn pollen than in leaves/stems therefore the risk to butterflies (e.g. Monarch) through pollen drift onto their food plants (e.g. milkweed for Monarch) is diminished. |In relation to population variance, sample sizes in lab and field tests (e.g. of earthworms) are sometimes too low to detect even large effects. Insecticidal crops containing the gene for Bacillus thuringiensis (Bt)-toxin kill beneficial organisms such as bees, ladybirds, lacewings & butterflies (e.g. through pollen). The Bt plant remains falling to the ground are harmful to earthworms and other members of soil fauna. Bt toxins are secreted into soil from Bt plant roots and are toxic to lepidoptera in the soil (Stotzky, et al. Nature 402, 480 (1999)). The specific targeting and elimination of one insect pest has led to other pestiferous insect species moving into the ecological niche created by the disappearance of the first species. Getting rid of one problem simply created another. If Bt toxin transgenes spread to wild relatives of crop plants the wild plants may also develop resistance to insect herbivores. This could lead to the affected wild plants becoming invasive weeds.| The problem of resistance to Bt toxin and other toxins engineered into crops can be countered by planting suitably sized ‘refuges’ of a non-GM variety of the crop at suitable intervals within the crop. The interbreeding of the wild population with the Bt-exposed potentially resistant population will dilute out the genetic trait and thus prevent it building up.|Putting the Bt toxin gene in the crop exposes the pest to the toxin for longer, thus allowing natural genetic resistance to the toxin to develop in the pest. So-called refuge systems do not work, partly because breeding cycles in the differing pest populations are not synchronized. Refuges of up to 40% of the acreage are having to be recommended and this is not practical or popular for farmers. The build up of Bt toxin resistance threatens to render ineffective an insecticide long used by organic agriculture. Increased use of biopesticides in transgenic crops deprives the ecosystem of one of its natural pest controls thereby putting at risk its ability to restore equilibrium after being upset by abnormal conditions. | Helps solve the problem of world hunger by creating varieties which will make more efficient utilization of scarce land and give higher yields because of better pest resistance, nutrient utilization etc.|World hunger will not be solved by technological means. It is a problem of inequitable distribution of wealth and corrupt governments. Reduces yields (e.g. cotton, soybeans and sugar beet in some areas).| If herbicide resistance spreads to weed populations it can be combated with another herbicide with a different active ingredient. The ecological and agricultural threat of a GM plant is no more than a non-GM invasive (exotic) species such as kudzu or purple l oosestrife. Although improved crop yields can be engineered by genetically modifying plants, there is ecological concern over whether these plants are likely to persist in the wild in the event of dispersal from their cultivated habitat. The results of a long-term study of the performance of transgenic crops in natural habitats on four different crops (canola seed, potato, maize and sugar beet) which were grown in 12 different habitats and monitored over a period of 10 years show that in no case were the genetically modified plants found to be more invasive or more persistent than their conventional counterparts. (M. J. CRAWLEY, S. L. BROWN, R. S. HAILS, D. D. KOHN & M. REES. Biotechnology: Transgenic crops in natural habitats Nature 409, 682 – 683 (2001)  © Macmillan Publishers Ltd)|Enhances spread of herbicide resistance to wild weed populations because the necessary genes are in the pollen, which can then pollinate wild relatives of the crop plant. This could create â₠¬Ëœsuperweeds’ especially if ‘gene stacking’ of several different transgenes occurs. Spread of transgenes is also caused by birds, animals & machinery carrying the seed to other locations (e.g. canola seed on Ailsa Craig isle, 10 miles from Scottish mainland) Increased weediness of GM crops is already beginning to show. In 1999, in Alberta, Canada canola seed volunteers (unwanted crop plants coming up the following year) resistant to three different herbicides have been discovered. A series of chemical and DNA tests confirm the weeds in farmer Tony Huether’s field near Sexsmith are resistant to Roundup ®, Liberty ® and Pursuit ® herbicide chemicals. Invasive species of plants can remain relatively unproblematic in a region for many years and then suddenly take a hold so much so that they become an economically significant nuisance. For this reason, the ecological impact of GM crops will be difficult to predict in the long term, i.e. over several decades. | Most cultivars are unlikely to survive amongst wild plant populations and those with herbicide resistance that escape will have no advantage from the herbicide resistance trait unless that particular herbicide is used. Such volunteers can be controlled with other herbicides.|Transgenic herbicide resistant cultivars could escape into the wild and become problematic ‘volunteers’ in agriculture. These volunteers will require increased use of more toxic herbicides.| Is a sustainable agriculture, because it reduces chemical inputs as well as fuel inputs for farm machinery.|Unsustainable — based on greed not need. Helps chemical agriculture to proliferate. The only sustainable agriculture for the future is organic (including biodynamic & permaculture).| Quicker and more precise than traditional breeding.|Breeding takes place outside the proper c ontext, i.e. in the laboratory, therefore the crops are so weakened that they need to have the environment of the laboratory (soil sterilization, artificial fertilizers and pesticides) brought to them in the field. Transgenic lines are unstable and can lead to crop failures (e.g. GM cotton in USA).| A greater range of distinct disease-resistant varieties can be created so that the farmer has a wide choice and can plant a mixture of several varieties of the same crop in the same field to insure against disease attack. Disease resistance traits can be rapidly introduced to cultivars, e.g. rice, thus keeping ahead of the changing pattern of disease in a particular locality.|Because of the huge investment in GM crops, the necessarily increased emphasis on single high-yielding varieties reduces genetic diversity within the crop itself. This can lay the crop open to massive losses when disease strikes.| Novel drought and salt-tolerant cultivars can be created (important for Third World Co untries).|Sustainable organic plant breeding can develop novel varieties properly suited to a locality perfectly satisfactorily.| Any royalties or technology fees are more than compensated for by advantages including higher yields and easier, therefore less expensive, husbandry.|No seed saving by the farmer is permitted. The farmer has to pay royalties to the biotech company. This undermines a traditional agricultural practice and particularly threatens peasant farming in developing countries. GM crops add to the tendency of modern chemical agriculture to undermine the autonomy of farmers and turn them into tractor drivers or machine minders for large transnational corporations.| New varieties are tested for toxicity more than any crop plants have ever been in the past, therefore they are likely to be safer. Jimmy Clark, a professor of ruminant nutrition in Animal Sciences at the University of Illinois at Urbana-Champaign, reviewed the results from 23 research experiments, which wer e conducted over the past four years at universities throughout the United States, Germany and France. In each study, separate groups of chickens, dairy cows, beef cattle and sheep were fed either genetically modified corn or soybeans or traditional corn or soybean as a portion of their diet. Each experiment independently confirmed that there is no significant difference in the animals’ ability to digest the genetically modified crops and no significant difference in the weight gain, milk production, milk composition, and overall health of the animals when compared to animals fed the traditional crops. Clark concluded, â€Å"Based on safety analyses required for each crop, human consumption of milk, meat and eggs produced from animals fed genetically modified crops should be as safe as products derived from animals fed conventional crops.† Clark added that approximately 70% of the genetically modified soybeans produced in the world and 80% of the genetically modified corn produced in the United States are used as animal feed. â€Å"Since these genetically modified crops were grown beginning in 1996, they have been fed to livestock and no detrimental effects have been reported,† Clark said. (University of Illinois at Urbana-Champaign, News Release, Apr il 2001).|Increases herbicide residues in the food because the herbicide is applied later in the growing season and closer to harvest | The issue of spread of antibiotic resistance from GM crops containing antibiotic resistance marker genes is unproven. If it is a problem at all it is likely to be small compared with the induction of antibiotic resistance through profligate use of antibiotics in animal nutrition, veterinary and medical practice.|Spreads antibiotic resistance to microorganisms in the environment, and then to pathogenic bacteria.| More profit for the farmer, seed producer and biotech company shareholder.|No demonstrable benefit to the consumer. | Crops producing ‘nutraceuticals’ can be engineered, i.e. food additives that have a nutritional benefit bordering on a pharmaceutical benefit, e.g. modified edible oils. The vitamin content of plants can be enhanced by GM. Plants which previously did not contain a particular vitamin can now be made to produce lar ge amounts of it (e.g. Vitamin A ‘golden’ rice). The aim of the GM Vitamin A rice project is not to achieve ideal levels of vitamin A intake through this source but to augment the extremely low intakes which lead to blindness and death of hundreds of thousands of people a year (Prof. Dr. Ingo Potrykus, statement, February 2001). |In 1999, Deutsche Bank issued a report advising investors to avoid investing in GM crop technology (agribiotech). A balanced diet of fresh fruit & vegetables plus cereals and protein is all that is necessary. ‘Nutraceuticals’ are a sticking plaster (band aid) attempt to remedy fundamentally unhealthy diets. Existing food sources provide adequate daily intakes of vitamins provided they are eaten in sufficient amounts and the vitamins are not destroyed in the processing or cooking. Vitamin-enhanced GM plants are an unnecessary technical solution to a problem, which does not exist. Even with Vitamin A GM rice a normal daily intake of 300 gram of rice would, at best, provide 8% percent of the vitamin A needed daily.| The ‘killer genes’ of the technology protection system (‘terminator technology’) allows the seed producer’s intellectual property (patent) to be protected by a biological rather than litigious method.|No seed saving by the farmer is permitted. The farmer has to pay royalties to the biotech company. This undermines a traditional agricultural practice and particularly threatens peasant farming in developing countries.| The increased cho ice of modern high-yielding cultivars to farmers allows diversification to keep ahead of economic, climatic and plant disease trends.|The possibility of further globalization of crop varieties that GE offers through the introduction of traits necessary for introduction into new regions of the globe erodes cultural diversity – i.e. traditionally, different crops and varieties are grown by different cultures. Genetic engineering works towards global uniformity, i.e. globalization of Western/Northern culture. There used to be far greater choice of crop varieties for farmers, sometimes hundreds of varieties of a particular crop in a given region, but this diversity is falling at an alarming rate because of the industrialization of farming under pressure from the agrochemical industry. This will be accelerated by biotech agribusiness.| GE allows the creation of plants that produce vaccines, pharmaceuticals or enhanced pharmaceutical raw materials. |GE is already used to produce ph armaceuticals in microorganisms in the much safer containment conditions of biotechnology factories. It should not be taken out into the environment thus putting the environment at risk. In any case, much of the pharmaceutical production, which would be created, is designed to treat diseases caused by industrialization and urbanization, which could be better treated not by a genetic fix but by changing lifestyles and environment.| Novel food crops are tested for genetic stability (breeding true), ‘substantial equivalence’, nutritive properties, toxicity and allergenicity. It is well known that conventional breeding can introduce increased levels of natural plant toxins into a new variety or can modify its digestibility or nutritiousness. Furthermore, certain organic crops have been shown to have higher levels of toxic substances, e.g potatoes.|GE introduces unpredictable toxic or allergenic effects into food plants (e.g. Brazil nut gene in transgenic soybeans). ‘Substantial equivalence’ is a political-commercial concept rather than a scientific one. GM plants are not genetically stable. For instance, the number of copies of an inserted gene changes through later generations of the GM plant. | This technology is completely new to the insurance industry. It is natural that insurers will be cautious about it. However, when they realize that the risks are no worse than with introduced ali en species that are dealt with by conventional methods of weed control, the problem of insurance will disappear. No amount of research under containment conditions will reveal how a GM plant will behave when grown en masse in the open field.|As the degree of escape of genes from GM crops is unpredictable, they can’t be recalled once they have escaped and they could multiply in the wild, some insurance underwriters have stated that such risks are uninsurable. There should be a moratorium on experiments in the open until the safety of the GM plant is fully tested under containment conditions. | Plant pathogens need not be used in making GM plants. The genes can be blasted into plant cells using a ‘gene gun’, which fire microparticles of metal coated with the DNA of interest.|Plant pathogens such as Agrobacterium tumefaciens (literally ‘cancer causing’) are used to shuttle genes into plants and viral gene sequences such as cauliflower mosaic virus promo ter are used to make the genes express themselves once in the plant. The pathogens could recombine with their natural equivalents in the plant thus risking unpredictable outbreaks of plant disease.| Bioprospecting has gone on since very ancient times. There is no reason why an organism, which just happens to be at a particular location of the globe, should be in the sole ownership of the people living there. Agreements can be entered into in order to protect the traditional usages of indigenous peoples.|Steals genetic commons from peasant farmers and indigenous peoples (biopiracy by the rich North, e.g. neem tree & basmati rice).| Biopolymers can be produced in GM plants allowing the manufacture of biodegradeable plastics (e.g. PHBV, Biopol), which are also sustainable because they are not made from fossil fuels. |No comment|

Greek and Roman Culture Essay examples - 1240 Words

Greek and Roman culture, although similar, are very different and interesting. Since the Romans adopted culture from the Greeks, many traditions are the same. When the Romans conquered the Hellenistic cities, they became fascinated with the idea of a Greek style of doing things. All things Greek were now considered popular. This is how much of the Greek way of life made its way into the Roman society. The first part of culture that the Romans adopted was the Greek art. Scores of Greek paintings were stolen from Greece and imported into the Roman Empire. Roman artists began adopting the Greek style of art, from the emotional intensity to the great detail. â€Å"In many cases, it is very difficult to distinguish between Hellenistic†¦show more content†¦Greek didn’t have the technologies to make these roads. Another thing Rome was good at was making bridges. If Rome had to cross a river to get to a battle, they built a well-made bridge and marched their soldie rs across it. The Greeks, although advanced, were not advanced enough to be able to build these great wonders quickly and efficiently. The Romans and Greek houses were similar, yet different. The Roman houses usually composed of many stories, but many of the Greek houses didn’t, due to their lack of concrete. The Romans had many rooms in their houses, usually each for a specific purpose. Greeks, on the other hand, had many of their rooms dedicated to gods, and only a few rooms. Many Greeks had their houses made out of pebbles, clay, or mud, which had to be kept up, since it would wear away and disintegrate. Greek houses were planned around a courtyard with a garden and statues in it. â€Å"In the modern mind (at least in the modern American mind) Greek and Roman culture and mythology are classed together. 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Biographical Sketch Dr. Vimala Pillari - 1471 Words

Biographical Sketch Dr. Vimala Pillari, the author of Scapegoating in Families, is well known in the social work professional community. As a Licensed Clinical Social Worker she has published over 8 books that have become widely known along with several articles. Currently serving as Dean of the Whitney M. Jr. School of Social Work, Dr. Pillari has also served as Dean and Director for two other accredited universities. In addition to her education, she is part-time social worker at Children’s Hospital in Buffalo, NY and was a part time social worker at the Family Agency of Tidewater in Norfolk. Having to grapple with and being exposed to scapegoating in families, as she has worked with many unfortunate child-victims and adult survivors of severe scapegoating, she now draws on her experience to explore and inform others of this phenomenon in families. Summary of Contents Scapegoating in families is a book written to shed light on the prevalence of such in today’s family dynamic. While the concept is not new, it is one that is not discussed as much as it should be. Thus the information in this book explores family dynamics in terms of scapegoating in families and identifies patterns of how it works and why it happens. In doing so this literature considers the multidimensional aspects of victims of scapegoating and their dysfunctional families (Pillari, 1991, second cover). In demonstrating that scapegoating is an intergenerational phenomenon Dr. Pillari provides detailed