WorldWideScience

Sample records for cho chiiki shin

  1. Regional new energy vision for Soryo Town, Hiroshima Prefecture; 2001 nendo Soryo cho chiiki shin energy vision. Chiiki shin energy vision sakutei tou jigyo

    NONE

    2002-02-01

    A regional new energy vision was formulated for the town for 'contributing to the solution of environmental problems' and 'contributing to the reenergization of the regional community through utilizing resources in the region.' The final energy consumption of the town is 103,315 times 10{sup 3} MJ, with the transportation sector accounting for a percentage higher than the nation-wide average. The town is characterized in that it has but a small number of manufacturers, offices, or stores. As for new energy, it was estimated that 9117 times 10{sup 3} MJ/year would be gained by methanol produced from cultured weed, 5696 times 10{sup 3} MJ/year by medium/small-scale hydropower generation, 5,233 times 10{sup 3} MJ/year by photovoltaic power generation, and so forth. New energy introduction projects were discussed, which included medium/small-scale hydropower generation at a counter dam; the introduction of photovoltaic power into principal public facilities, general households, and agricultural facilities; introduction of solar heat appliances into principal public facilities and general households; introduction of clean energy automobiles into the town office and general households; and the introduction of pellet-fired stoves into principal public facilities and general households. (NEDO)

  2. Report on energy saving vision in Santo-cho region; Santocho chiiki sho energy vision hokokusho

    NONE

    2001-02-01

    An energy-saving vision was decided on in Santo-cho region in Hyogo Prefecture, with its outline reported. This town is such that about 80% of the region is mountains, forests and wilderness and that aging is advancing at the rate above that of Hyogo Prefecture or the national average. Nearly entire energy of the town is dependent on the supply from outside. The energy consumption is somewhat increasing as a whole, with that of the people's livelihood/domestic sector and of transportation sector are rising. In the classification of fuels, electricity is growing in consumption. As an energy-saving vision, it aimed principally at personal surroundings in which every one got into the habit of saving energy continuously without being forced. The basic plan for the energy conservation drive consisted of inducement to an energy-saving life style, energy conservation to be spread by the next generation children, continuation of energy saving activity rooted in the region, and promotion of energy conservation as a basis for introducing new energy. The diffusion and enlightenment for children destined to lead the next generation were defined as a particularly important assignment, as was the promotion of energy conservation and environmental education. (NEDO)

  3. Report of regional new energy vision for Kannami Town; 2001 nendo Kannami cho chiiki shin energy vision hokokusho

    NONE

    2002-03-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Kannami Town, Shizuoka Prefecture, surveys and studies were conducted involving the energy consumption structure of the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The town consumes approximately 5-million GJ/year, with the industrial sector consuming 47%, the transportation sector 29%, and the residential and commercial sector 26%. When a comparison is made, the percentages of the residential/commercial and transportation sectors are relatively higher than the national average. Projects for new energy introduction were studied, which involved photovoltaic power generation for the town office, community hall, primary and junior high schools, nursery schools, and general residences; solar heat utilization for public facilities, greenhouses, and general residences; wind power generation for sightseeing facilities, public gardens, and emergency evaluation areas; utilization of sewage waste heat for the sewage purification center; utilization of hot spa waste heat in the urban area; a biomass-fueled power plant; medium- and mini-scale hydroelectric power generation for the drinking water treatment plant; refuse-fueled power generation at refuse incineration facilities; use of clean-energy automobiles; natural gas-fueled cogeneration for public organizations; and so forth. (NEDO)

  4. Report on new energy vision at Hinase Town area; 2001 nendo Hinase cho chiiki shin energy vision hokokusho

    NONE

    2002-03-01

    With an objective of accelerating introduction of and uplifting consciousness on new energies at Hinase Town in Hyogo Prefecture, investigations and discussions were given on the energy demand, quantity of new energies available, and the new energy introduction projects for the town, thus a new energy vision was established. The estimated amount of energy consumption in Hinase Town is 242,814,320,000 kcal in fiscal 2000. The amount of carbon dioxide discharge associated therewith is 76.57 t-CO2/year. Regarding the energy consumption by departments, the industrial department consumes the most, followed by the transportation department, civil household department, and civil business department. The new energy introduction projects have discussed servicing of the bio-gas plant utilizing organic wastes and cogeneration, the new energy introduction projects for the Natural Energy Island at Kakui Island and the ocean ranch consisted of the use of solar heat water warmer, solar cell powered street lights, photovoltaic power generation, and electric cars for sightseeing visitors, the Eco-school promotion project for primary schools, introduction of energy saving devices into the town office, and the new energy proliferation and enlightenment project. (NEDO)

  5. Report on establishing new energy vision at Furen Town area; 2001 nendo Furen cho chiiki shin energy vision sakutei hokokusho

    NONE

    2002-03-01

    With an object of accelerating introduction of and uplifting consciousness on new energies at the city of Furen in Hokkaido, investigations and discussions were given on the energy demand, quantity of new energies in existence, and new energy introduction projects of the city, thus a new energy vision was established. The estimated amount of energy consumed in Furen Town is 119,846 x 10{sup 3} kWh. By departments, the transportation department has the highest consumption ratio, followed by general household. Energy types are kerosene, followed by heavy oil, electric power and LPG in the decreasing order. The important new energy introduction projects have discussed the ''Snow Storehouse Workshop'' being a low temperature rice storage facility in Bibai City, which performs cold storage and shipment adjustment of agricultural products, through-the-year agriculture to perform house cultivation by using rice straws and rice husks as the heat energy source, adjustment of temperatures of paddy fields by using calorific power of rice husks, electric power generation and heat supply by using heat of combustion of chips from lumber factories, electric power generation and heat supply by using livestock excreta (bio-gas), and introduction of new energies into schools and parks. (NEDO)

  6. Regional new energy vision for Kobuchizawa Town. Eco-energy town - Kobuchizawa; 2001 nendo Kobuchisawa cho chiiki shin energy vision hokokusho. Eco energy town Kobuchisawa

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Kobuchizawa Town, Yamanashi Prefecture, surveys and studies were conducted on the trend of energy consumption in the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The town consumes 635,021 GJ/year in energy, with the industrial sector consuming 34.6%, the transportation sector 24.8%, the residential sector 21.8%, and the commercial sector 18.7%. The energy sources are electric power, kerosene, LPG, gasoline, gas oil, and heavy oil, as named in the descending order in terms of amount. Studies were made about new energy introduction projects, which included the use of photovoltaic power for outdoor lights, primary schools, nursery schools, the flower park (Fiore Kobuchizawa), mountain huts, and for the Kannondaira sightseeing spot; mini-scale hydroelectric power generation for the maintenance of the Otaki spa and existing water channel utilities; wind power generation for improvement on mountain huts; introduction of clean-energy vehicles; introduction of cogeneration (natural gas); and a business for effectively utilizing biomass. (NEDO)

  7. Regional new energy vision formulation for Kadogawa Town, Miyazaki Prefecture. Report; 2001 nendo Miyagi ken Kadokawa cho chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-02-01

    For promoting the introduction of new energy and for elevating people's consciousness of such in Kadogawa Town, Miyazaki Prefecture, surveys and studies were conducted concerning the energy demand in the town, the amount of new energy resources in existence, and new energy introduction plans, and then a vision was formulated. The town's energy demand is estimated to be 1,197.4-million MJ/year, which includes 44.1% for the transportation sector, 22.9 for the residential sector, 22.7% for the industrial sector, and 10.3% for the people's livelihood-related business sector. The energy demand is covered 62.7% by oil-based fuels, 22.7% by electric power, and 13.4% by LP gas. Projects discussed for new energy introduction included the introduction of photovoltaic power into the town office, primary schools, and a comprehensive workers' welfare center; introduction of a solar hot water supply system and hybrid power-lighted street light into a seaside park; introduction wind power into Motoyama Park; mini-scale hydroelectric power on the Isuzu river; introduction of clean energy vehicles into the town office; power generation fueled by methane gas from livestock excrement; and introduction of natural gas fueled cogeneration into a resort house. (NEDO)

  8. Regional new energy vision formulation survey for Ochi Town. Report; 2001 nendo Ouchi cho chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-02-01

    For promoting the introduction of new energy and for elevating people's consciousness of such in Ochi Town, Saga Prefecture, surveys and studies were conducted involving the amount of energy needed by the town, the amount of new energy resources in existence, and new energy introduction programs, and a vision was formulated. The demand for energy of the town is estimated at 613,575 times 10{sup 9} J, with the industrial sector responsible for 47.7%, the transportation sector for 26.6%, and the residential and commercial sector for 25.7%. Electric power, heavy oil, gas oil, gasoline, and kerosene are demanded, named in the descending order in terms of magnitude of demand. Key projects were discussed for new energy introduction, which included the introduction of photovoltaic power into a culture exchange center; nighttime illumination of Mikaeri Waterfall by micro-scale hydroelectric power; installation of a water purification system for the Ikisa Dam using photovoltaic power and micro-scale hydroelectric power; introduction of photovoltaic power into a comprehensive sports park and the Ochi Junior High School; and large-scale agricultural production and processing plant operation using photovoltaic power. (NEDO)

  9. - From point, line to plane - . New energy vision at Osafune Town area; 2001 nendo Osafune cho chiiki shin energy vision (Ten sen soshite men he)

    NONE

    2002-02-01

    With an objective of accelerating introduction of and uplifting consciousness on new energies at Osafune Town in Okayama Prefecture, investigations and discussions were given on the energy consumption structure, status of new energies available, and the new energy introduction projects for the town, thus a new energy vision was established. The estimated amount of energy consumed in Osafune Town is 1,443,467 GJ/year. By departments, the industrial department accounts for 41%, the transportation department for 40%, the civil household department for 13% and the civil business department for 6%. Energy types are accounted for by petroleum at 81%, and electric power at 19%. The important new energy introduction projects discussed as the first step include 'Cooperative projects by residents and the administration', and 'Wide area introduction of new energies' into public facilities, as the second step, 'New energy networking' targeted at mutual coordination, 'Information network' for new energies and energy saving, and as the third step, 'Acceleration of proliferation at general levels' beginning with housing, and 'Creation of green energy experiencing park' at the Osafune Beautiful Forest. (NEDO)

  10. Regional new energy vision formulation project for Takarabe Town. Report; 2001 nendo Takarabe cho chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-02-01

    For promoting the introduction of new energy and for elevating people's consciousness of such in Takarabe Town, Kagoshima Prefecture, surveys and studies were conducted concerning the energy consumption profile of the town, the amount of new energy resources in existence, and new energy introduction plans, and then a vision was formulated. The town's energy consumption is estimated at 879.350-million MJ/year, with the transportation sector responsible for 46.6%, the industrial sector for 23.0%, the residential sector for 18.7%, and the people's livelihood-related business sector for 11.7%. Oil-based fuels occupy 70.4%, electric power 18.9%, and LP gas 8.4%. New energy introduction projects were studied, which included the introduction of photovoltaic power into the town office; photovoltaic power and hybrid power-lighted light for an oasis-on-the-road project and school facilities; introduction of cogeneration, wind power, and photovoltaic power into a hot spa health center and a water-friendly park; small-scale hydropower in the Okawara valley; wind power generation on Mount Shiraga and Jingaoka; introduction of clean energy vehicles for official use; and compost manufacturing and power generation using livestock waste. (NEDO)

  11. Regional new energy vision formulation project for Yobuko Town. Fiscal 2001 report; 2001 nendo Yobuko cho chiiki shin energy vision sakutei jigyo hokokusho

    NONE

    2002-02-01

    For promoting the introduction of new energy and for elevating people's consciousness of such at Yobuko Town, Saga Prefecture, surveys and studies were conducted involving the amount of energy needed by the town, the amount of new energy resources in existence, and new energy introduction programs, and a vision was formulated. The demand for energy of the town is estimated at 105,040 drums/year in terms of crude oil - 37% in electric power, 34% in fuel oil A, 13% in gas oil, and 8.1% in gasoline. As for the presence of new energy resources, it is deemed that wind and solar energies will be richly available. Studies were conducted for new energy introduction programs, which included the introduction of photovoltaic power into a nursing home for aged people, primary and junior high schools, and a water reservoir; street lamps lighted by a hybrid power system and a solar cell clock for the plaza in front of the harbor; introduction of wind power into the little islands of Ogawa and Kabe; installation of a solar energy collector for the nursing home for aged people; and the use of clean energy vehicles for official business. It is estimated that the efforts will bring about a 1,256.6 t-C reduction in carbon dioxide emissions. (NEDO)

  12. Report on new energy vision planning operation at Yachiyo Town; 2001 nendo Yachiyo cho chiiki shin energy vision sakutei gyomu hokokusho

    NONE

    2002-02-01

    With an object of accelerating introduction of and uplifting consciousness on new energies at Yachiyo Town in Hyogo Prefecture, investigations and discussions were given on the energy consumption of the town, quantity of new energies in existence, and the new energy introduction projects, thus a new energy vision was established. Yachiyo Town consumes energy annually at 99,966 Gcal. By departments, the transportation department accounts for 46%, the industrial department for 24%, the civil household department for 16%, and the civil business department for 14%. By energy types, petroleum accounts for 69%, and electric power for 31%. The new energy introduction projects that have been discussed include introduction of photovoltaic power generation into the town office, public halls, primary and middle schools, and photovoltaic and micro-wind power generation for street lights, photovoltaic and wind power generation in the Garten Yachiyo, a city and farm town culture exchange facility, micro-hydroelectric power generation in Noma River, bio-mass energy utilizing forest resources and fabric wastes, clean energy fueled automobiles as public use cars, school buses and welfare buses, and small wind power and large photovoltaic power generation at Mt. Noma. (NEDO)

  13. Survey for working out a new energy vision of the Tateshina Town area; 2001 nendo Tateshina machi shin energy vision sakutei chosa hokokusho. Chiiki shin energy vision sakutei tou jigyo

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and heightening the awareness of new energy in Tateshina Town, Kita-saku County, Nagano Prefecture, study was made of the existence amount of new energy, energy consumption amount in the town, project for the concrete introduction, etc., and a 'new energy vision of the area' was worked out. According to the sample survey, in the energy supply/demand structure of Tateshina Town, electricity was 32.2%, kerosene 23.9%, gasoline 17.1%, light oil 11.2%, heavy oil 10.3%, etc. By sector, the commercial/residential sector was 57.1%, transportation sector 25.9% and industrial sector 17.0%. The demand in FY 2010 is expected to increase by 8.9%-22.0% in FY 2010. For the project on new energy introduction, the following were studied: supply of new energy information, support of the introduction to town people, energy utilization of forest resource, preparation of an eco-museum in the Shin-jin'nai forest park, promotion of the tourism using new energy, study of energy utilization of stockbreeding waste, introduction to administrative facilities, etc. (NEDO)

  14. Survey report. New energy vision formulation project for Osa Town, Okayama Prefecture; 2001 nendo Osa cho shin energy vision sakutei tou jigyo chosa hokokusho. Chiiki shin energy vision sakutei tou jigyo

    NONE

    2002-02-01

    A New Energy Vision was formulated for Osa Town after investigations about energy demand, new energy reserves, and their available amounts. The town consumes approximately 50.6-billion kcal in total, with automobiles consuming a half thereof as fuel. Studies were made about new energy reserves and recoverable amounts in the town, and then an estimation was made that 16.2 times 10{sup 5} kWh/year would be recoverable from photovoltaic energy, 1.58 times 10{sup 5} kWh from small-scale hydropower, 8.79 times 10{sup 6} kWh from ligneous biomass, and 1.96 times 10{sup 5} kWh from livestock biomass, and that 40 MW, for example, would come from wind power, subject to change depending on the wind power generator system to be built. Projects for new energy introduction were discussed, which involved the use of biodiesel fuel for official vehicles, installation of solar hot water showers in primary and junior high schools, application of photovoltaic/wind power to crime prevention lights and street lamps, snow melting systems for roads utilizing geothermal energy, use of heat from thinnings and ligneous waste, wind power facilities, small-scale hydropower facilities, introduction of new energy appliances into sightseeing facilities, and the introduction of photovoltaic power or the like into public facilities. (NEDO)

  15. Shin Splints

    ... dancers and military recruits. Medically known as medial tibial stress syndrome, shin splints often occur in athletes who ... and might progress to a stress reaction or stress fracture. When to see a doctor Consult your doctor ...

  16. Protect and bring up forest of Kuse Town. New energy vision at Kuse Town area; 2001 nendo Kuse cho chiiki shin energy vision. Kuse no mori to machi wo mamori sodateru

    NONE

    2002-03-01

    With an objective of accelerating introduction of and uplifting consciousness on new energies at Kuse Town in Okayama Prefecture, investigations and discussions were given on the current situation of energy demand, quantity of new energies available, and the idea of introducing new energies, thus a new energy vision was established. The estimated amount of energy consumption in Kuse Town is 304,768 MWh/year, and the amount of carbon dioxide discharge associated therewith is 22,300 t-C. Regarding the energy consumption by departments, the industrial department accounts for 40%, followed by the transportation department accounting for 30%. The basic new energy introduction ideas have discussed town resident enlightenment and town resident participation projects, bio-mass energy utilization, such as bark combustion plant, procurement of wood resources remaining in forest, ligneous pellet and transportation fuel manufacture, utilization of edible oil into diesel fuel, introduction of photovoltaic power generation at public facilities and places of refuge, introduction of solar heat utilization system into public facilities and homes, large size wind power generation project, introduction of small size wind power generation into road illumination and places of refuge at disaster, small hydro-energy utilization system, and introduction of clean energy fueled automobiles and fuel cells. (NEDO)

  17. New energy vision of the Chuo town area. Creation of the town of good rapport and the environment; 2001 nendo Okayama ken Chuo cho chiiki shin energy vision. Fureai to junkan no machidukuri

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Chuo Town, Okayama Prefecture, an investigational study was conducted of the state of energy utilization of the town, project for new energy introduction, etc., and a new energy vision was worked out. The energy consumption amount in Chuo Town was estimated at 68,482 drums in terms of oil drum, and the amount of CO2 emitted from these was 13,287 t-CO2. To reduce this, the introduction of new energy and energy conservation were studied. As a result, the following were indicated as measures to be originally taken: introduction of solar light/solar heat use equipment to general households; installation of the regional cooperation power plant; each household's keeping of a household account book for the environment; introduction of energy-saving navigation device; activities for preservation of forest/terraced paddy field; ticket for exchange of forest volunteer work for products derived from the forest; campaign for composting of garbage using corrugated cardboard; recovery/recycle of waste cooking oil; full-of-flower gardening 500 movement; regionally consistent environmental education of parent/child participating type; introduction of micro hydraulic turbine; manufacture of products from thinnings by biomass lumber drying; expansion of special products of the area; method of the environmental management, etc. (NEDO)

  18. Report on the project for working out the new energy vision of the Shime town area; 2001 nendo Shime cho chiiki shin energy vision sakutei tou jigyo chosa hokokusho

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Shime Town, Fukuoka Prefecture, an investigational study was conducted of the state of energy consumption of the town, existence amount of new energy, project for new energy introduction, etc., and a vision was worked out. The rate of electric power demand of the town consisted of 44% of the residential sector such as general household, the highest, 30% of the business sector such as business office and 21% of the industrial sector such as plant. The demand from the industrial sector was on the decrease. The sales amount of domestic use gas almost leveled off. As the project for new energy introduction, the following were studied: introduction of photovoltaic power system to the Shime town office/townspeople center/elementary school/junior high school; introduction of solar heat utilization system to the welfare center for old people; introduction of wind power/photovoltaic power hybrid street light to school road/cycling road/playground; heat pump space heating/cooling using temperature different energy of the water of mine roadway; introduction of clean energy vehicle to the town office; introduction of photovoltaic power generation/solar heat utilization/hybrid car to general houses. (NEDO)

  19. Report on investigation of new energy vision at Hamanaka Town area. Investigation theme 'Investigation on commercialization of bio-gas facility at Hamana Town'; 2001 nendo Hamanaka cho chiiki shin energy vision sakutei tou chosa hokokusho. Chosa tema Hamanaka cho baiogasu shisetsu jigyoka chosa

    NONE

    2002-02-01

    In the 'new energy vision at Hamanaka Town area' planned in the previous fiscal year, investigations and discussions were given on commercialization of the 'bio-gas plant (joint type) utilizing livestock excreta, kitchen refuse, fishery-based wastes, dairy factory washing sludge, and sewage sludge'. The investigations were carried out for the joint type bio-gas plant on the local citizens' intention and participation, examples in Europe, examples in Japan, introduction of overseas examples of technological levels of the plant to be introduced, operating conditions of factories in Japan, and systems to be employed. In addition, discussions were given on economic evaluation on the business, and the nuclear organization of the business and operation, and effects of promoting the business. Issues in promoting the business included the fund supporting policies, assurance of operation rates in the severely cold season, maximization of electric power and thermal efficiencies, assurance of kitchen refuse resources, assurance of commission expense in the revenue aspect, assurance of major stockholders in the operating organization, assurance of the systems in the operation aspect, stocks of the new company to be possessed by Hamanaka Township residents, and acquisition of performance guarantee. (NEDO)

  20. Shin Splints 101: Explaining Shin Splints to Young Runners

    Newlin, Dana; Smith, Darla S.

    2011-01-01

    Shin splints are a common but often confusing injury. Sources disagree on both the cause of the injury and the anatomical source of the pain. Some blame shin splints on foot pronation, footstrike pattern, or arch height. Regardless of what causes the condition, it affects many runners, beginning in some at a young age. Young runners often have…

  1. Shin splints - self-care

    ... to make sure you do not have a stress fracture. You will also be checked to make sure you do not have ... Pain - shins - self-care; Anterior tibial pain - self-care; Medial tibial stress syndrome - self- ...

  2. Shin splints--a literature review.

    Bates, P

    1985-01-01

    "Shin splints" is not a specific diagnosis. It is merely a descriptive term that describes chronic exertional shin pain in an athlete. The evidence seems clear that shin splint pain has many different causes and this reflects the variation in the anatomy. It would be preferable to describe shin splint pain by location and aetiology, for example, lower medial tibial pain due to periostitis or upper lateral tibial pain due to elevated compartment pressure. This would aid communication between p...

  3. FY 2001 new energy vision of the Kin Town area. Initial stage survey. Town with plenty of heart, happiness, good living environment and vitality in harmony with nature; 2001 nendo Kin cho chiiki shin energy vision shoki dankai chosa. Shizen to no chowa ni yoru kokoro yutaka de akaruku sumiyoi katsuryoku aru machi

    NONE

    2002-02-01

    For working out a new energy vision in Kin Town, Okinawa Prefecture, study as an initial stage survey was conducted of the state of energy supply/demand of the town, existence amount of new energy, possibility of new energy introduction, etc. The energy consumption amount of Kim Town was estimated at 10.71 x 10{sup 10} kcal/y. By sector, it consisted of 5.10 x 10{sup 10} kcal in the transportation sector, 3.51 x 10{sup 10} kcal in the commercial/residential sector and 1.66 x 10{sup 10} kcal in the industrial sector. The rate of energy source was 55.3% of petroleum-base fuel, 40.6% of electric power and 4% of LP gas. The existence amount of new energy was estimated at 39,373 MWh, which is equivalent to 77.8% of the electric power consumption amount of the town. The existence amount of new energy is broken down into 31.4% of wind power generation, 30.3% of solar energy, 12.5% of photovoltaic power generation, 3.0% of waste utilization, etc. As to the program assuming the new energy introduction, a lot of themes were cited such as the introduction of photovoltaic power generation/wind power generation to around the hog raising complex, utilization of wind power/biomass energy in the stockbreeding industry and introduction of hybrid car to official vehicle. (NEDO)

  4. New energy vision of the Daito town area. Creation of a green energy town - Aiming at realizing the zero-emission society; 2001 nendo Daito cho chiiki shin energy vision. Green enenrgy town no sozo - Zero emisshon shakai no jitsugen wo mezashite

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Daito Town, Shimane Prefecture, an investigational study was conducted of the energy demand of the town, existence amount of new energy, project for new energy introduction, etc., and a new energy vision was worked out. The energy demand amount of the town was estimated at 141,437 x 10{sup 6} kcal, and the amount of CO2 emitted from this was 15,787 t-C. By sector, the energy demand amount consisted of 38% in the transportation sector, 37% in the commercial/residential sector and 26% in the industrial sector. As the project for new energy introduction, the following were studied: formation of the zone for learning new energy; introduction of solar system/thermoelectric supply system to the center for providing school lunch; introduction of thermoelectric supply system by stockbreeding biomass gas turbine; introduction of large wind power system; introduction of micro hydraulic power system/heat pump using waste hot water as heat source to tourist facilities/townspeople exchange facilities; introduction of photovoltaic power system/solar system/cogeneration system/solar car to public facilities; district thermoelectric supply using wood biomass. (NEDO)

  5. New energy vision of the Kumano town area. Making of an eco-town that was promoted by both residents and the administration; 2001 nendo Kumano cho chiiki shin energy vision. Jumin to gyosei ga ittai to natta ecotownka no suishin

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Kumano Town, Hiroshima Prefecture, an investigational study was conducted of the energy supply/demand of the town, existence amount of new energy, project for new energy introduction, etc., and a new energy vision was worked out. The energy consumption amount of the town in FY 2000 was estimated at 215,882 Gcal/y. The rate of energy source was 32.9% of electric power, 27.3% of gasoline, 12.4% of kerosene and 11.4% of light oil, etc. As the project for new energy introduction, the following were studied: introduction of photovoltaic power/micro wind power generation to park; introduction of solar energy use hot water supply system to health center; solar energy utilization at facilities of the heated swimming pool; introduction of photovoltaic power generation to library; utilization of the spring from JR tunnel (drinking water/small hydraulic power generation); small hydraulic power generation using the water over-flowing from the Sakamo-oike (pond); installation of the street light using photovoltaic power/micro wind power hybrid generation at shopping street; introduction of clean energy vehicle to official vehicle. (NEDO)

  6. Shin splints--a literature review.

    Bates, P

    1985-01-01

    "Shin splints" is not a specific diagnosis. It is merely a descriptive term that describes chronic exertional shin pain in an athlete. The evidence seems clear that shin splint pain has many different causes and this reflects the variation in the anatomy. It would be preferable to describe shin splint pain by location and aetiology, for example, lower medial tibial pain due to periostitis or upper lateral tibial pain due to elevated compartment pressure. This would aid communication between physicians and also direct therapy more accurately. Images p132-a PMID:3907743

  7. Report on new energy vision in Koushida Town area; Koushidacho chiiki shin energy vision hokokusho

    NONE

    2001-03-01

    A new energy vision was established in the Koushida Town area in Miyagi Prefecture. The population of Koushida Town has leveled off in recent years at 20,245 persons in fiscal 2000. However, number of people working outside the town is increasing, and the ratio of the daytime population is decreasing slowly. Agriculture in this town depends mainly on production rice of such brands as 'Hitomebore' and 'Sasanishiki', wherein the agricultural production amount is 3 billion 260 million yen in fiscal 1998. Commercial activities are decreasing its power with the annual retail sales amount of 17 billion 800 million yen, while the product shipping amount from the attracted factories is on the increasing trend, at 25 billion 700 million yen in 1998. The energy consumption is 526 times 10{sup 9} kcal in total, accounted for by the business department at 42.0%, the transportation department at 32.3%, and the consumer department at 25.7%. The annual utilizable quantity of new energies amounts to 28,816 times 10{sup 6} kcal, made available from solar power, wind power, bio-mass, and refuses, which is about 5.5% of the final energy consumption of the town. The new energy introducing project includes introduction of photovoltaic power generation into the town office building, facilities for interchange of citizens and schools, conversion of the automobiles used in the official organizations into clean energy fueled automobiles, the Eco-town Plan for the east side of Koushida Station, and the project of utilizing bio-mass supplied from the livestock market. (NEDO)

  8. New energy visions for the Town of Ichinohe district; Ichinohecho chiiki shin energy vision

    NONE

    2001-02-01

    The new energy visions are drawn for Town of Ichinohe, Iwate Pref. The town's population has been generally decreasing since 1975, from 21,433 to 17,906 in 1995. The town consumed energy totaling 288,691Gcal in 1998, of which the transportation sector accounts for 55%, followed by the domestic, commercial and industrial sectors, in this order, the industrial sector accounting for only 7%. The estimated new energy sources available to the town are wind power, power generated by temperature difference at rivers or using snow, solar energy, biomass energy and energy from wastes, in the order of quantity, totaling 137GWh/y as electrical power and 45.6Tcal/y as heat. The priority projects include making Ichinohe Primary School an eco-school, construction of a biomass demonstration plant in the Okunakayama district, introduction of a boiler firing wood chips at Okunakayama Heights, construction of a wind power mill at Takamori Heights, and introduction of new energies by the citizens. (NEDO)

  9. Report on new energy vision in Koushida Town area; Koushidacho chiiki shin energy vision hokokusho

    NONE

    2001-03-01

    A new energy vision was established in the Koushida Town area in Miyagi Prefecture. The population of Koushida Town has leveled off in recent years at 20,245 persons in fiscal 2000. However, number of people working outside the town is increasing, and the ratio of the daytime population is decreasing slowly. Agriculture in this town depends mainly on production rice of such brands as 'Hitomebore' and 'Sasanishiki', wherein the agricultural production amount is 3 billion 260 million yen in fiscal 1998. Commercial activities are decreasing its power with the annual retail sales amount of 17 billion 800 million yen, while the product shipping amount from the attracted factories is on the increasing trend, at 25 billion 700 million yen in 1998. The energy consumption is 526 times 10{sup 9} kcal in total, accounted for by the business department at 42.0%, the transportation department at 32.3%, and the consumer department at 25.7%. The annual utilizable quantity of new energies amounts to 28,816 times 10{sup 6} kcal, made available from solar power, wind power, bio-mass, and refuses, which is about 5.5% of the final energy consumption of the town. The new energy introducing project includes introduction of photovoltaic power generation into the town office building, facilities for interchange of citizens and schools, conversion of the automobiles used in the official organizations into clean energy fueled automobiles, the Eco-town Plan for the east side of Koushida Station, and the project of utilizing bio-mass supplied from the livestock market. (NEDO)

  10. New energy introduction guidebook for Chugoku area; Shin energy donyu guidebook. Chugoku chiiki

    NONE

    2001-03-05

    A guidebook was edited for the purpose of assisting introduction of new energies. Sections consist of Section 1, new energies available in the Chugoku area; Section 2, procedure for new energy introduction; Section 3, assistance institutions by energy; and Section 4, the status of new energy introduction in the Chugoku area, the future introduction targets, and referential materials. Section 1, upon defining the new energies, summarized features of photovoltaic power generation, solar heat utilization, wind power generation, wastes power generation, cogeneration, fuel cells, clean energy fueled automobiles, and introduction records up to the year 2000. Section 2 summarizes the general roles of local governments in the introduction, processing procedures for individual programs, related regulations, and summary of the assistance institutions. Section 3 puts into order the assistance institutions by energy. Section 4 summarizes precautions, targets and records in introduction by each new energy. The referential materials include a list of the Japanese assistance institutions, and subsidy institutions related to new energies for local governments in the Chugoku area. (NEDO)

  11. New energy vision in Daito Town area; Daitocho chiiki shin energy vision

    NONE

    2001-02-01

    A new energy vision was established in the Daito Township area in Iwate Prefecture. The energy consumed by Daito town as a whole in fiscal 1998 is about 213,685 two-hundred-kL drums, accounted for by the consumer department at 35.7%, the transportation department at 33.3%, and the industrial department at 31.0%. Carbon dioxide emission amount is estimated to be 109,647 t-CO2. General households in the town consume fossil fuel largely for room heating, whose energy conservation or substitution by new energies is important. The utilizable quantity of new energies in the town include solar energy, forest bio-mass, agricultural resource bio-mass, livestock excreta bio-mass, and refuses, whose total quantity is estimated to be 23,161 drums. The new energy utilization project includes enlightenment activities by introducing a photovoltaic power generator and a small wind power generator into Daito Middle School and the Murone Plateau Natural Energy Park, introduction of solar heat generated hot water supply devices for public facilities and town-operated houses, a discussion on a pellet production line in the wood processing industrial complex, and introduction of pellet boilers into public facilities. (NEDO)

  12. New energy vision of the Gotsu city area; 2001 nendo Gotsu shi chiiki shin energy vision

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Gotsu City, Shimane Prefecture, an investigational study was conducted of the energy consumption amount of the city, existence amount of new energy, project for new energy introduction, etc., and a new energy vision was worked out. The energy consumption amount of the city was estimated at 1,558,022 Gcal. By sector, the energy consumption amount consisted of 80.0% in the industrial sector, 11.2% of in the transportation sector, 5.8% in the residential sector and 3.0% in the business sector. In the industrial sector, the main energy consumption industries were the paper manufacturing industry and tile industry. As the project for new energy introduction, the following were studied: project for the wind farm where a lot of wind power generators are installed; park as a symbol having 1 or 2 wind power generators; introduction of solar energy use water warmer/photovoltaic power generation to public facilities; system for promotion of the introduction of solar energy use water warmer/photovoltaic power generation to households; project on CO2 reduction by installation of LNG station; refuse/waste power generation project; project on utilization of the waste heat from tile makers. (NEDO)

  13. New energy vision in Gamagori City area (Aichi Prefecture); Gamagorishi chiiki shin energy vision

    NONE

    2001-03-01

    With an objective of early realization of a district society harmonized with the natural environment, a vision was established as a guideline to achieve systematic and effective introduction of new energies, with cooperation from citizens, business entities, and the administration. The activity achievements were put into order by the following six items: 1) movements in introducing new energy systems, 2) identification of the characteristics of the district and major relevant measures, 3) identification of the current status and characteristics of the energy demand structure in the district, 4) estimation of existence and utilizable quantity of new energies, 5) needs of introduction of new energies in the area, and 6) introduction possibilities, realization measures, and proliferation acceleration measures. Item 6 is composed of considerations given from the result of the initial stage investigation, basic ideas, targets of the introduction, introduction policies, and promotion of energy countermeasures. Plans were made on a solar heat hot water supply system, a photovoltaic power generation system, a wind power generation system, and introduction of clean energy fueled automobiles. Annual supply of 221,790 GJ was estimated. The major components are the automobiles, solar heat hot water system, and power generation systems. (NEDO)

  14. New energy vision of Oshimizu Town; 2001 nendo Oshimizu machi chiiki shin energy vision

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Oshimizu Town, Ishikawa Prefecture, an investigational study was conducted of the energy consumption amount of the city, existence amount of new energy and project for new energy introduction, and a vision was worked out. The energy consumption amount of the city was 3.02 x 10{sup 11} kcal, out of which electric power was 1.53 x 10{sup 11} kcal, petroleum 1.33 x 10{sup 11} kcal, and LPG 1.59 x 10{sup 10} kcal. As the project for new energy introduction, the following were studied: introduction of photovoltaic power generation to the general center/Oshimizu Junior High School/general household; solar energy utilization for the general center/general household; introduction of wind power generation to the Azuma Line of forest road; biomass power generation project using cattle feces/hog feces; RDF project for waste from Oshimizu Town; introduction of clean energy vehicle (hybrid car/electric car). The amount of new energy introduction from these was estimated at 1,716kL in crude oil conversion, which is equal to 5.3% of the energy demand amount. (NEDO)

  15. New energy vision of the Kosaka town area; 2001 nendo Kosaka machi chiiki shin energy vision

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Kosaka Town, Akita Prefecture, an investigational study was conducted of the energy demand of the town, existence amount of new energy, project on new energy introduction, etc., and a new energy vision was worked out. The amount of energy demand of the town was 2,442,464 GJ/y. The energy demand consists of C heavy oil (about 1/3) and electric power (about 1/3) in the industrial sector, which are outstanding, and others including kerosene (about 12%) in the commercial/residential sector and light oil (about 10%) and gasoline (about 7%) in the transportation sector. The CO2 emissions from these are 60,000 t-C/y. As the project on the new energy introduction, study was made on the introduction of solar energy/snow cold energy space cooling to the insurance welfare center/interchange center, introduction of the photovoltaic power generation to the Korakukan area/elementary school/junior high school, introduction of the snow melting system using soil heat to town street/sidewalk, use of clean energy vehicle to public vehicle, use of ligneous biomass energy, etc. (NEDO)

  16. New energy vision in Kitakata City area; Kitakatashi chiiki shin energy vision

    NONE

    2001-02-01

    A new energy vision was established in Kitakata City in Fukushima Prefecture. The population of Kitakata City in 2000 was 37,490, not having experienced extreme increase or decrease. Population employed in the tertiary industry occupies the largest ratio at 36.9%, followed by the secondary industry. The energy demand of the entire city is about 3300 trillion Joule, and carbon dioxide emitted therefrom is estimated about 77,000 t-C annually. Calculation of new energy amount in existence reveals about 4190 trillion Joule annually, which corresponds to about 1.3 times the energy demand quantity of the city. The new energy introducing project includes introduction of photovoltaic power generation into the public hall and schools, utilization of solar heat for greenhouses, introduction of solar heat and wind power hybrid power generation into the Green Tourism experiencing facilities, parks, street lights, and evacuation places, introduction of small to medium hydropower generation plants into Kitanogo, Kuranoyu and Happo-Ike, utilization of cold heat of snow, introduction of clean energy fueled automobiles into the public organizations, utilization of bio-mass energy, wastes burning power generation and heat utilization. (NEDO)

  17. Regional new energy vision for Sakurae town; Sakuraecho chiiki shin energy vision

    NONE

    2001-02-01

    The town is situated in a mountainous region typical of the Iwami district, Shimane Prefecture, rich in natural energy resources with as many as four hydroelectric power stations in service. Such local features were taken into account fully when a regional new energy vision was formulated for showing the town the course to follow for promoting the introduction of new energy which is environmentally friendly. The results of related efforts are described in eight chapters, which are (1) the outline of the vision, (2) survey of the trend of new energy, (3) description of Sakurae town, (4) townspeople's awareness of energy matters, (5) rate of energy consumption, (6) available amount of new energy, (7) basic policy for introducing new energy, and (8) projects for introducing new energy. Part (8) covers a photovoltaic project, wind power project, ligneous biomass energy introduction project, and the introduction of clean energy vehicles. Under the ligneous biomass energy introduction project, business profitability is discussed of a scrap wood fueled power plant. (NEDO)

  18. New energy vision in Iwaizumi Township area; 2000 nendo Iwaizumicho chiiki shin energy vision

    NONE

    2001-02-01

    A new energy vision was established in the Iwaizumi Township area in Iwate Prefecture. The heat energy consumed by each household in Iwaizumi Town is 20.5 two-hundred-liter drums, while transportation (railways, buses, trucks and private cars) consumes 12.2 drums annually, and the electric energy field 4.7 drums annually. As the town as a whole, the ratio of utilization as heat energy is relatively high. Therefore, new energy introduction should be focused importantly on use of solar heat hot water supply device and high-level utilization of forestry fuels. The new energy introduction project includes introduction of bio-mass energy to produce heat energy and electric power using livestock excreta, cut woods, thinned woods, and kitchen refuses as the resources, structuring a new energy model for the 'Communication Land Iwaizumi', activation of the central urban area by introducing micro hydropower generation, assistance to proliferation of new energies to town-operated houses, households, and business entities, introduction of new energies into the disaster prevention center, schools, and insurance, medical and welfare facilities, introduction of clean energy automobiles, and uplift of consciousness of town people. (NEDO)

  19. New energy vision of the Yokohama town area; 2001 nendo Yokohama machi chiiki shin energy vision

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Yokohama Town, Aomori Prefecture, an investigational study was conducted of the structure of energy demand of the town, existence amount of new energy, introduction of new energy, etc., and a new energy vision was worked out. The population of Yokohama Town was 5,508 as of October 2000, which is slightly decreasing. The number of household was unchanged, though. The amount of energy demand was estimated at 21,368 kL/y in crude oil conversion. The energy demand consisted of petroleum-base energy such as kerosene, light oil, heavy oil and gasoline (about 70%), electric power (20%) and LPG (10%). In the action plan for new energy introduction, the following were studied: wind power generation of which the plan has already been worked on by a private company (15 x 1,750kW class generator), introduction of the photovoltaic power generation to Yokohama Junior High School, hothouse vegetable cultivation using exhaust heat from hot spring, and 'Yokohama Rape Blossoms Eco Project' that mainly includes the agricultural land protection/oil squeezing from rapeseed/recycle of waste cooking oil as fuel, for which rape blossoms as one of the most important resources in Yokohama Town are used. (NEDO)

  20. Regional new energy vision for Fukuoka city; Fukuokashi chiiki shin energy vision

    NONE

    2001-02-01

    A regional new energy vision has been formulated for Fukuoka city, Fukuoka Prefecture, which will serve as a basic guide to the promotion of new energy introduction which is one of the measures for coping with environmental problems. The vision is described in five chapters of (1) the local characteristics of Fukuoka city, (2) energy situation in Fukuoka city, (3) basic program, (4) high priority plans, and (5) efforts to enhance the vision toward realization. Discussed as high priority plans are the introduction of new energy into public buildings above all others, high efficiency utilization of refuse-derived energy, utilization of new energy at water supply and drainage facilities, utilization of water temperature difference energy, introduction of clean energy vehicles, promotion of a new energy industry, and the extension of assistance for encouraging citizens and industrialists to adopt new energy. In extending assistance for encouraging the introduction of new energy, studies are made about measures for introducing new energy into individual residences and about a citizenry-involving new energy adopting industry (offering a site to the power generation industry) under the basal conditions that information be supplied sufficiently and that the citizenry and administration cooperate with each other in driving forward the vision. (NEDO)

  1. New energy vision of the Ehime prefecture area; 2001 nendo Ehime ken chiiki shin energy vision

    NONE

    2002-03-01

    For the contribution to environmental preservation and stable supply of energy, 'a new energy vision of the Ehime prefecture area' was worked out which is to be a guide for new energy introduction using photovoltaic power, wind power, etc. In the Uma area, the following were proposed: introduction of waste energy cogeneration to the paper manufacturing industry; study of introduction of small wind turbine to household and business establishment. In the Niihama/Saijo area, introduction of cogeneration to business establishment; creation of the processing/assembly industry for materials/parts of equipment of photovoltaic power generation/wind power generation. In the Imabari area, introduction of natural gas cogeneration to business establishment, creation of the industry for equipment/parts of photovoltaic power generation. In the Matsuyama area, introduction of cogeneration to business establishment; utilization of biomass energy; creation of the industry of production/processing of resin for solar module use. In the Yawatahama/Oozu area, utilization of wind energy for agriculture/forestry, fisheries and tourism fields; study of introduction of biomass energy. In the Uwajima area, utilization of wind power energy for agriculture/fisheries, fisheries and tourism fields; introduction of biomass energy. (NEDO)

  2. New energy vision of the Noogata city area; 2001 nendo Noogata shi chiiki shin energy vision

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Noogata City, Fukuoka Prefecture, an investigational study was conducted of the energy demand amount of the city, existence amount of new energy, project for new energy introduction, etc., and a vision was worked out. The energy consumption amount of the city was estimated at 4,825.4 x 10{sup 6} MJ/y. It consisted of 47.1% in the industrial sector, 26.1% in the commercial/residential sector and 24.9% in the transportation sector. The rate of energy source was 65.7% of petroleum-base energy and 25.1% of electric power. As the project for new energy introduction, the following were studied: introduction of wind power generation/photovoltaic power generation to the flower park at the foot of Mt. Fukuchi and Nakanoshima park on the sandbank of the Onga river; introduction of photovoltaic power generation to library. Moreover, as future models of the introduction, the potential study was made on the following: installation of the stockbreeding waste biogas plant at the compost center; installation of fuel cell system using digestion gas from night soil treatment facilities; installation of the natural gas cogeneration system in the project on redevelopment of the urban area, etc. (NEDO)

  3. Regional new energy vision for Hita City; 2001 nendo Hita shi chiiki shin energy vision

    NONE

    2002-02-01

    For promoting the introduction of new energy and for elevating people's consciousness of such in Hita City, Oita Prefecture, surveys and studies were conducted concerning the energy supply/demand trend in the city, the amount of new energy resources in existence, and new energy introduction plans, and a vision was formulated. The city's energy consumption is estimated at 16,216,376 GJ, with the industrial sector responsible for 78.23%, the transportation sector for 11.35%, and the residential and commercial sector for 9.73%. It is in the public sector, general households, forestry, and hog raising that new energy will be utilized. Key projects for new energy introduction were discussed, which covered a residence incorporating a combination of new energies such as solar heat for water heating and photovoltaic power; lumber drying facilities utilizing forestry generated biomass; cogeneration fueled by methane gas from livestock excrements and garbage; photovoltaic power for schools, the city office, etc.; use of photovoltaic power, temperature difference energy, and methane gas at purification centers, etc.; use of clean energy vehicles for official use; etc. (NEDO)

  4. New energy vision of the Akita city area; 2001 nendo Akita shi chiiki shin energy vision

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Akita City, an investigational study was conducted of the energy demand of the town, existence amount of new energy, project on new energy introduction, etc., and a new energy vision was worked out. The population of Akita City was 317,868 as of December 31, 2000, which is continuously on the increase. The amount of energy demand of the whole city was 990,000 kL/y. The energy demand consists of fossil fuels such as petroleum, coal and propane gas (77.7%) and electric power (22.3%). The CO2 emissions from these are 2,908,000 t-CO2. As the project on the new energy introduction, study was made on the installation of the photovoltaic power system and wind power system at base centers and introduction of the waste power system at the Goshono Works. As the model project, study was made on the introduction of the photovoltaic power generation and wind power generation to street light and public facilities, road snow melting/space heating and cooling using soil heat, introduction of clean energy vehicles, BDF introduction using waste cooking oil and rate blossoms, etc. (NEDO)

  5. Report on regional new energy vision for Kahoku town; Kahokucho chiiki shin energy vision hokokusho

    NONE

    2001-03-01

    A regional new energy vision has been formulated for Kahoku town, Kochi Prefecture, which features the introduction of recoverable energy harmonious with nature and friendly to human life and environments. The results of activities conducted in this connection are compiled into nine chapters, which cover (1) Kahoku-town's local characteristics relative to new energy, (2) survey of townspeople's awareness of new energy, (3) amounts of new energy in existence, (4) basic policy toward introduction, (5) study of concrete measures for promoting introduction, (6) study of prioritized projects, (7) establishment of target amounts for introduction, (8) coordination with energy conservation measures, and (9) a system for thrusting forward the regional new energy vision toward embodiment. Chapter (7) comprises the way of setting target values for introduction; study of the target values to be achieved by the industry, administration, and citizenry, respectively; and a conclusion on the target values. Studies are conducted in the chapter about the introduction of solar energy, wind power generation, small scale hydroelectric power generation, cogeneration, clean energy vehicles, and the like. It is predicted that 960 kl/year will be introduced in fiscal 2010 in terms of oil, which accounts for 12% of energy consumption. (NEDO)

  6. New energy vision in Tochigi Prefectural area; Tochigiken chiiki shin energy vision

    NONE

    2001-03-01

    With an objective to structure a circulation type society that imposes less load on the environment, a new energy vision was established in the Tochigi Prefectural area. This plan is an overall plan to promote introduction of new energies, and at the same time serves as a guideline when prefectural residents, business entities and the administration attempt to introduce new energies. The plan was prepared upon viewing the year 2010. The new energy introduction quantity in 2010 was estimated to be 470,000 kL as converted into crude oil (4.7% of the consumption in fiscal 1997). The plan document covers the following five fields: 1) the basic conception of the vision establishment, 2) the current status and problems surrounding the energies, 3) the basic directionality in introducing new energies, 4) policy systems and role sharing toward introducing new energies, and 5) case studies and case presentation. Item 4 is composed of policy systems for new energy introduction, projects to be implemented with emphasis, roles of working organizations, and institutions for promotion. The projects to be executed with emphasis consist of five items including initiative introduction into facilities utilized by the prefectural residents. (NEDO)

  7. New energy visions for the Town of Ichinohe district; Ichinohecho chiiki shin energy vision

    NONE

    2001-02-01

    The new energy visions are drawn for Town of Ichinohe, Iwate Pref. The town's population has been generally decreasing since 1975, from 21,433 to 17,906 in 1995. The town consumed energy totaling 288,691Gcal in 1998, of which the transportation sector accounts for 55%, followed by the domestic, commercial and industrial sectors, in this order, the industrial sector accounting for only 7%. The estimated new energy sources available to the town are wind power, power generated by temperature difference at rivers or using snow, solar energy, biomass energy and energy from wastes, in the order of quantity, totaling 137GWh/y as electrical power and 45.6Tcal/y as heat. The priority projects include making Ichinohe Primary School an eco-school, construction of a biomass demonstration plant in the Okunakayama district, introduction of a boiler firing wood chips at Okunakayama Heights, construction of a wind power mill at Takamori Heights, and introduction of new energies by the citizens. (NEDO)

  8. Report on new energy vision in Fudai Village area; Fudaimura chiiki shin energy vision hokokusho

    NONE

    2001-03-01

    A new energy vision was established in the Fudai Village area in Iwate Prefecture. The population of Fudai Village is decreasing from the peak of 4,103 persons in 1985. Its basic industry is fishery and agricultural industries, with the fish and marine product raising industry particularly active. The energy consumption in Fudai Village is, in the decreasing order, the transportation field (48.5%), the social and household field (26.5%), social industry, and manufacturing industry. Homes consume room heating energy at a greater extent. Energies are supplied by fuel oil at 53,554 Gcal (79.3%) and electric power at 11,551 Gcal (17.1%). The new energy introducing project has discussed introduction of cogeneration into the Kurosaki House, introduction of photovoltaic power generation and solar heat utilization systems, and introduction wind power generation as a tourism symbol. Discussions were also given on conversion of the automobiles used in the official organizations into clean energy automobiles. Further discussions were given on introduction of small new energy facilities, such as hybrid illumination, small wind power generation, and photovoltaic power generation, into the village office, the station square, the fish market, facilities in the fishing port, the children's hall, and the Road Station. (NEDO)

  9. Regional new energy vision for Yakumo village; Yakumomura chiiki shin energy vision

    NONE

    2001-02-01

    A regional new energy vision has been formulated for Yakumo village, Shimane Prefecture, which abides with the village doctrines of introducing new energy for quality life and building a community advanced in terms of environmental protection. Yakumo village, which has a population of approximately 6,800 engaged mainly in agriculture or forestry, consumed 5 times 10{sup 5} GJ in energy in fiscal 1998. The results of activities conducted in this connection are summarized in nine chapters which are (1) the trends of affairs surrounding new energy, (2) Yakumo village's local characteristics as seen from the new energy viewpoint, (3) collection of information on new energy measures taken by local governments, (4) estimation of demand for energy in Yakumo village, (5) estimation of the existing amount and available amount of new energy in Yakumo village, (6) study of the basic concept and basic policy of the vision, (7) study of new energy introduction, (8) approach to the issue by thinking, and (9) reference materials. In concrete terms, discussion is held about introduction into public facilities of photovoltaic power generation and water heaters, wind power generation, cogeneration, and waste heat utilization. For the sake of gardening at public facilities, for instance, the introduction of photovoltaic power generation is taken up. (NEDO)

  10. Regional new energy vision for Aogaki town; Aogakicho chiiki shin energy vision

    NONE

    2001-02-01

    A regional new energy vision has been formulated as part of Phase 4 of the Town Development Program of Aogaki town, Hyogo Prefecture, calling for 'building a town, comfortable to live in and full of hope, by utilizing rich natural environments.' The results of activities conducted in this connection are summarized in five chapters, which are (1) the description of the new energy vision, (2) Aogaki-town's energy characteristics, (3) basic plan, (4) driving forward of prioritized plans, and (5) toward the driving forward of the new energy vision. Chapter (4) comprises the goal of the formulation of the prioritized plans, the definition of the prioritized plans, and the establishment of individual projects. Established as the individual projects are the introduction of new energy linked to the inhibition of carbon dioxide gas emissions, use of wind energy for multiple purposes, public facilities preceding others in the introduction of new energy, building of environmentally symbiotic residences utilizing new energy, utilization of new energy at recreation facilities, and the popularization of and enlightening about new energy and its introduction through the participation of the local population. (NEDO)

  11. New energy vision in Mobara City area (Chiba Prefecture); Mobarashi chiiki shin energy vision

    NONE

    2001-02-01

    With the saying {sup M}obara, you find good when you live in it' as the target, the 'new energy vision for Mobara City area' was established. The vision is intended to utilize natural gas existing around the city, and utilize energy saving and new energy technologies. Specifically, the vision was established by a discussion committee composed of people of experience and academic standing, related business entities, and organizations, who took into consideration the natural environment, status of energy existence, and economic activities. The activity achievements were put into order by the following seven items: 1) the situation surrounding new energies, 2) district characteristics and viewpoints of introducing new energies, 3) energy demand, 4) existence quantity of new energies, 5) actual status of new energy introduction, and result of hearing investigations, 6) directionality and conception of introducing new energies, and 7) specific project objectives related to new energy introduction. Five projects were set up, including introduction of natural gas fueled automobiles, natural gas utilizing cogeneration and fuel cells, and effective utilization of wastes and bio-mass. (NEDO)

  12. New energy vision in Shioya Town area (Tochigi Prefecture); Shioyacho chiiki shin energy vision

    NONE

    2001-02-01

    A new energy vision in Shioya Town area was established with objectives to make clear what is to be tackled by the administration and the residents in their respective standings, and elevate their consciousness to the environmental preservation and consciousness regarding the energy problems. The target for new energy introduction quantity in fiscal 2010 was set as CO2 emission quantity reduction to about 3,900 t-c/year, a reduction of 5% of the quantity in fiscal 1999. The activity achievements were put into order by the following seven items: 1) summary of implementing investigations, 2) basic district characteristics, 3) the current status and estimation of energy consumption, 4) estimated calculation on possibility of new energy utilization, 5) discussions and problems on possibilities of the introduction as seen from the district characteristics, 6) the new energy vision, and 7) the future assignments. Item 6 is composed of the introduction target, measures for introduction to achieve the target in fiscal 2010, and development of the leading projects. The projects include small to middle size hydropower generation, photovoltaic and wind power hybrid type power generation, and introduction of clean energy fueled automobiles. (NEDO)

  13. New energy vision in Murone Village area; 2000 nendo Muronemura chiiki shin energy vision

    NONE

    2001-02-01

    A new energy vision was established in the Murone Village area in Iwate Prefecture. The population of the village as of 1995 was 6,552 persons, which has continued with slow decrease in the last decade, revealing remarkable departure from agriculture. Energy consumption in this village per person is 11.5 two-hundred-liter drums, and carbon dioxide discharge per person is 5.6t-CO2. This is about 60% of the national average, because of no existence of thermal power plants and factories. The existing quantity of new energies is occupied by bio-mass as the largest because more than 70% of the village area is forest, followed by solar beam and heat. However, in the case of forest, since the energy is actually utilized only from the felled woods, the utilizable quantity is considerably small, whereas the availability of solar beam and heat is higher. With regard to introduction of new energies, the efforts of the introduction thereof are directed to introduction into primary schools, the village office, the special nursing home for elderly people, and the meal providing center for middle schools, in addition to proliferation and enlightenment of household solar systems, and photovoltaic power generation systems. Furthermore, discussions were given on introduction of a pellet burning power plant utilizing waste woods. (NEDO)

  14. Regional new energy vision for Joetsu City; 2001 nendo Joetsu shi chiiki shin energy vision

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Joetsu City, Niigata Prefecture, surveys and studies were conducted involving energy consumption of the city, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The city consumes 3,593 times 10{sup 9} kcal in energy, with the industrial sector responsible for 47.5%, the transportation sector 28.1%, and the residential/commercial sector 24.3%. As for the energy type, oil based fuel accounts for 58.3%, electric power 26.8%, and town gas 8.6%. The amount of carbon dioxide emissions due to energy consumption is estimated at 1,160 kt-CO2. Studies conducted for new energy introduction covered the supply of photovoltaic power to a project on municipal housing construction and a project on promoting the buildup of environment preservation facilities; the installation of a photovoltaic power generation system for a Citizens' Forest buildup project; the installation of a wind power generation system at the Minowadai Square of Recreation and Relaxation for the citizens; the installation of eco-stations to deal with gasoline, propane gas, and natural gas; the extension of financial support for the purchase of low-pollution vehicles; and so forth. (NEDO)

  15. Regional new energy vision for Tadami Town; 2001 nendo Tadami machi chiiki shin energy vision

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Tadami Town, Fukushima Prefecture, surveys and studies were conducted involving the energy demand characteristics of the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. Though there is not a great change through the year in town's demand for power, more power is necessary in winter for heating and lighting. As for demand for gas and oil, demand for heating oil reaches its peak in winter. New energy introduction projects were discussed, which would utilize snow for the dynamization of industries, and studies were conducted about the selection of items to be stored in snow rooms, selection of proper cold heat utilization systems, care to be taken in the designing of facilities, installation of snow rooms, development of specialty goods proper to the town, and so forth. Eco-school development was also studied as an environmental education project harmonizing with the local circumstances. Discussed for the fostering of welfare were an energy business for a Welfare House, snow handling measures linked to new energy utilization systems, and the like. (NEDO)

  16. Bone scanning in Shin Splint evaluation

    Dumont, M.; Lamoureux, F.; Lamoureux, J.; Danais, S.; Lacoste, P.; Duranceau, J.

    1983-01-01

    Jogging is increasingly popular; in U.S.A., in 1979 more than 25 millions people were considered joggers. Lesions of the locomotive system of the lower limbs are very frequent in athletes and the Shin Splint syndrome, for one, is very frequent. However this precise clinical entity, usually X-Ray negative, is ill-known. A bone scan study in a series of 30 athletes suffering from Shin Splints is presented. The bone scans being positive and typical were highly useful in confirming the clinical diagnosis. Moreover, follow-up studies were done in a number of these patients and results correlated well with the clinical evolution [fr

  17. The effectiveness of shin guards used by football players.

    Tatar, Yasar; Ramazanoglu, Nusret; Camliguney, Asiye Filiz; Saygi, Evrim Karadag; Cotuk, Hasan Birol

    2014-01-01

    In football, injuries from opponent contact occur commonly in the lower extremities. FIFA the world's governing body for football requires players to wear shin guards. The aim of this study was to compare the protective effectiveness of polypropylene based shin guards with custom-made carbon fiber ones. Three commercial polypropylene shin guards (Adidas Predator™, Adidas UCL™, and Nike Mercurial™) and two custom-made carbon fiber shin guards were examined. The experimental setup had the following parts: 1) A pendulum attached a load cell at the tip (CAS Corp., Korea) and a fixed prosthetic foot equipped with a cleat to simulate an attacker's foot. 2) An artificial tibia prepared by condensed foam and reinforced by carbon fibers protected with soft clothing. 3) A multifunctional sensor system (Tekscan Corp., F-Socket System, Turkey) to record the impact on the tibia. In the low impact force trials, only 2.79-9.63 % of the load was transmitted to the sensors. When comparing for mean force, peak force and impulse, both carbon fiber shin guards performed better than the commercial ones (Adidas Predator™, Adidas UCL™, and Nike Mercurial™) (p = 0.000). Based on these same parameters, the Nike Mercurial™ provided better protection than the Adidas Predator™ and the Adidas UCL™ (p = 0.000). In the high impact force trials, only 5.16-10.90 % of the load was transmitted to the sensors. For peak force and impulse, the carbon fiber shin guards provided better protection than all the others. Carbon fiber shin guards possess protective qualities superior to those of commercial polypropylene shin guards. Key PointsShin guards decrease the risk of serious injuries.Carbon shin guards provide sufficient protection against high impact forces.Commercially available Polypropylene based shin guards do not provide sufficient protection against high impact forces.

  18. New energy vision of the Tono City area; 2001 nendo Tonoshi shin energy vision. Chiiki shin energy vision sakutei tou jigyo

    NONE

    2002-02-01

    As a leading plan to reconsider the resource consumption in large quantities and to change the society to a circulation type, a new energy vision of the Tono City area was worked out in Tono City, Iwate Prefecture. In this vision, FY 2002 was set as the first fiscal year, and FY 2010 as the target year. From the comprehensive judgement based on the objective availability, technical evaluation, civic needs, etc., biomass energy, wind energy, solar energy, etc. were made the main energy in new energy introduction. As model projects, studied were the introduction of solar energy utilization facilities to Kamigo Elementary School, introduction of the photovoltaic power generation to Aozasa Nursery School and disaster prevention base facilities, project on the wind power generation using large wind turbines, biomass alcohol plant, wood pellet production, etc. The target amount of new energy introduction was 2,640 kW in photovoltaic power generation, 19,000 kW in wind power generation, etc. The CO2 reduction amount including that in heat utilization, clean energy car, etc. was 41,374 t-CO2/y. (NEDO)

  19. Regional new energy vision formulated for Murakami City; 2001 nendo Murakami shi chiiki shin energy vision sakutei tou jigyo. Murakami shi shin energy puran

    NONE

    2002-03-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Murakami City, Niigata Prefecture, surveys and studies were conducted involving energy consumption in the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The annual energy consumption of the city is estimated at 78,000 kL in terms of oil. The residential/commercial sector consumes 24,000 kL, the industrial sector 28,000 kL, and the transportation sector 26,000 kL, with the three sectors demanding similar amounts. The carbon dioxide emitted by the sectors is estimated at 223,000 t-CO2/year. New energy introduction projects were discussed, which included hot water supply, heating, road heating, and power generation, all these utilizing heat from the Senami hot spa; biogas fueled power generation and BDF (bio-diesel) bus operation in the Senami hot spa district; the introduction of a hybrid wind power/photovoltaic power generation system into Ogata Seaside Park; the introduction of a mini-scale hydroelectric power generation system into the Kamikaifu district; a large-scale wind power generation business at the Iwafune district; the introduction of wind power and photovoltaic power generation facilities into primary and junior high schools; the use of clean energy vehicles for official use; and so forth. (NEDO)

  20. Tohoku Town developing with new energy. New energy vision of the Tohoku town area; 2001 nendo Tohoku machi chiiki shin energy vision. Shin energy de hiraku Tohoku machi

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Tohoku Town, Aomori Prefecture, an investigational study was conducted of the potential utilization of new energy, etc., and a vision was worked out. The population of Tohoku Town was 10,662 according to the results of the national census taken in 2000, which is slightly decreasing. The energy consumption amount of Tohoku Town was estimated at 280,977 Gcal/y, and the consumption of fossil fuel in the commercial/residential sector stands out. This is because a lot of kerosene is consumed in the area where it is cold. As measures to be taken for the new energy vision, the following were cited: initiative introduction of new energy to public facilities/preparation of a system toward the promotion of introduction, supply of thermal energy to townspeople/creation of the living environment using new energy, new energy use to activities of agricultural production/energy use of stockbreeding resource/energy use of forestry resource/potential study of the wind power generation, spread/education for townspeople/proprietors, preparation of places of bodily sensation/exchanges of new energy, training/support of activities of townspeople themselves, etc. (NEDO)

  1. The Effectiveness of Shin Guards Used by Football Players

    Yasar Tatar

    2013-03-01

    Full Text Available In football, injuries from opponent contact occur commonly in the lower extremities. FIFA the world’s governing body for football requires players to wear shin guards. The aim of this study was to compare the protective effectiveness of polypropylene based shin guards with custom-made carbon fiber ones. Three commercial polypropylene shin guards (Adidas Predator™, Adidas UCL™, and Nike Mercurial™ and two custom-made carbon fiber shin guards were examined. The experimental setup had the following parts: 1 A pendulum attached a load cell at the tip (CAS Corp., Korea and a fixed prosthetic foot equipped with a cleat to simulate an attacker’s foot. 2 An artificial tibia prepared by condensed foam and reinforced by carbon fibers protected with soft clothing. 3 A multifunctional sensor system (Tekscan Corp., F-Socket System, Turkey to record the impact on the tibia. In the low impact force trials, only 2.79-9.63 % of the load was transmitted to the sensors. When comparing for mean force, peak force and impulse, both carbon fiber shin guards performed better than the commercial ones (Adidas Predator™, Adidas UCL™, and Nike Mercurial™ (p = 0.000. Based on these same parameters, the Nike Mercurial™ provided better protection than the Adidas Predator™ and the Adidas UCL™ (p = 0.000. In the high impact force trials, only 5.16-10.90 % of the load was transmitted to the sensors. For peak force and impulse, the carbon fiber shin guards provided better protection than all the others. Carbon fiber shin guards possess protective qualities superior to those of commercial polypropylene shin guards.

  2. Report on new energy vision establishment in Oga City area; Ogashi chiiki shin energy vision sakutei hokokusho

    NONE

    2001-02-01

    A new energy vision was established in Oga City in Akita Prefecture. The population of the city is continuing the decreasing trend since 1955 to 30,462 persons as of 2000. The number of employed people by industry is in the trend of decreasing in the primary industry, leveling off in the second industry, and increasing in the tertiary industry. The annual energy demand in this city is 838,000 MWh in electric power and 302 times 10{sup 13} J, accounted for by the consumer department at about 44.4%, the transportation department at about 28.3% and the industrial department at 27.3%. The carbon dioxide emission amount therefrom is estimated 68,100 t-C. The expected usable quantity of new energies in the city would be available in solar heat as the largest source, followed by solar beam and wind power, whose amount is estimated 136.41 times 10{sup 13}J annually in total. The new energy introducing project includes introduction of wind power generation into Namahage Line and its vicinity and the Ikari area, manufacture of fuel from refuses, introduction of clean energy fueled automobiles into the cars used by the city office, introduction of photovoltaic into the general gymnasium and primary and middle schools, and solar heat utilization at the general gymnasium. (NEDO)

  3. New energy vision of the Gushikami Village area; 2001 nendo Gushikami son chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Gushikami Village, Okinawa Prefecture, an investigational study was conducted of the energy demand of the village, existence amount of new energy, plan for new energy introduction, etc., and a vision was worked out. The energy demand of Gushikami Village was estimated at 640.2 x 10{sup 6} MJ/y. By sector, it consisted of 37.1% in the transportation sector, 32.0% in the commercial/residential sector and 30.8% in the industrial sector. The rate of energy source was 73.7% of petroleum-base fuel and 26.3% of electric power. As the plan for new energy introduction, the following were studied: introduction of photovoltaic power generation to the village office/museum/elementary school/junior high school, introduction of solar heat utilization facilities to the meal providing center/gymnasium, introduction of wind power generation to the sports park, project of wind power generation on the sugar cane hill (600kW x 6 turbines), cogeneration by composting of stockbreeding waste and methane, introduction of clean energy car to official vehicle, introduction of natural gas vehicle, installation of experimental plant for conversion of sugar cane to methanol, etc. (NEDO)

  4. Fiscal 1999 edition. Guidebook for introducing new energies in Kinki District; Kinki chiiki shin energy donyu guide book

    NONE

    2000-09-01

    The captioned guidebook is composed of the following subjects: (1) the current status and problems of new energies (the energy status and the current status of new energies in Japan, and approaches to new energies in Japan); (2) the status of introduction and works in Kinki District (the outline of introduction and works in Kinki District on photovoltaic electric power generation, solar heat utilization, wind power generation, wastes power generation, clean energy fueled automobiles, cogeneration, fuel cells, unused energies, and other reproducible energies); (3) NEDO related supportive institutions (new energy introduction promotion projects by NEDO, and the outline of different supportive institutions); (4) new energy introduction flow (total flow leading to the introduction, and introduction flow by new energies); (5) the grand new energy prize in Kinki District (what is the grand new energy prize? and cases of prizes awarded in Kinki District); (6) new energy related supportive institutions (a list of the related institutions, and the outline of different supportive institutions); and (7) new energy consultation windows and contacts (consultation windows and contacts for general new energy aspects and the government related supportive institutions, and consultation windows by new energies). The grand new energy prize is awarded by New Energy Foundation as the 'institution for public recognition of new energy devices of the 21st century type'. (NEDO)

  5. Report on a regional new energy vision establishment in Yatsuka Township; Yatsukamachi chiiki shin energy vision sakutei jigyo hokokusho

    NONE

    1999-03-01

    Surveys and discussions were given on establishing a new energy vision in Yatsuka Township in Shimane Prefecture. For the survey on available amount of natural energies (sunlight and wind), all-weather insolation required for solar energy power generation was analyzed by using the NEDO information, and wind was analyzed based on observations at Matsue Meteorological Observatory, Sakai-minato Observatory, and Nakaura Water Gate. In discussing the system, power generation amount in the Matsue area was estimated for the case when solar cells of single crystal silicon plus amorphous hybrid type HIP-H48B1 are used. For wind power generation, power generation amount was calculated from observation values derived at the Nakaura water gate when wind mills with diameters from 2 to 10 m are used. Furthermore, calculations were performed on power amount generated uniformly by using a hybrid system using solar energy power generation and wind power generation. As a result of the discussions, a plan was established to introduce the new energies by means of a solar and wind energy hybrid power generation system to the greenhouse 'Green Stellar' symbolizing the Japan's largest peony production, being capable of year-round peony cultivation. The system is planned to be introduced to the town office building and other public organizations sequentially according to annual programs. (NEDO)

  6. Survey report on establishing a regional new energy vision in Gohoku Village; Gohokuson chiiki shin energy vision sakutei chosa hokokusho

    NONE

    1999-03-01

    A regional new energy vision was established in Gohoku Village in Kochi Prefecture. Significance of this vision as a guideline for new energy introduction lies in preservation of natural environment, development promotion of the district, and building of a village strong against disasters. According to the discussion on energy availability in Gohoku Village, the expected usable amount will be about 5.13 million kWh/year by using solar energy power generation, about 2.25 billion kcal/year by utilizing solar heat, about 42 million kWh/year by utilizing wind energy, about 1.07 million kWh/year by using a small hydroelectric power plant, and about 31 million kcal/year by using wastes energy. The important new energy introduction projects are planned for introducing solar energy power generation, solar heat utilization, wind power generation, and small hydroelectric power generation in the area of 'Green Park Hodono' to make it a new energy model park (an ecology park), introducing solar energy power generation and solar heat utilization in the health and welfare center, day-service center, and special aged nursing home, and introducing clean energy fueled automobiles in official use. (NEDO)

  7. Fiscal 1999 edition. Guidebook for introducing new energies in Kinki District; Kinki chiiki shin energy donyu guide book

    NONE

    2000-09-01

    The captioned guidebook is composed of the following subjects: (1) the current status and problems of new energies (the energy status and the current status of new energies in Japan, and approaches to new energies in Japan); (2) the status of introduction and works in Kinki District (the outline of introduction and works in Kinki District on photovoltaic electric power generation, solar heat utilization, wind power generation, wastes power generation, clean energy fueled automobiles, cogeneration, fuel cells, unused energies, and other reproducible energies); (3) NEDO related supportive institutions (new energy introduction promotion projects by NEDO, and the outline of different supportive institutions); (4) new energy introduction flow (total flow leading to the introduction, and introduction flow by new energies); (5) the grand new energy prize in Kinki District (what is the grand new energy prize? and cases of prizes awarded in Kinki District); (6) new energy related supportive institutions (a list of the related institutions, and the outline of different supportive institutions); and (7) new energy consultation windows and contacts (consultation windows and contacts for general new energy aspects and the government related supportive institutions, and consultation windows by new energies). The grand new energy prize is awarded by New Energy Foundation as the 'institution for public recognition of new energy devices of the 21st century type'. (NEDO)

  8. Basic survey report on regional new energy vision for Ehime Prefecture; Ehimeken chiiki shin energy vision kiso chosa hokokusho

    NONE

    2001-03-01

    A new energy vision has been formulated to establish guidelines for popularizing and utilizing new energy which generates but a small amount of environmental impact. The results of surveys conducted in this connection are described in five chapters, which cover (1) the trend of new energy, (2) current situation of new energy in Ehime Prefecture, (3) calculation of the existing amount and available amount of new energy, (4) calculation of the introducible amount of new energy, and (5) the basic policy toward the introduction of new energy. Part (4) covers solar energy, wind energy, waste-derived energy, temperature difference energy, minor scale hydroelectric power generation, biomass energy, and like others. It states that a total of 540,000 kl/year will be available in terms of oil, equivalent to approximately 6% of Ehime Prefecture's 1997 energy consumption, and that it is expected that in fiscal 2010 photovoltaic/passive solar energy utilization, cogeneration, and wind power will yield 152,000, 314,000, and 27,000 kl/year respectively. (NEDO)

  9. Regional new energy vision formulation for Nichinan City. Fiscal 2001 report; 2001 nendo Nichinan shi chiiki shin energy vision sakuteisho

    NONE

    2002-03-01

    For promoting the introduction of new energy and for elevating people's consciousness of such in Nichinan City, Miyazaki Prefecture, surveys and studies were conducted concerning the energy demand of the city, the amount of new energy resources in existence, and new energy introduction plans, and then a vision was formulated. The city's energy demand is estimated to be approximately 4,028-million MJ/year, which includes 46% for the transportation sector, 30% for the people's livelihood-related business sector, 17% for the residential sector, and 7% for the industrial sector. The energy demand is covered 62% by oil-based fuels and 30% by electric power. Projects discussed for new energy introduction included the introduction of photovoltaic power into a lifelong learning center, a comprehensive center for health and welfare, and primary schools; introduction of solar hot water supply systems into school meal providing facilities; introduction of medium-size wind power systems (five systems); medium- or small-scale hydroelectric power generation on the Sakatani river; use of street light lighted by photovoltaic/wind hybrid power systems; introduction of clean energy vehicles; power generation utilizing livestock excrement; introduction of photovoltaic, hydroelectric, and methane gas power generation into a sewage treatment plant; and the use of RPF (refuse paper and plastic fuel) at the Nichinan plant of Oji Paper Co., Ltd. (NEDO)

  10. FY 2001 Report of regional new energy vision for Futaba Town; 2001 nendo Futaba machi chiiki shin energy vision hokokusho

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Futaba Town, Fukushima Prefecture, surveys and studies were conducted involving the amount of energy consumed by the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The annual energy consumption of the town is 113,049 times 10{sup 6} kcal, with automobiles consuming 47.2%, manufacturing industries 19.8%, households 18.9%, and commerce 12.6%. The amount of carbon dioxide due to the consumption is estimated at 9,999 times 10{sup 3} kg-C in total. Studies were made about new energy introduction projects toward 2010, which involved the introduction of photovoltaic power generation systems and solar heat utilization systems into four public facilities and 214 general residences; introduction of nine clean energy vehicles into public facilities and 181 into general residences; a wind power station near the seashore; a mini-scale hydroelectric power system for irrigation; introduction of new energy for illuminating sightseeing roads and parks, and so forth. (NEDO)

  11. Investigation report on establishment of new energy vision in Toyohashi area; Toyohashi chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2001-02-01

    The 'new energy vision for Toyohashi (Aichi Prefecture) area' was established from the viewpoint that proliferation of new energy use, which is less familiar and higher in cost, requires induction by urban and environmental measures, and consistency with district characteristics. The achievements were put into order as the report on the Toyohashi area new energy vision and the investigation on the vision establishment. The vision is composed by the following four items: 1) basic matters concerning the vision, 2) ideas and policies of the vision, 3) major measures for introducing new energies, and 4) promotion of the vision. Item 3) presents the introduction measures, introduction project, and measure development by areas. Considerations on the introduction measures include enlightenment for proliferation, expansion in information provision, development of preferential treatments, initiative introduction into public facilities, and the measures to strengthen cooperative works by citizens, business entities, and the administration. The following four projects were set: introduction of wastes power generation into the Resource Production Center (wastes treatment plant), improvement of the surplus heat utilization facilities, and introduction of wind power generation into the General Agricultural Park. (NEDO)

  12. Investigation report on establishment of new energy vision in Toyohashi area; Toyohashi chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2001-02-01

    The 'new energy vision for Toyohashi (Aichi Prefecture) area' was established from the viewpoint that proliferation of new energy use, which is less familiar and higher in cost, requires induction by urban and environmental measures, and consistency with district characteristics. The achievements were put into order as the report on the Toyohashi area new energy vision and the investigation on the vision establishment. The vision is composed by the following four items: 1) basic matters concerning the vision, 2) ideas and policies of the vision, 3) major measures for introducing new energies, and 4) promotion of the vision. Item 3) presents the introduction measures, introduction project, and measure development by areas. Considerations on the introduction measures include enlightenment for proliferation, expansion in information provision, development of preferential treatments, initiative introduction into public facilities, and the measures to strengthen cooperative works by citizens, business entities, and the administration. The following four projects were set: introduction of wastes power generation into the Resource Production Center (wastes treatment plant), improvement of the surplus heat utilization facilities, and introduction of wind power generation into the General Agricultural Park. (NEDO)

  13. Fiscal 2000 report on regional new energy vision for Tanoura town; 2000 nendo Tauracho chiiki shin energy vision hokokusho

    NONE

    2001-03-01

    A new energy vision has been formulated to make an environmentally friendly community of Tanoura town, Kumamoto Prefecture, which is a 6,000-people town facing Shiranui Bay and consisting of seashore, the streets, and a mountainous district. The amount of new energy in existence, when converted into electric power, will be 10 GWh/year accounting for approximately 20% of 1999 power consumption. The types of new energy subjected to study are solar energy, wind energy, biomass energy, refuse-derived energy, and geothermal energy, with solar energy surpassing the rest. In the new energy vision, the roles of the administration, citizens, and industrialists are made obvious; new energy introduction projects are established for the introduction of photovoltaic power, solar heat, and wind power into Hot Spring Center; and clean energy vehicles will be acquired to serve as official vehicles. The new energy vision booklet comprises six chapters, with chapter 1 dealing with the basic philosophy of vision formulation. (NEDO)

  14. Plan to promote new energy introduction in Niigata Prefecture area; Niigataken chiiki shin energy donyu suishin keikaku

    NONE

    2001-03-01

    The 'Plan to promote new energy introduction in Niigata Prefecture area' was established to leave the conditions that everybody can live with affluence and comfort in the next generation. The plan lasts for ten years until 2010. Upon identifying the district characteristics of Niigata Prefecture, and based on the results of investigations on new energy existence quantity, utilization possibility thereof, and consciousness of residents of the prefecture, considerations were given that the plan shows the basic policy to promote proliferation of the new energies, and serves as the guideline for practical implementation. The plan document is composed of the following four items: 1) the foreword, 2) the current status of energies, 3) new energies expected of introduction, and 4) basic measures. The energy consumption was estimated to increase to 1.345 times that of fiscal 1990 in the year 2010, the increase being mainly in consumer and household use. The targeted quantity for new energy introduction was set to 90,000 kl annually as converted to petroleum. Expected new energy applications would include photovoltaic power generation, snow energy and solar heat utilization, and cogeneration utilizing natural gas. (NEDO)

  15. CHO Quasispecies—Implications for Manufacturing Processes

    Florian M. Wurm

    2013-10-01

    Full Text Available Chinese hamster ovary (CHO cells are a source of multi-ton quantities of protein pharmaceuticals. They are, however, immortalized cells, characterized by a high degree of genetic and phenotypic diversity. As is known for any biological system, this diversity is enhanced by selective forces when laboratories (no sharing of gene pools grow cells under (diverse conditions that are practical and useful. CHO cells have been used in culture for more than 50 years, and various lines of cells are available and have been used in manufacturing. This article tries to represent, in a cursory way, the history of CHO cells, particularly the origin and subsequent fate of key cell lines. It is proposed that the name CHO represents many different cell types, based on their inherent genetic diversity and their dynamic rate of genetic change. The continuing remodeling of genomic structure in clonal or non-clonal cell populations, particularly due to the non-standardized culture conditions in hundreds of different labs renders CHO cells a typical case for “quasispecies”. This term was coined for families of related (genomic sequences exposed to high mutation rate environments where a large fraction of offspring is expected to carry one or more mutations. The implications of the quasispecies concept for CHO cells used in protein manufacturing processes are significant. CHO genomics/transcriptomics may provide only limited insights when done on one or two “old” and poorly characterized CHO strains. In contrast, screening of clonal cell lines, derived from a well-defined starting material, possibly within a given academic or industrial environment, may reveal a more narrow diversity of phenotypes with respect to physiological/metabolic activities and, thus, allow more precise and reliable predictions of the potential of a clone for high-yielding manufacturing processes.

  16. Investigation of shinning Spot Defect on Hot-Dip Galvanized Steel Sheets

    Yonggang, Liu; Lei, Cui

    2014-01-01

    Shinning spot defects on galvanized steel sheets were studied by optical microscope, scanning electron microscope(SEM), Energy Dispersive Spectrometer (EDS) and Laser-Induced Breakdown Spectroscopy Original Position Statistic Distribution Analysis (LIBSOPA) in this study. The research shows that the coating thickness of shinning spot defects which caused by the substrate defect is much lower than normal area, and when skin passed, the shinning spot defect area can not touch with skin pass roll which result in the surface of shinning spot is flat while normal area is rough. The different coating morphologies have different effects on the reflection of light, which cause the shinning spot defects more brighter than normal area

  17. Bone lesions from overload: shin splint and stress fracture

    Una Gorospe; Jon Andoni; Isla Gallego, Concepcion; Santana Borbones, Aranzazu; Perera Romero, Carmen; Allende Riera, Ana J

    2005-01-01

    There are many stress injuries in the lower extremities due to exercise, and the case we present is an example of two injuries which may present in children or young adults who train excessively. The patient complains of pain and tightness on exercise. The underlying pathology is probably rupture of insertion fibres of the tibial and soleus (Sharpey's fibres) muscles. Probably, there is also periostitis and myositis. Stress fractures and shin splints are often present at the same time in different stages, and both are typical pathologies due to excessive training. Scintigraphy allows identification and early management of shin splints and stress fractures (au)

  18. Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics

    Kumar, Amit; Baycin-Hizal, Deniz; Wolozny, Daniel

    2015-01-01

    -SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO-CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential...

  19. Tibial Stress Injuries: Decisive Diagnosis and Treatment of "Shin Splints."

    Couture, Christopher J.; Karlson, Kristine A.

    2002-01-01

    Tibial stress injuries, commonly called shin splints, often result when bone remodeling processes adopt inadequately to repetitive stress. Physicians who are caring for athletic patients must have a thorough understanding of this continuum of injuries, including medial tibial stress syndrome and tibial stress fractures, because there are…

  20. Misonidazole-glutathione conjugates in CHO cells

    Varghese, A.J.; Whitmore, G.F.

    1984-01-01

    Misonidazole, after reduction to the hydroxylamine derivative, reacts with glutathione (GSH) under physiological conditions. The reaction product has been identified as a mixture of two isomeric conjugates. When water soluble extracts of CHO cells exposed to misonidazole under hypoxic conditions are subjected to HPLC analysis, misonidazole derivatives, having the same chromatographic properties as the GSH-MISO conjugates, were detected. When CHO cells were incubated with misonidazole in the presence of added GSH, a substantial increase in the amount of the conjugate was detected. When extracts of CHO cells exposed to misonidazole under hypoxia were subsequently exposed to GSH, an increased formation of the conjugate was observed. A rearrangement product of the hydroxylamine derivative of misonidazole is postulated as the reactive intermediate responsible for the formation of the conjugate

  1. Mechanisms of oxygen radiosensitization in CHO cells

    Whillans, D.W.

    1981-01-01

    A model is presented for repair and fixation pathways when CHO cells are irradiated in the presence of O 2 . This analysis predicts that an increase in the repair path such as has been postulated for addition of a radioprotective sulfhydryl should increase OER/sub max/ in porportion to k prime, the new repair rate constant and also increase K with k prime. Any radiosensitizer which mimics the action of O 2 simply increases k prime 2 , so that the OER/sub max/ decreases at 1/k prime 2 but K increases as k prime 2 . These predictions have been tested in mammalian CHO cells making use of a Clark-type oxygen probe with defined conditions to ensure that O 2 is not depleted by radiation or cellular consumption, and so O 2 levels are known with accuracy. In a complementary study, the technique of rapid-mixing was used to measure the rate of development of O 2 sensitization in these same cells. By a variation of this rapid-mixing approach, the rate of diffusion into these cells has also been measured independently. Neither the dependence of OER on O 2 concentration nor the development of radiosensitivity with time of incubation in O 2 gives evidence in CHO cells for two components of sensitization indicative of two sites or two mechanisms of action, as seen in some V79 sublines. 13 references, 4 figures

  2. Features of Computerized Procedure System of Shin-Kori unit 5 and 6

    Seong, Nokyu; Jung, Yeonsub; Sung, Chanho

    2016-01-01

    The Computerized Procedure System (CPS) is one of the Man Machine Interface (MMI) resources of Main Control Room (MCR) of the Advanced Power Reactor 1400 (APR1400). The CPS has been continuously improved since it was installed in Shin-Kori unit 3 and 4. The Korea Hydro Nuclear Power Central Research Institute (KHNP CRI) has found the points of improvement of CPS through CPS centered Human Factors Engineering Verification and Validation (HFE V and V) and Operating Experience Review (OER) of reference power plant. This paper shows the main features of CPS of Shin-Kori 5 and 6 unit. This paper shows the main features of CPS of Shin-Kori 5 and 6. These are some of improvements of CPS. This prototype of CPS currently is implementing in CRI. The respective function can be more detailed after testing the prototype. These features will be applied to Shin-Kori 5 and 6 CPS after HFE V and V

  3. Features of Computerized Procedure System of Shin-Kori unit 5 and 6

    Seong, Nokyu; Jung, Yeonsub; Sung, Chanho [KHNP CRI, Daejeon (Korea, Republic of)

    2016-10-15

    The Computerized Procedure System (CPS) is one of the Man Machine Interface (MMI) resources of Main Control Room (MCR) of the Advanced Power Reactor 1400 (APR1400). The CPS has been continuously improved since it was installed in Shin-Kori unit 3 and 4. The Korea Hydro Nuclear Power Central Research Institute (KHNP CRI) has found the points of improvement of CPS through CPS centered Human Factors Engineering Verification and Validation (HFE V and V) and Operating Experience Review (OER) of reference power plant. This paper shows the main features of CPS of Shin-Kori 5 and 6 unit. This paper shows the main features of CPS of Shin-Kori 5 and 6. These are some of improvements of CPS. This prototype of CPS currently is implementing in CRI. The respective function can be more detailed after testing the prototype. These features will be applied to Shin-Kori 5 and 6 CPS after HFE V and V.

  4. Foot posture in basketball players with history of the shin splint

    Saeed Forghany

    2013-07-01

    Full Text Available Introduction: Shin splint is one of the common injuries in most athletes. Although the relationship between abnormal foot posture and shin splint has been reported previously but, the relation between foot posture and shin splint has not been well documented. The aim of this study was to explore the relationship between foot postures in basketball players and the history of shin splint. Materials and Methods: Thirty Iranian male basketball players who had experience of shin splint during last three months were participated in this study. Foot Posture Index (FPI-6 was used as the measure of foot posture. Talar head palpation, curvature at the lateral malleoli, inversion/eversion of the calcaneus, prominence in the region of the talonavicular joint, congruence of the medial longitudinal arch, abduction /adduction of the forefoot on the rear foot were 6 items which were assessed with FPI in the standing position. Foot posture was defined as ‘normal’, ‘supinated’ or ‘pronated. Data were collected and analyzed by SPSS, version16. Results: Most participants showed abnormal foot posture (%80. Fifty-three percent of subjects had pronated foot (%53 and 10% did hyper-pronation. The foot postures of 17% of participants were in supination. The results of this study did not show a significant difference in foot posture between right and left foot (P > 0.05. Conclusion: abnormal foot posture were common (%80 in basketball players with the history of shin splints. These findings could support this idea that the footwear and orthotic prescription both can prevent and treat basketball players with history of shin splints. Keywords: Foot posture, Shin splints, Basketball

  5. Notes on breeding sharp-shinned hawks and cooper’s hawks in Barnwell County, South Carolina

    Mark Vukovich; John C. Kilgo

    2009-01-01

    Breeding records of Accipiter striatus (Sharp-shinned Hawks) in the southeastern US are scattered and isolated. We documented a Sharp-shinned Hawk and Accipiter cooperii (Cooper’s Hawk) nest while conducting a telemetry study on Melanerpes erythrocephalus (Red-headed Woodpeckers) in Barnwell County, SC in 2006 and 2007. We report the first known nest of a Sharp-shinned...

  6. The Products of the Thermal Decomposition of CH3CHO

    Vasiliou, AnGayle; Piech, Krzysztof M.; Zhang, Xu; Nimlos, Mark R.; Ahmed, Musahid; Golan, Amir; Kostko, Oleg; Osborn, David L.; Daily, John W.; Stanton, John F.; Ellison, G. Barney

    2011-04-06

    We have used a heated 2 cm x 1 mm SiC microtubular (mu tubular) reactor to decompose acetaldehyde: CH3CHO + DELTA --> products. Thermal decomposition is followed at pressures of 75 - 150 Torr and at temperatures up to 1700 K, conditions that correspond to residence times of roughly 50 - 100 mu sec in the mu tubular reactor. The acetaldehyde decomposition products are identified by two independent techniques: VUV photoionization mass spectroscopy (PIMS) and infrared (IR) absorption spectroscopy after isolation in a cryogenic matrix. Besides CH3CHO, we have studied three isotopologues, CH3CDO, CD3CHO, and CD3CDO. We have identified the thermal decomposition products CH3(PIMS), CO (IR, PIMS), H (PIMS), H2 (PIMS), CH2CO (IR, PIMS), CH2=CHOH (IR, PIMS), H2O (IR, PIMS), and HC=CH (IR, PIMS). Plausible evidence has been found to support the idea that there are at least three different thermal decomposition pathways for CH3CHO: Radical decomposition: CH3CHO + DELTA --> CH3 + [HCO] --> CH3 + H + CO Elimination: CH3CHO + DELTA --> H2 + CH2=C=O. Isomerization/elimination: CH3CHO + DELTA --> [CH2=CH-OH] --> HC=CH + H2O. Both PIMS and IR spectroscopy show compelling evidence for the participation of vinylidene, CH2=C:, as an intermediate in the decomposition of vinyl alchohol: CH2=CH-OH + DELTA --> [CH2=C:] + H2O --> HC=CH + H2O.

  7. Museum of water, green and the sun - Marumori. New energy vision of the Marumori Town area; 2001 nendo Marumori machi shin energy vision hokokusho. Chiiki shin energy vision sakutei tou jigyo (Mizu to midori nto taiyo no hakubutsukan Marumori)

    NONE

    2002-03-01

    In Marumori Town, Miyagi Prefecture, a 'new energy vision of the area' was worked out which indicated the assessment of the energy reserved in the town and the course of new energy to be determined in the future. The energy consumption amount of Marumori Town in FY 1999 was estimated at 47,535 kL in crude oil conversion. By sector, the energy consumption amount was the largest in the order of the industrial sector, the transportation sector and the commercial/residential sector. The availability of new energy in Maruyama Town was estimated at approximately 480,585 GJ, of which the solar energy utilization was the largest, 53.1%. The photovoltaic power generation was the next largest, 25.4%, followed by the temperature difference energy (underground water), 8.9%, clean energy car, 7.8%, etc. As a plan for the first introduction of new energy, the following were studied: introduction of photovoltaic power generation facilities to the Marumori Town Office, introduction of new energy to public facilities around the Fudoson Park, introduction of wind power generation facilities to the town-run pasture, construction of the Marumori-type biogas plant, construction of a mini hydroelectric power plant, introduction of clean energy car to public vehicles, introduction of new energy facilities to the area of Kawa-no-Eki (station of the river), etc. (NEDO)

  8. Fiscal 2001 survey report on regional new energy vision formulation at Kyonan Town; 2001 nendo Kyonan machi shin energy vision sakutei chosa hokokusho. Chiiki shin energy vision sakutei tou jigyo (Watashitachi ga tsukuru machi, subete ga koen no machi)

    NONE

    2002-03-01

    A regional new energy vision was formulated at Kyonan Town, Chiba Prefecture, which includes a new energy introduction plan, an action program for the plan, and the like, intended to promote enlightenment/popularization relative to new energy and environmental matters. Kyonan Town consumes 18.69 times 10{sup 10} kcal of energy, with oil accounting for 65.4%, LPG for 6.0%, and electricity 28.6%, the statistics disclosing that the town has an oil-dependent energy consumption structure. The new energy likely to be introduced will comprise 13,574 times 10{sup 10} kcal/year in solar energy, 336,187 MWh/year in wind power, 17,170 kWh/year in small-scale hydroelectric power, 0.57 times 10{sup 10} kcal/year in refuse fueled power, and 0.60 times 10{sup 10} kcal/year in biomass fueled power. The town's new energy introduction project centers on photovoltaics, passive solar heat utilization, wind power, small-scale hydroelectric power, and aims to introduce the new energies into public facilities such as the Eco Garden and Katsuyama Primary School, and into kindergartens, nursery schools, shopping malls, and so forth. A plan was also worked out for adopting clean-energy vehicles for official use. (NEDO)

  9. New energy vision for Nomura town. Fiscal 2000 survey report on regional new energy vision formulation project for Nomura town, Ehime Prefecture; Nomuracho shin energy vision. 2000 nendo Nomuracho chiiki shin energy vision sakutei nado jigyo chosa hokokusho

    NONE

    2001-02-01

    An effort is being exerted to build a town which will generate but a little environmental impact, with symbiosis with nature established as the highest priority target of the town administrative activities. Under the Nomura town new energy vision, a basic policy and measures are established toward the promotion of new energy introduction, with local features, existence of new energy resources, and the feasibility of new energy adoption taken into consideration. Surveys were made and the results are compiled into seven chapters, which involve (1) the trend of new energy, (2) local features, (3) existing amount and available amount of new energy, (4) basic policy toward the introduction of new energy, (5) main projects for the introduction of new energy, (6) details of the basic policy toward the introduction of new energy, and (7) the Nomura Town Deliberation Committee on New Energy Vision Formulation. Chapter (6) covers the assessment of new energy introduction feasibility, promotion of concretized introduction projects, public relations/enlightening/popularizing activities, and assistance for new energy introduction. Studied for introduction are photovoltaic power generation, solar heat utilization, wind power generation, biomass power generation, and the like. (NEDO)

  10. New energy vision of the Yokaichi city area. New energy society to be created by all citizen - Yokaichi City; 2001 nendo Youkaichi shi chiiki shin energy vision. Minna no chikara de tsukuru shin energy shakai Youkaichi

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Yokaichi City, Shiga Prefecture, an investigational study was conducted of the energy demand amount of the city, existence amount of new energy, project for new energy introduction, etc., and a new energy vision was worked out. The energy consumption amount of the city was estimated at approximately 1,004,638 drums in terms of oil drum (200L). By sector, the energy consumption amount was 66.1% in the industrial sector, 17.4% in the transportation sector and 16.5% in the commercial/residential sector. The rate of energy source was 62.2% of fossil fuel, etc., and 37.9% of electric power. As the project for new energy introduction, the following were studied: promotion of introduction of solar energy collector/photovoltaic power generation to general households; introduction of photovoltaic power generation/solar hot water supply space heating system to public facilities; road preparation by installation of street light using photovoltaic power generation; installation of micro wind power generator at the disaster-prevention base; introduction of biomass energy such as wood pellet stove to public facilities; project of rape blossoms (cultivation of rape/production and consumption of rape oil/recovery of waste cooking oil/BDF). (NEDO)

  11. Organo-Zintl-based superatoms: [Ge9(CHO)3] and [Ge9(CHO)

    Reddy, G. Naaresh; Jena, Puru; Giri, Santanab

    2017-10-01

    A systematic study, based on density functional theory and different hybrid functionals for exchange-correlation potential, shows that the electron affinities of organo-zintl clusters [Ge9(R)n] [R = CHO; n = 1, 3] are close to that of chlorine (3.6 eV) and iodine (3.0 eV). A detailed study of the molecular orbitals of these complexes, when compared to those of Al13-, Cl- and I-, confirm that they behave as superatoms, mimicking the chemistry of halogens. This study expands the scope of superatoms by including a new class of pseudo-halogens based on ligated organo-Zintl ions.

  12. Regional new energy vision formulation survey for Makurazaki City. Fiscal 2001 report; 2001 nendo Makurazaki shi chiiki shin energy vision sakutei tou chosa hokokusho

    NONE

    2002-02-01

    For promoting the introduction of new energy and for elevating people's consciousness of such in Makurazaki City, Kagoshima Prefecture, surveys and studies were conducted concerning the energy demand of the city, the amount of new energy resources in existence, and new energy introduction plans, and then a vision was formulated. The city's energy demand in fiscal 1999 is estimated at approximately 3,206 TJ, with the industrial sector demanding 1,820 TJ, the residential and commercial sector demanding 701 TJ, and the transportation sector demanding 684 TJ. Energy consuming industries in this prefecture are fisheries industry and food industry. Pilot projects for new energy introduction were discussed, which included the introduction of photovoltaic power into the city hall, primary and junior high schools, a school meal providing center, and a city-managed hospital; installation of hybrid power-lighted light at the city hall, along the Kedo river, and in the Hinokami park; introduction of a solar heat utilization system into a nursing home for elderly people; introduction of wind power into education facilities; conversion of waste of cooking oil into diesel fuel; introduction of clean energy vehicles into public service; and new energy projects to be contracted to the private sector. (NEDO)

  13. Report on the project on working out the new energy vision of Hisai City; 2001 nendo Hisai shi chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Hisai City, Mie Prefecture, an investigational study was conducted of the energy demand structure, existence amount of new energy and project for new energy introduction, and a vision was worked out. The energy consumption amount of the city was 21,500 lorries in the industrial sector, 11,200 lorries in the transportation sector, 2,700 lorries in the business sector and 2,400 lorries in the residential sector in terms of tank-lorry loaded oil. As features, the following were cited: growth in the transportation sector, mostly in automobiles, was great; growth in the electric power consumption in the residential sector was great. As the project for new energy introduction, the following were supposed and studied in terms of the economical efficiency and environmental acceptability: introduction of cogeneration to hospital; introduction of ligneous biomass power generation to hospital; substitution of wood burning boiler for A heavy oil boiler for hot water supply use; introduction of photovoltaic power generation for home use and business use; introduction of solar heat use hot water supply device. (NEDO)

  14. Report on the survey for working out the new energy vision of the Urugi village area; 2001 nendo Urugi mura chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it at Urugi Village, Nagano Prefecture, an investigational study was conducted of characteristics of the natural environment/characteristics of social economy of the village, existence amount of new energy, project for new energy introduction, etc., and a vision was worked out. Urugi Village is 800 meters above sea level and in the riches of the Nature being surrounded by mountains on all sides. The population of the village was 734 as of March 1993, which is decreasing by 14-15/y. The aging rate was very high, 32.3%. As the project for new energy introduction, the following were studied: 10kW class photovoltaic power generation facilities to be installed at Furusato-kan and by the road of Hiraya Pass, 2.5kW class wind power generation facilities to be installed at Hiraya Pass for education of new energy, installation of a 10kW class mini hydroelectric power plant supplying power to the auto-camping site, stockbreeding biomass cogeneration which conducts fermentation of the methane from animal feces and garbage generated in Urugi Village, ligneous biomass cogeneration, etc. (NEDO)

  15. Report on investigation new energy vision establishment in Zao Town area in fiscal 2000; 2000 nendo Zaocho chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2001-02-01

    A new energy vision was established in the Zao Town area in Miyagi Prefecture. The population of Zao Town is 14,240 persons as of 2000, continuing the slow decreasing trend since 1986. The energy consumption is accounted for by the industrial department at about 50% of the total consumption, followed by the transportation department at about 30%, and the consumer department at about 20%. Discussions on the new energy utilization possibility revealed 30,905 MWh annually by photovoltaic power generation, and 34,630Gcal annually by solar heat utilization. Discussions were also given on power wind generation, small to medium hydropower generation, utilization of hot spring heat, and others. The new energy introducing project includes introduction of photovoltaic power generation into such public facilities as the public hall and athletic park, private sector operated houses and business facilities, introduction of wind power generation into the surrounding areas of the Zao Heart Land and skiing areas, snow melting utilizing hot spring heat, conversion of the automobiles used in the official organizations into clean energy fueled automobiles, installation of bio-mass plants, illumination of parks and streets utilizing photovoltaic and wind power generation systems, and micro hydropower generation in small to medium size rivers. (NEDO)

  16. Regional new energy vision for Kakegawa City. Formulation survey report; 2001 nendo Kakegawa shi chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Kakegawa City, Shizuoka Prefecture, surveys and studies were conducted involving the energy demand of the city, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The city consumes approximately 11,638 GJ/year, with the industrial sector consuming 4,896 GJ, the residential/commercial sector 3,608 GJ, and the transportation sector 3,134 GJ. Energy types are electric power, gas oil, heavy oil, gasoline, kerosene, LPG, and town gas, as listed in the descending order in terms of consumption rate. The amount of carbon dioxide resulting from the consumption is estimated at 673,214 t-CO2. Projects for new energy introduction were studied, which involved photovoltaic power generation for the general-purpose gymnasium, kindergartens, the school meal center, and meeting places; introduction of solar heat utilization systems into public facilities; financial support system for the purchase of solar heat utilization apparatuses; refuse-fueled power generation and heat utilization at a new cleaning center; utilization of biogas from garbage and stock raising waste; utilization of woody resources; clean-energy vehicles; and so on. (NEDO)

  17. Regional new energy vision formulation project for Ukiha Town. Fiscal 2001 report; 2001 nendo Ukiha machi chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such in Ukiha Town, Fukuoka Prefecture, surveys and studies were conducted involving the amount of energy needed by the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The energy consumption of the town is estimated at 158,886 drums/year in terms of crude oil - 56.2% in electric power, 19.5% in gasoline, 10.5% in gas oil, 6.9% in kerosene, 4.2% in fuel oil A, and so forth. Projects for introducing new energies were studied, which included the introduction of photovoltaic power systems into public facilities, gymnastic halls, and comprehensive welfare centers; introduction of a large wind power system (1,500 kW) in the vicinity of the Michinoeki roadside service spot; installation of a power plant fueled by woody biomass (scrap wood, saw dust, and the like); introduction of clean energy vehicles for official use; installation of photovoltaic/wind hybrid power-lighted street lamps in the shopping area; installation of light traps for agriculture burned by photovoltaic power; and financial aids for encouraging the installation of photovoltaic power generation systems in general residences. (NEDO)

  18. Fiscal 2000 survey report on regional new energy vision formulation for Kawanabe town, Kagoshima Prefecture; 2000 nendo Kawabecho chiiki shin energy vision sakutei nado chosa hokokusho

    NONE

    2001-03-01

    A regional new energy vision has been formulated for Kawanabe town, Kagoshima Prefecture, to be participated in by inhabitants and beneficial to both natural environment protection and inhabitants. The results of the survey are described in six chapter which involve (1) the basic philosophy of vision formulation, (2) outline of new energy, (3) environmental characteristics and townspeople's awareness, (4) energy situation and the amount of new energy existing in Kawanabe town, (5) regional new energy vision for Kawanabe town region, and (6) future tasks. After a study of the existing amount of new energy, energy demand, and people's requests, it is concluded that photovoltaic power generation, wind power generation, hot spring heat utilization, small scale water energy, and refuse-derived energy be the types to be preferably introduced into the town. Projects are established and concrete plans are prepared thereunder, which concern the reinforcement of public relations and enlightening activities, installation of a wind power system as one of enlightening activities, study of Kawanabe town's own subsidy system for encouraging the introduction of photovoltaic power systems and solar heaters for domestic use, and the introduction of photovoltaic power systems into public facilities such as school houses. In conclusion, it is hoped that, since financial difficulties will be a strong factor to impede new energy introduction, the subsidy system will be a solid one. (NEDO)

  19. Report on the project for working out the new energy vision of the Iwasaki village area; 2001 nendo Iwasaki mura chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it at Iwasaki Village, Aomori Prefecture, an investigational study was conducted of the energy demand of the town, existence amount of new energy, introduction of new energy, etc., and a new energy vision was worked out. The population of Iwasaki Village was 2,845 as of October 2000, which decreased approximately 30% in the past 20 years. The energy demand of the whole village was about 280,000 GJ in amount (about 7,202 kL/y in crude oil conversion), consisting of kerosene, light oil, heavy oil, gasoline, electric power and LPG. As viewed by sector, the rate of energy demand in the commercial/residential sector was comparatively high. As the project for new energy introduction, the following were studied: wind power generation around the Yamamori plain/Iwasaki village office; snow melting system using soil heat exchanger on the road in front of elementary school, photovoltaic power generation for public facilities (elementary and junior high schools/house of recreation and relaxation for old people/day nursery)/Tsugaru Semi-national Park/general houses; wave power generation at the Iwasaki fishing port; introduction of clean energy vehicles to public facilities; utilization of ligneous biomass energy, etc. (NEDO)

  20. Let People Bathe in Clean Energy. Regional new energy vision for Matsuyama Town; 2001 nendo Matsuyama machi chiiki shin energy vision. Toumeinal energy wo sosoide

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Matsuyama Town, Yamagata Prefecture, surveys and studies were conducted involving the amount of energy needed by the town, the amount of new energy resources in existence, and new energy introduction projects, and then a vision was formulated. The town demands 120,407-million kcal/year in energy comprising 56.8% from oil based fuels, 39.2% from electric power, and 4.1% from LP gas. As for consumption, 35.6% is consumed by households, 28.9% by industries, 21.3% by transportation, and 14.2% by commerce. The amount of carbon dioxide due to the consumption is estimated at 28,000 t-CO2/year. Key projects for new energy introduction were discussed, which included an eco-town project for introducing photovoltaic power generation systems, passive solar heat utilization systems, clean energy vehicles, and so forth, into public facilities; an eco-agriculture project for utilizing wind power generation and livestock excreta energy; an eco-park project for exhibiting new energies to the public; and an eco-school pilot model project. (NEDO)

  1. Regional new energy vision formulation project for Kanai Town. Fiscal 2001 report; 2001 nendo Kanai machi chiiki shin energy vision sakutei tou jigyo

    NONE

    2002-03-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Kanai Town, Niigata Prefecture, surveys and studies were conducted involving energy demand of the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The town demands 541,062 GJ/year in energy, with the residential/commercial sector requiring approximately 46%, the industrial sector approximately 30%, and the transportation sector approximately 25%. The energy types are kerosene, heavy oils, gas oil, electric power, gasoline, and LPG, mentioned in the descending order in terms of magnitude of requirement. The resultant amount of carbon dioxide is estimated at 11,449 t-C. New energy introduction projects were discussed, which included photovoltaic power generation for primary and junior high schools, the town office, and the Dorin stock farm; a solar heat utilization system for the school meal center; wind power generation for Hometown Kimpoku, primary schools, and the Dorin stock farm; mini-scale hydroelectric power generation on the Shimpo river; use of forestry biomass (pellet stoves) at nursery schools; introduction of clean-energy automobiles for public use; and the introduction of cogeneration into Sado Hospital. (NEDO)

  2. Report on results of the survey for drawing new energy visions for the Town of Shiranuka district; Shiranukamachi chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2001-03-01

    The new energy visions are drawn for Town of Shiranuka. Utilization of solar energy is at the top of the prospective new energy reserves for the town, followed by utilization of heat of wastes, biomass energy (in the stock raising industry), and photovoltaic power generation. The expected recoverable new energies account for approximately 35% of the total annual energy consumption of the town. The basic guidelines for introduction of new energies are drawing the priority projects which place importance on environmental education and bringing up talented persons, development of the soft policies for supporting the citizens and related entrepreneurs, and introduction of new energies for promoting new industries. The priority projects include hard ones, e.g., foundation of Eco-school Shiranuka, Eco-park Kushiro/Shiranuka (biomass energy supply center) and Koima new energy park, and soft ones, e.g., establishment of the new energy grand prize awarded by PR brochure ''Shiranuka'', foundation of the Shiranuka fund for introduction of new energies, construction of a Shiranuka Citizens' power station, and rent-a-car project for eco-car test runs. (NEDO)

  3. Report on new energy vision establishment project in Higashi Naruse Village area; Higashinarusemura chiiki shin energy vision sakutei nado jigyo hokokusho

    NONE

    2001-02-01

    A new energy vision was established in the Higashi Naruse Village in Akita Prefecture. The population of the village is continuing the decreasing trend since 1950 to 3,390 persons as of 2000. The annual energy demand in this village is about 320,000 GJ, accounted for by the consumer department at about 40%, the transportation department at about 36% and the industrial department at about 24%. The carbon dioxide emission amount therefrom is estimated 6,300 t-C annually. The expected usable quantity of new energies in the village would be available, in the decreasing order, in the wind power as the most promising source, forestry bio-mass, snow cold heat energy, agricultural bio-mass, and solar beam (heat). The total quantity would be 514,330 GJ annually in heat utilization, and 85,305 MWh in electric power. The new energy introducing project includes introduction of photovoltaic and wind power generation into Kurikoma Mountain Villa, introduction of wind power generation into the Junes skiing area and Oyanagi Natural Park, introduction of photovoltaic power generation and heat utilization into primary and middle schools, introduction of clean energy fueled automobiles into the village office, and introduction of photovoltaic power generation into general houses. (NEDO)

  4. Fiscal 2000 survey report on regional new energy vision formulation for Kito village; 2000 nendo Kitoson chiiki shin energy vision sakutei nado chosa hokokusho

    NONE

    2001-02-01

    A regional new energy vision has been formulated for Kito village, Tokushima Prefecture, in the hope that it will trigger off an effort to build a mentally affluent community. Kito village is a 2,000-strong village situated in a mountainous region, and consumed 1.3 times 10{sup 5} GJ in energy in fiscal 1999. The results of activities conducted in this connection are summarized in four chapters which are (1) the outline of survey, (2) local characteristics, (3) existing amount of new energy, and (4) plans for promoting new energy introduction and energy conservation. Discussed as plans for promoting new energy introduction and energy conservation are a business of commercialization feasibility survey, plot to construct a natural energy operated school building, and a plan for promoting energy conservation. Discussed in relation to commercialization feasibility survey are a small scale hydroelectric power generation utilizing a sand-trap dam, small scale hydroelectric power generation utilizing waste water from a fish farm, regional heat supply business utilizing ligneous biomass, manufacture of ligneous pellet fuel, and so forth. In relation to the promotion of energy conservation, a drive will be launched to positively bring photovoltaic power generation into daily life. (NEDO)

  5. Report on results of the survey for drawing new energy visions for the Town of Hamanaka district; Hamanakacho chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2001-02-01

    The new energy visions are drawn for Town of Hamanaka, Hokkaido. The town had a population of 7,866 in 1995, which compares with 11,915 in 1960, aging as a whole with reduced number of new born babies. The town has been holding the Japan's top town in tangleweed production and core site in the Kushi-Ne dairy belt. Biomass energy (excreta and woody resources) is at the top of the prospective new energy reserves for the town, which total 88,741Gcal/y, followed by utilization of solar energy and heat exhausted from garbage incineration. The priority projects selected for introduction of new energies include a biogas plant which produces organic compost and biogas energy by anaerobic fermentation of excreta discharged from domestic animals, raw garbage and fishery wastes; system for utilizing waste heat from a stoker type incinerator burning domestic garbage, which satisfies the dioxin emission standard; and system for utilizing low-temperature heat including energy of snow and ice, which uses only a limited extent of the conventional energy and free of chlorofluorocarbon gases. (NEDO)

  6. Report on investigation of new energy vision planning at Tomi Village; 2001 nendo Tomi son chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-02-01

    With an objective of accelerating introduction of and uplifting consciousness on new energies at Tomi Village in Okayama Prefecture, investigations and discussions were given on the energy demand, quantity of new energies available, and the new energy introduction projects for the town, thus a new energy vision was established. The amount of energy demand in Tomi Village is 64,261 GJ/year in fiscal 1999. By departments, the transportation department accounts for 45.2%, the consumer department for 28.3% and the industrial department for 26.5%. Energy types are accounted for by petroleum at 87.5%, and electric power at 12.5%. The new energy introduction projects have discussed introduction of photovoltaic power generation system and solar heat hot water making system into nursery schools, primary and middle schools, the public hall, the Tomi Communication Center, the village office, and clinics, small size wind power generation system into primary and middle schools, ligneous bio-mass combustion system and middle size wind power generation system into the camping site, low-drop electric power generation system into Shiraga Valley, street lights powered by photovoltaic and small power wind generation systems, and clean energy fueled automobiles for public use. (NEDO)

  7. New energy vision of the Sen'nan city area. Outline; 2001 nendo Sen'nan shi chiiki shin energy vision (Gaiyo ban)

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Sen'nan City, Osaka Prefecture, a new energy vision was worked out, and a version of the outline was made. The policy on new energy introduction was described as follows: to make a great use of solar energy; to promote the spread of low-emission car and other new energy; to construct a system for spread that is connected to cooperation with citizen and education/enlightenment. Concretely, the following were cited: introduction of photovoltaic power generation to the city office/elementary school/junior high school; study of preferential treatment for introduction of solar energy; introduction of low-emission car to official vehicle; study of preferential treatment for introduction of low-emission car; promotion of car sharing by low-emission car; introduction of natural gas cogeneration and fuel cell to the city office; installation of street light using small wind power generation; potential study of small- and medium-size power generation; support for class for new energy experience at elementary/junior high school; potential study of utilization of biomass energy such as bamboo charcoal; study of preferential treatment for new energy to be given to citizen and enterprises in the city; construction/support of energy utilization system using waste, etc. (NEDO)

  8. New energy vision at Sabae City. Toward realization of 'environmental international city'; 2001 nendo Sabae shi chiiki shin energy vision. Kankyo kokusai toshi Sabae no jitsugen ni mukete

    NONE

    2002-03-01

    With an object of accelerating introduction of and uplifting consciousness on new energies at Sabae City in Fukui Prefecture, a new energy vision was put into order based on the result of the initial stage investigation having been performed in the previous fiscal year. Solar light and solar heat energies will be introduced positively into public facilities, and at the same time works will be implemented to accelerate the proliferation thereof into general households. The target of introduction by the year 2010y is set to 2,600 kW by photovoltaic power generation, and the introduction thereof is planned to schools, nursery schools, municipality operated houses, and unattended facilities. With regard to solar heat water warmer, the present proliferation rate of about 7.3% will be raised to 15%. Regarding wastes generated from industrial areas, efficient energy extraction by thermal recycling is intended on the assumption of suppression of wastes generation, their re-use, and recycling. Systems may include the bio-gas system, cogeneration, and recovery of energies from plastics wastes. For acceleration of proliferation of new energies in general, promotion of introduction will be achieved on mini-wind power generation, small hydroelectric power generation, and fuel cell automobiles. (NEDO)

  9. Regional new energy vision formulation project for Ukiha Town. Fiscal 2001 report (Summary); 2001 nendo Ukiha machi chiiki shin energy vision sakutei tou jigyo hokokusho (Gaiyo ban)

    NONE

    2002-02-01

    For contributing to the preservation of the global environment through new energy application and energy conservation, efforts were made to work out a new energy vision for Ukiha Town, Fukuoka Prefecture. Town's population was 17,482 in fiscal 2000, which showed a tendency to decline slowly. The town demands 158,886 drums/year in terms of crude oil - 56.2% in electric power, 19.5% in gasoline, and 10.5% in gas oil. As for the presence of new energy resources, wind power comes in first in terms of magnitude, and woody biomass energy is also found in abundance since lumbering prospers in the town. Named as candidate projects in the efforts to introduce new energies were the embodiment of practical size wind power systems in the hilly part; heat utilization and power generation using biomass such as scrap wood from lumber mills; introduction of photovoltaic power into public facilities; use of clean energy vehicles by the town office; photovoltaic power generation and solar heat utilization at welfare facilities and public gymnastic halls; street lamps lighted by hybrid power systems in the shopping area and at the Michinoeki roadside service spot; use of new energies in agriculture; financial aids to encourage photovoltaic power into general residences; etc. (NEDO)

  10. FY 2001 new energy vision of Shiramine Village. Creation of new energy; 2001 nendo Shiramine mura chiiki shin energy vision. Onkotakushin energy no sozo

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Shiramine Village, Ishikawa Prefecture, an investigational study was conducted of the energy demand amount of the village, existence amount of new energy and project for new energy introduction, and a vision was worked out. The energy demand amount of the village was 155,970 GJ/y. As viewed by sector, the energy demand amount consisted of 43.9% in the industrial sector, 30.0% in the commercial/residential sector and 26.1% in the transportation sector. The rate of energy source was 79.0% of petroleum, 18.7% of electric power and 2.2% of gas. In the project for new energy introduction, the following were cited and studied: as the project for forest/snow resource utilization in the Nishiyama area, production/storage of charcoal using thinnings/wood chips, and food preservation using snow; as the project for experiencing/learning the nature using existing facilities, introduction of photovoltaic power system/solar heat use water warmer to Shiramine Elementary School/Shiramine Junior High School; as the project for functionally reinforcing tourist attractions, 'park for learning new energy' by lighting using solar light/wind power, etc. (NEDO)

  11. Report on the survey for working out the new energy vision of the Yamagata city area; 2001 nendo Yamagata machi chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Yamagata City, Ibaraki Prefecture, an investigational study was conducted of the energy consumption amount of the city, existence amount of new energy and project for new energy introduction, and a vision was worked out. The energy consumption of the city in FY 1999 was 178.8 billion kcal in amount and was broken down into 76.5% of petroleum, 18.8% of electric power and 0.85% of LPG. As viewed by sector, the energy consumption consisted of 37.9% in the transportation sector, 35.2% in the industrial sector and 26.8% in the commercial/residential sector. As the project for new energy introduction, the following were studied: biomass cogeneration/greenhouse using waste heat from hot spring/photovoltaic power generation/wind power - photovoltaic power hybrid street light/biomass vehicle in the Santanoyu/Eco-Zone formation project; introduction of photovoltaic power generation/solar heat utilization to gymnasium of junior high school/multi-purpose cultural center/municipal dwelling house/elementary school/junior high school/central public hall in the new energy project for public facilities; introduction of clean energy vehicle, etc. (NEDO)

  12. Report on the survey for working out the new energy vision of the Shiga village area; 2001 nendo Shiga mura chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it at Shiga Village, Nagano Prefecture, an investigational study was conducted of the energy demand in the village, existence amount of new energy, project for new energy introduction, etc., and a vision was worked out. The energy consumption amount in FY 2000 in Shiga Village was approximately 50.2 billion kcal and was broken down into 54.7% of petroleum, 38.3% of electric power, 6.3% of LPG and 0.75% of others. As the project for new energy introduction, the following were studied: installation of photovoltaic power generation panels to the new office under construction, introduction of photovoltaic power/wind power hybrid power generation to Klein Garten Club House, installation of the solar system to hot spring facilities in the village, project on low-temperature wood drying by smoking using sawdust/wood chips, methanol production using thinnings as raw materials and the use for automobile fuel/cogeneration, methane fermentation from animal feces/house garbage and the use for cogeneration, etc. (NEDO)

  13. Fiscal 2000 survey report on regional new energy vision formulation for Oki town; 2000 nendo Okicho chiiki shin energy vision sakutei nado chosa hokokusho

    NONE

    2001-02-01

    Efforts are in progress to build a resource circulation type town symbiotic with natural environments at Oki town, Fukuoka Prefecture, which is an agricultural community where creeks run in every direction. A new energy vision has been worked out for the town, which centers about a plan for building a resource circulation type community. The survey results are summarized in six chapters dealing with (1) the survey outline, (2) local conditions relative to energy, (3) energy supply/demand structure, (4) amount of new energy in existence, (5) records and analysis of inhabitants' approach to energy conservation, and (6) a new energy introduction plan. The new energy introduction plan consists of four items involving a short term plan or a key project, a project for medium/long term surveys or feasibility study for the commercialization of new energy, plans for reducing energy consumption, and so forth. Discussed in relation to the short term plan are the introduction of photovoltaic power generation into public facilities, installation of a photovoltaic power station to be jointly utilized by the inhabitants, establishment of a grant system for the installation of solar water heaters for domestic use, creation of an additional subsidy system for domestic photovoltaic power generation, and the like. (NEDO)

  14. Fiscal 2000 report on regional new energy vision formulation project for Yunomae town, Kumamoto prefecture; 2000 nendo Yunomaecho chiiki shin energy vision sakutei nado jigyo hokokusho

    NONE

    2001-02-01

    A regional new energy vision has been formulated for Yunomae town, Kumamoto Prefecture, from the standpoint that local autonomies are required to work out measures compatible with Government energy policies and thereby to wrestle with environmental measures being undertaken across the world. The results of activities conducted for this purposed are stated in six chapters, which cover (1) the meaning of regional new energy vision formulation and its position, (2) outline of Yunomae town, (3) energy and environments, (4) energy situation in Yunomae town, (5) Yunomae town's regional new energy vision, and (6) plans for promoting the embodiment of the vision. In chapter (5), basic policies toward new energy utilization, overall plan, leading projects, and energy conservation measures are stated. The types of new energy taken up for discussion are photovoltaic power, wind power, biomass power, refuse-derived energy, and hydraulic energy. Projects are subjected to study, which relate to photovoltaic power generation, solar heat utilization, small scale hydroelectric power, small scale wind power, and the introduction of clean energy vehicles. It is expected that these projects when carried out will cover approximately 10% of the current energy consumption of the town. (NEDO)

  15. Survey of regional new energy vision formulation for Shimizu town. Report on initial stage survey; Shimizucho chiiki shin energy vision sakutei chosa. Shoki dankai chosa hokokusho

    NONE

    2001-02-01

    Shimizu town, Hokkaido, is a 11,000-strong town, located in the northwestern part of the Tokachi plains and consuming 1.7 times 10{sup 4} Gcal in energy. Discussion is held for new energy which will make the most of the local characteristics of the town while the basics of town building are considered and confirmed. The results of the survey are summarized in four chapters which are (1) the purpose of regional energy vision formulation, (2) local characteristics, (3) state of new energy in existence, (4) and the course to follow for the introduction of new energy (draft). Chapter (4) is constituted of the purpose of new energy introduction and the basic policy, and the course along which new energy will be adopted. Proposed in relation with the course of new energy introduction are the effective utilization of biomass resources, utilization of cold heat energy (ice) involving agriculture, introduction of a symbolic small scale water energy system, introduction of a symbolic wind energy system, extensive introduction of solar energy, and the deployment of popularizing and enlightening activities. A project is mentioned of experimentally introducing biogas facilities that makes use of farm animals' excretion. (NEDO)

  16. Fiscal 2000 basic survey report for vision formulation. Regional new energy vision for Sabae city, Fukui prefecture; Sabaeshi chiiki shin energy vision. 2000 nendo sakutei kiso chosa hokokusho

    NONE

    2001-03-01

    Sabae city, Fukui Prefecture, has worked out a regional new energy vision for which the inhabitants, industrialists, and administrators combined their efforts to build a daily life related culture, friendly to environments and peculiar to the locality. The fruits of their activities are summarized in five chapters, which are (1) the basic philosophy, (2) survey of the amounts of new energy in existence, (3) survey of the amount of energy demanded, (4) various surveys conducted with the participation of the citizenry, (5) and summarization of important topics. Discussed in chapter (2) is the basic policy toward reckoning the amounts of new energy in existence, such as photovoltaic energy, solar heat energy, wind energy, hydraulic energy, temperature difference energy, and other energy resources remaining to be utilized. It states that they, when converted into electric power, 2.0 times 10{sup 8} kWh/year will be available and, when converted into heat energy, 2.92 times 10{sup 11} kcal/year will be available, accounting for approximately 30% of the city's energy consumption. When the energy resources are broken down by type, it is found that solar energy, wind energy, and refuse-derived energy are in existence aplenty. (NEDO)

  17. Basic research report for drawing up regional new energy vision (provisional name) for Gunma Prefecture; Gunmaken chiiki shin energy vision (kasho) sakutei kiso chosa hokokusho

    NONE

    1999-03-01

    In view of relatively good solar radiation that the prefecture enjoys and of a result of the investigations of this time conducted into the amount of new energy in existence, it is concluded that a fairly large amount of solar energy is collectable as the regional new energy for Gunma Prefecture. As for wind power energy, since there is no proper location in the prefecture where the annual wind speed average of not less than 5m/s is available, the value generally accepted as necessary for wind power generation, it is inferred that conditions in the prefecture in general are disadvantageous when it comes to wind power generation. As for hydraulic energy of small and medium dimensions, a result of the investigations shows that in the prefecture this energy is next to solar energy in terms of the amount expected to be collectable. In this prefecture, the prefectural project bureau makes use of the rich water resources and generates electric power, and supplied approximately 850-million kWh in fiscal 1998. Since it is feared that it will be difficult to find new sites for power generation in the area now remaining to be developed, however, prudence should be exercised in estimating the expected collectable amount of hydraulic energy. (NEDO)

  18. Fiscal 2000 report on regional new energy vision formulation project for Yoron town; 2000 nendo Yoroncho chiiki shin energy vision sakutei nado jigyo hokokusho

    NONE

    2001-02-01

    A regional new energy vision has been formulated for Yoron town, Kagoshima Prefecture, which will serve as guidelines for building an Environmentally Friendly Island. Yoron town is a town of approximately 6,000 inhabitants as of fiscal 2000, its energy consumption is equivalent to 61,427 drums in terms of crude oil, and it receives electric power from Yoron Power Plant of Kyushu Electric Power Co., Inc. The results of activities conducted in this connection are summarized in seven chapters, which are (1) coping with new energy vision formulation (2), energy supply and demand, (3) estimation of existing amount of new energy, (4) basic policy toward new energy introduction, (5) projects for introducing new energy, (6) existing energy systems, and (7) reference materials. Named as projects are the construction of a water supply power source (for storage pump) for the Furusato district; power source for farmland irrigation pumps; power sources for the integrated sports park and municipal library; power source for a farmyard compost center; wind power, photovoltaic power, and combined power generation facilities for streetlights; and the utilization of bagasse. (NEDO)

  19. Report for fiscal 2000 on establishment of new energy vision in Fujino Town; 2000 nendo Fujino chiiki shin energy vision sakutei nado hokokusho

    NONE

    2001-03-01

    With an objective to show specifically the efforts of working on new energies in Fujino Town in Kanagawa Prefecture, the existence quantity of new energies centering on the sun and woods was made clear to discuss the possibilities of utilizing them. Furthermore, investigations were performed on the energy demand and supply structure and possibilities of introducing new energies, and the specific new energy introduction plan was established. The activity achievements were put into order by the following six items: 1) the basic conception of the new energy vision, 2) various conditions of Fujino Town related to new energies, 3) energy demand and supply structure, 4) utilizable quantity of new energies, 5) toward the introduction of new energies, and 6) specific new energy facilities to discuss the introduction. Item 6 includes facilities to be built by defining model areas, facilities to discuss the full-swing introduction into facilities managed by the township, facilities to be discussed of full-swing introduction and to be composed in relation with the overall plan including large size hot water supply devices used at the spa, firewood boilers, a heat recovery system, and a large-scale photovoltaic power generation system. (NEDO)

  20. Report on regional new energy vision formulation project for Kahoku town, Kumamoto Prefecture; Kahokucho chiiki shin energy vision sakutei nado jigyo hokokusho

    NONE

    2001-02-01

    A regional new energy vision has been formulated for Kahoku town, Kumamoto Prefecture, for the realization of an energy resource circulation type regional community in which people earnestly strive to be symbiotic with nature. The results of related activities are shown in six chapters, which are (1) the prerequisites to the plan, (2) outline of the Kahoku town region, (3) surveys of the existing amount and expectedly available amount of new energy, (4) feasibility of new energy introduction, (5) assessment of new energy introduction plans and their effect, and (6) tasks to discharge, and policies to follow, for new energy introduction. In chapter (5), new energy introduction plans and introduction policies are shown. Plans are made for the incorporation of photovoltaic power facilities, small hydroelectric power facilities, and bamboo chip boilers into public facilities; the installation of 750 kW class wind power facilities; and the adoption of hybrid vehicles to serve as official special vehicles. It is expected that these plans when implemented will bring about a 1,400 t-c/year reduction in CO2 emissions. As for the introduction of wind power generation, the dimensions are determined by taking into consideration the prerequisites to a NEDO (New Energy and Industrial Technology Development Organization) grant. (NEDO)

  1. Report on the project for working out the new energy vision of the Genkai town area; 2001 nendo Genkai machi chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Genkai Town, Fukuoka Prefecture, an investigational study was conducted of the estimated amount of energy demand of the town, existence amount of new energy, project for new energy introduction, etc., and a vision was worked out. The energy consumption amount of the town in FY 1999 was estimated at 834 TJ/y. By sector, it consisted of 299.31 TJ in the industrial sector, 282.08 TJ in the transportation sector and 252.78 TJ in the commercial/residential sector. By energy source, the rate of light oil was the highest, followed in order by gasoline, electric power, kerosene, LPG, etc. As the project for new energy introduction, the following were studied: preparation of disaster-prevention type photovoltaic power generation at shelter, etc.; introduction of photovoltaic power generation to kindergarten, elementary school, etc.; introduction of photovoltaic power generation to street light, tourist information center, etc.; introduction of wind power generation to public facilities; use of the waste cooking oil made into diesel fuel for official vehicle; introduction of cogeneration to the health welfare hall; solar light/heat utilization system for residential/industrial use; wind power generation business for business establishment. (NEDO)

  2. Report of survey for formulating regional new energy vision for Okuma Town; 2001 nendo Okuma machi chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-03-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Okuma Town, Fukushima Prefecture, surveys and studies were conducted involving the amount of energy needed by the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The town demands 29,769 times 10{sup 7} kcal/year in energy comprising 44.5% for the industrial sector, 31.4% for the transportation sector, and 24.1% for the residential/commercial sector. As for energy source types, 25.7% is covered by electric power, 20.0% by gasoline, 16.2% by gas oil, and 15.3% by heavy oil, with the oil based sources responsible for 71.2%. New energy introduction projects were discussed, which included the introduction of photovoltaic power generation for a newly built children's hall, residential complexes, town managed housing, street lights and road signs, and for some other public facilities; introduction of cogeneration into enterprises present in the eastern industrial park and into hotels; and the introduction of natural gas fueled cogeneration. Studies were also made about public information, public relations, and support systems for implementation in Okuma Town. (NEDO)

  3. Dicty_cDB: CHO774 [Dicty_cDB

    Full Text Available m cDNA clone 58915 5', mRNA sequence. 42 13 1 DN493309 |DN493309.1 X077H09.3pR Populus wood cDNA library Populus tremula x Popul...CH (Link to library) CHO774 (Link to dictyBase) - - - Contig-U12145-1 - (Link to Or...iginal site) - - CHO774Z 618 - - - - Show CHO774 Library CH (Link to library) Clone ID CHO774 (Link to dicty...osome 3. 42 13 1 CV435371 |CV435371.1 58915.1 Suspension culture Solanum tuberosu...manei cDNA 5, mRNA sequence. 34 2.1 2 AP004054 |AP004054.3 Oryza sativa (japonica culti

  4. Engineered CHO cells for production of diverse, homogeneous glycoproteins

    Yang, Zhang; Wang, Shengjun; Halim, Adnan

    2015-01-01

    Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferas...

  5. Methods for modeling chinese hamster ovary (cho) cell metabolism

    2015-01-01

    Embodiments of the present invention generally relate to the computational analysis and characterization biological networks at the cellular level in Chinese Hamster Ovary (CHO) cells. Based on computational methods utilizing a hamster reference genome, the invention provides methods for identify...

  6. Conservative treatment of congenital false joint of shin in newborns and infants

    O. V. Shchokin

    2017-04-01

    Full Text Available Congenital false joint of shin occurs in 1 case per 28 000 - 190 000 live births, but it is hard-to-treat and disabling disease. Objective: Improving treatment outcomes, reducing the number of surgical interventions for the treatment of congenital false joint of shin. Materials and Methods. The method is carried out using staged plaster casts with the gradual correction of deformities. When axis of the leg approximates to the normal one, axial load is added. First, it is done by tapping on the heel. When the child was 7-8 months old he was put on the legs and taught to walk. The plaster bandage is replaced by "Scotch cast" and "soft cast" bandage. The treatment lasted up to reaching of clinical effect – correction of shin deformation and absence of pathological mobility. Clinical effect must be confirmed by roentgenography which must demonstrate filling of false joint zone with bone tissue and restoration of intramedullary canal. During all the period of treating alternate courses of electrophoresis with calcium chloride, medical mud extracts and magnetic therapy are conducted. In the period from 1995 till 2015 in the Regional Zaporizhzhia Children Clinical Hospital 4 children (6 shins aged from 1 to 7 months with false joint of shin were treated using proposed method. Results and discussion. All 4 children (6 shins treated in clinic with proposed conservative method showed filling of false joint zone with bone tissue, restoration of intramedullary canal and significant extension of axis of the shin. The load on the leg in early terms results in compression of bone fragments (as in compression-distraction osteosynthesis, magnetic therapy, electrophoresis with calcium chloride and medical mud extracts promote active functioning of the muscles that improves regional blood supply and improves osteogenesis. Conclusions. Using the proposed method of treatment of congenital false joint of shins in newborns and infants can allow avoiding surgical

  7. Notes on breeding sharp-shinned hawks and Cooper’s hawks in Barnwell County, South Carolina.

    Vukovich, Mark; Kilgo, John, C.

    2009-07-01

    Abstract - Breeding records of Accipiter striatus (Sharp-shinned Hawks) in the southeastern US are scattered and isolated. We documented a Sharp-shinned Hawk and Accipiter cooperii (Cooper’s Hawk) nest while conducting a telemetry study on Melanerpes erythrocephalus (Red-headed Woodpeckers) in Barnwell County, SC in 2006 and 2007. We report the first known nest of a Sharp-shinned Hawk in Barnwell County, SC and the first report of Sharp-shinned Hawks preying upon Red-headed Woodpeckers. Thirteen of 93 (13.9 %) woodpeckers were killed by accipiters in the summers of 2006 and 2007. Large, contiguous forests managed for Picoides borealis (Red-cockaded Woodpeckers) may be used by breeding Sharp-shinned Hawks. The bright plumage, loud calls, and behavior of Red-headed Woodpeckers, particularly during the nestling stage, may make them conspicuous prey for accipiters.

  8. Postirradiation properties of a UV-sensitive variant of CHO

    Wood, R.D.; de Veciana, M.; Presson-Tincknell, B. (California Univ., Berkeley (USA). Lawrence Berkeley Lab.)

    1982-08-01

    A UV-hypersensitive mutant of Chinese hamster ovary (CHO) cells, termed 43-3B, has been used in a comparative study with the wild type CHO in order to determine the involvement of repair in several postirradiation phenomena. 43-3B has the same growth rate and chromosome number as the wild type CHO-9. It is hypersensitive to UV irradiation. 43-3B shows only about 17% of the UV-stimulated unscheduled DNA repair synthesis of CHO-9 as measured by autoradiography. When breaks in supercoiled chromatin are measured after UV by the nucleoid sedimentation method, the mutant appears to be capable of carrying out only limited incision. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line, suggesting that the removal of UV-induced replication blocks by excision repair is the most important factor in allowing recovery of UV-inhibited DNA synthesis. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild type CHO-9, but little recovery was seen in 43-3B. This indicates that excision repair capability can also be important in split-fluence recovery.

  9. The apoptosis of CHO cells induced by X-rays

    Lu Zhaohong; Zhao Jingyong; Zhu Mingqing; Shi Xijin; Wang Chunlei

    2004-01-01

    The work is to study the mechanism of toxic effects on reproductive system and apoptosis of Chinese hamster ovary (CHO) cells induced by X-rays. CHO cell was exposed to X-rays 2 to 20 Gy. Apoptosis and morphological changes of the cells were observed by fluorescent microscopy and flow cytometry analyzer with double staining with Annexin V/PI. The apoptosis could be observed at 24, 48 and 72h after the exposure, but it was more obvious 48 and 72 h after the exposure. Rate of the apoptosis increased along with radiation dose were elevated. Some morphological changes, such as irregular agglomerate of chromatins, pycnosis and periphery distribution of nuclei, crescent-moon-like cells, small apoptosis body, were observed. Radiation results DNA damage in the CHO cells, and the damage cannot be repaired, hence the induced cell apoptosis. (authors)

  10. Glycoengineering in CHO cells: Advances in systems biology

    Tejwani, Vijay; Andersen, Mikael Rørdam; Nam, Jong Hyun

    2018-01-01

    are not well understood. A systems biology approach combining different technologies is needed for complete understanding of the molecular processes accounting for this variability and to open up new venues in cell line development. In this review, we describe several advances in genetic manipulation, modeling......For several decades, glycoprotein biologics have been successfully produced from Chinese hamster ovary (CHO) cells. The therapeutic efficacy and potency of glycoprotein biologics are often dictated by their post translational modifications, particularly glycosylation, which unlike protein synthesis....... Recently, CHO cells have also been explored for production of therapeutic glycosaminoglycans (e.g. heparin), which presents similar challenges as producing glycoproteins biologics. Approaches to controlling heterogeneity in CHO cells and directing the biosynthetic process toward desired glycoforms...

  11. Identification of a novel temperature sensitive promoter in cho cells

    Hesse Friedemann

    2011-05-01

    Full Text Available Abstract Background The Chinese hamster ovary (CHO expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature. Results Based on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37°C and 33°C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence. Conclusions This novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, a two to three-fold increase of basal productivity (already higher than SV40 promoter is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.

  12. Standardization of spedalized medical care to patients with shin fractures in multifield city hospital

    R. M. Tikhilov

    2013-01-01

    Full Text Available The aim of the investigation was development of science-based recommendations for increasing efficiency of operative treatment of adult patients with shin fractures in multiprofile city hospital. Investigation was made in Saint-Petersburg Alexandrovskiy City Hospital. Clinical material was presented by official hospital reports, individual medical documentation and results of direct survey of patients treated in this hospital in period 1999-2010 years. All patients had follow up treatment in outpatient department of this hospital. Information was completed following federal and local laws. Recommendations for standardization of modern specialized medical care of patients with shin fractures, based on methods of internal fixation, were performed. We took into consideration possibilities of conventional and minimally invasive fixation of closed and open fractures including politrauma injuries. Models of patients with shin fractures depending on method of internal fixation and list of basic diagnostic procedures and treatment were formed. Operations classifier of internal shin fractures fixation was developed. This classifier includes calculation of hospital costs in process of specialized medical care considering actual correction coefficients. Calculation of each surgical procedure component was performed. List and composition of instrument sets and expense materials for such operations were formed. Analisis of organizational, medico-technological, economica aspect and expert evaluation of clinical results of different methods of long bones fractures fixation have provided conceptual approach to treatment standardization. On this base we have developed medico-economical standards of long bones fractures treatment in city multiprofile hospital.

  13. Development of Severe Accident Management Strategies for Shin-Kori 3 and 4

    Lee, Youngseung; Kim, Hyeongtaek; Shin, Jungmin

    2013-01-01

    Shin-Kori units 3 and 4 are new reactors under construction as an APR 1400 type reactor. The plants which considered coping with severe accident from design phase are different from other operating plants in view of severe accident management strategies. The purpose of this paper is to establish optimal strategies for Shin-Kori 3 and 4. A scheme for optimized severe accident management was drawn up with the object of achieving core cooling, containment integrity, and decreased release of fission product. Shin-Kori units 3 and 4 are a new reactor and designed to add mitigating systems for coping with severe accident such as ECSBS, PAR, and CFS. Also the plants are reflected as a part of Fukushima followup measures The strategies of SAMG for Shin-Kori 3 and 4 were developed. The strategic approach was based on the concept of defense in depth. Firstly, strategies for core cooling were chosen such as RCS depressurization, injection to SG, injection to RCS, and injection to reactor cavity. Secondly, the plans for containment integrity were developed for controlling pressure and hydrogen in containment. Lastly, reduced release of fission product was considered for protection of the public after containment failure. The achieved strategies meet the needs of effective methods for severe accident management and enhancement of safety

  14. Perforate on CHO cell membranes induced by electromagnetic ...

    STORAGESEVER

    2009-06-17

    Jun 17, 2009 ... Key words: Electromagnetic pulse (EMP), atomic force microscope, CHO cell, cell membrane. INTRODUCTION .... of perforation ranges from 390 to 660 nm and the depth is. 392.95 nm. ... cell membrane perforations increased when both the field intensity and ..... Melatonin and a spin-trap compound block.

  15. Glycoengineering of CHO Cells to Improve Product Quality.

    Wang, Qiong; Yin, Bojiao; Chung, Cheng-Yu; Betenbaugh, Michael J

    2017-01-01

    Chinese hamster ovary (CHO) cells represent the predominant platform in biopharmaceutical industry for the production of recombinant biotherapeutic proteins, especially glycoproteins. These glycoproteins include oligosaccharide or glycan attachments that represent one of the principal components dictating product quality. Especially important are the N-glycan attachments present on many recombinant glycoproteins of commercial interest. Furthermore, altering the glycan composition can be used to modulate the production quality of a recombinant biotherapeutic from CHO and other mammalian hosts. This review first describes the glycosylation network in mammalian cells and compares the glycosylation patterns between CHO and human cells. Next genetic strategies used in CHO cells to modulate the sialylation patterns through overexpression of sialyltransfereases and other glycosyltransferases are summarized. In addition, other approaches to alter sialylation including manipulation of sialic acid biosynthetic pathways and inhibition of sialidases are described. Finally, this review also covers other strategies such as the glycosylation site insertion and manipulation of glycan heterogeneity to produce desired glycoforms for diverse biotechnology applications.

  16. Perforate on CHO cell membranes induced by electromagnetic ...

    Atomic force microscopy (AFM) has been used to visualize the morphological change on the surface of Chinese hamster ovary (CHO) cell membranes before and after electromagnetic pulses (EMP) irradiation. The results show that there were different sizes and shapes of membrane perforate (width ranging from 0.39 - 0.66 ...

  17. Method for reducing ammonium and lactate production in cho cells

    2018-01-01

    The present invention relates to modified producer cells for improved production of therapeutic proteins. Specifically, the inventors have found that removing genes involved in amino acid catabolism in Chinese Hamster Ovary (CHO) cells improves the cell growth and viability and likely also...

  18. Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness

    Sellick, C.A.; Croxford, A.S.; Maqsood, A.R.; Stephens, G.M.; Westerhoff, H.V.; Goodacre, R.; Dickson, A.J.

    2015-01-01

    Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC-MS) analytics to define the molecular loci by which two yield-enhancing feeds

  19. Accelerated and Rational Design of Improved CHO Cell Factories

    Grav, Lise Marie

    Recombinant production of therapeutic proteins provides huge benefits to human health and promises solutions to some of the most devastating and currently untreatable diseases in healthcare. Key to the development of new therapeutic proteins is to optimize and engineer living cells, namely cell...... of a number of novel tools is reported that aim to accelerate the construction of production cell lines for therapeutic proteins with optimal phenotypic attributes for industrial processes. Chinese hamster ovary (CHO) cells are the predominant production host for therapeutic proteins, and are the cell factory...... of interest in this thesis. The core of the thesis is revolved around the development and application of genome editing techniques that enable us to precisely engineer the genome of CHO cells by either rendering specific-targeted genes unfunctional or inserting new genes in precise genomic locations...

  20. Functional heterogeneity and heritability in CHO cell populations.

    Davies, Sarah L; Lovelady, Clare S; Grainger, Rhian K; Racher, Andrew J; Young, Robert J; James, David C

    2013-01-01

    In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing--new parental cell lines which are inherently more "fit for purpose." One-hundred and ninety-nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell-specific proliferation rate during extended deep-well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell-specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub-culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N-glycan processing were identified. Finally, for clonal populations most "evolved" by extended sub-culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N-glycosylation. Rapid-specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone-specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and

  1. Development of Neutronics Model for ShinKori Unit 1 Simulator

    Hong, JinHyuk; Lee, MyeongSoo; Lee, SeungHo; Suh, JungKwan; Hwang, DoHyun [KEPRI, Daejeon (Korea, Republic of)

    2008-05-15

    ShinKori-Unit 1 and 2 is being built in the Kori site which will be operated at 2815 MWt of thermal core power. The purpose of this paper is to report on the performance of the developed neutronics model of ShinKori Unit 1 and 2. Also this report includes the convenient tool (XS2R5) for processing the large quantity of information received from the DIT/ROCS model and generating cross-sections. The neutronics model is based on the NESTLE code inserted to RELAP5/MOD3 thermal-hydraulics analysis code which was funded as FY-93 LDRD Project 7201 and is running on the commercial simulator environment tool (the 3KeyMaster{sup TM} of the WSC). As some examples for the verification of the developed neutronics model, some figures are provided. The output of the developed neutronics model is in accord with the Preliminary Safety Analysis Report (PSAR) of the reference plant.

  2. An advanced NSSS integrity monitoring system for Shin-Kori nuclear units 3 and 4

    Oh, Y. G.; Kim, H. B.; Galin, S. R.; Kim, S. H.; Lee, S. J.

    2009-01-01

    The advanced design features of NSSS (Nuclear Steam Supply System) Integrity Monitoring System for Shin-Kori Nuclear Units 3 and 4 are summarized herein. During the overall system design and detailed component design processes, many design improvements have been made for the system. The major design changes are: 1) the application of a common software platform for all subsystems, 2) the implementation of remote access, control and monitoring capabilities, and 3) the equipment redesign and rearrangement that has simplified the system architecture. Changes give an effect on cabinet size, number of cables, cyber-security, graphic user interfaces, and interfaces with other monitoring systems. The system installation and operation for Shin-Kori Nuclear Units 3 and 4 will be more convenient than those for previous Korean nuclear units in view of its remote control capability, automated test functions, improved user interface functions, and much less cabling. (authors)

  3. An Advanced NSSS Integrity Monitoring System for Shin-Kori Nuclear Units 3 and 4

    Oh, Yang Gyun; Galin, Scott R.; Lee, Sang Jeong

    2010-12-01

    The advanced design features of NSSS (Nuclear Steam Supply System) Integrity Monitoring System for Shin-Kori Nuclear Units 3 and 4 are summarized herein. During the overall system design and detailed component design processes, many design improvements have been made for the system. The major design changes are: 1) the application of a common software platform for all subsystems, 2) the implementation of remote access, control and monitoring capabilities, and 3) the equipment redesign and rearrangement that has simplified the system architecture. Changes give an effect on cabinet size, number of cables, cyber-security, graphic user interfaces, and interfaces with other monitoring systems. The system installation and operation for Shin-Kori Nuclear Units 3 and 4 will be more convenient than those for previous Korean nuclear units in view of its remote control capability, automated test functions, improved user interface functions, and much less cabling.

  4. The software testing of PPS for shin Ulchin nuclear power plant units 1 and 2

    Kang, Dong Pa; Park, Cheol Lak; Cho, Chang Hui; Sohn, Se Do; Baek, Seung Min

    2012-01-01

    The testing of software (S/W) is the process of analyzing a software item to detect the differences between existing and required conditions to evaluate the features of the software items. This paper introduces the S/W testing of Plant Protection System (PPS), as a safety system which actuate Reactor Trip (RT) and Engineered Safety Features (ESF) for Shin Ulchin Nuclear Power Plant Units 1 and 2 (SUN 1 and 2)

  5. Treatment efficiency of patients with shin fracture after intraosseous blocked osteosynthesis by using the load dispenser

    Yu. V. Sukhin

    2017-08-01

    Full Text Available The purpose of research: еvaluation of the effectiveness of the device for determining the value of the load on the lower extremity while walking in real time with controlling and signalization of excessive and insufficient load. Materials and methods. Еlaborated and applied device, that allows to determine the load magnitude on the lower extremity in real time, and also to signal about excessive or weak load. The sensory block with the insole and the sensor is located in shoes, under patient's heel, and the main block is fixed on the shin with the help of the strap. Current value of the load on the leg is registered in real time. Received data is recorded in non-volatile memory. The system provides an opportunity to notify patient or doctor by email about the presence of a strong or weak load on the lower extremity, and also about the absence of load for a long period. Results. We used the loading batcher in 38 patients with the shin bones fractures, who were on inpatient treatment at the traumatology and orthopedics center in Odessa in the period from 1.5 to 12 months. The main group included patients, who used the load batcher on the lower extremity in rehabilitation period (transversal fracture of the shin bones diaphysis – 9 patients, oblique fracture – 11 patients. The control group consisted of patients, who didn't use the load batcher (10 patients with oblique fracture of the shin bones in the middle third, 8 patients with transversal fracture of both shin bones in the middle third. As a result of applying the device we succeeded to reduce the fracture fusion period for two weeks and avoid such complications as contracture of joint and fracture non-union. Conclusions. The device allows patients with traumatic consequences reaching the optimal load in rehabilitation period, avoiding excessive load on the lower extremity. The elaboration provides an opportunity to determine the statistics of the load and its transfer to the server

  6. The role of choline (Cho) in the diagnostics and differentiation of brain tumours with HMRS technique

    Sobiecka, B.; Urbanik, A.

    2009-01-01

    Background: The aim of the research was a comprehensive analysis of Cho concentration and Cho/Cr, NAA/Cho, NAA/Cho+Cr ratios for the purposes of the diagnostics and differentiation of brain tumours (the type of the pathological lesion in patients with brain tumours) with the use of HMRS technique. Material/Methods: The HMRS examinations were performed with the use of the MRI Signa Excite 1.5 T system, in PRESS technique (TR = 1500 ms, TE = 35 ms) and involved 100 patients with brain tumours (age range: 18 to 81 yrs, mean age 50.61). Spectra were taken from three different locations: tumour centre, the tumour edge and contralateral unchanged cerebral tissue. All patients underwent surgery followed by histopathological analysis, on the basis of which two groups were separated (benign tumours, malignant tumours - 50 cases each). Additionally, 30 healthy volunteers in the age of 20 to 79 years (mean age 40.8) were examined. Results: The comparison of the examined patients with the control group revealed significantly higher Cho concentrations in patients with brain tumours. The analysis of Cho concentration was also performed with consideration of the age factor (under and over 60 years of age). Significantly lower mean Cho concentrations were discovered in a group of patients under 60 years of age. The analysis of Cho concentrations and Cho/Cr ratios reveled statistical significance for two factors: voxel location factor and the type of the pathological lesion. The average of Cho concentration and Cho/Cr ratios were higher in the group of patients with malignant tumours. The highest Cho concentrations and Cho/Cr ratios were observed in the tumour centre. The relative NAA/Cho and NAA/Cho+Cr ratios were statistically significant when taking into consideration the voxel location factor only. The results received from contralateral normal cerebral tissue (the internal model) were compared with control group (the external model). Mean values of Cho concentration were

  7. Synthesis of human prolactin in Chinese hamster ovary (CHO) cells

    Soares, Carlos Roberto Jorge

    2000-01-01

    Three different eukaryotic expression vectors, based on the same selectable gene marker (dhfr), have been used for dhf- CHO cells transfection to rapidly isolate stable cell lines capable of secreting high levels of recombinant human prolactin (rec-hPRL). Two vectors, one codifying a human prolactin (p658-hPRL) and the other a tag-prolactin (p658-tagPRL), contain the complete hepatitis B virus-X (HBV-X) gene coding for a viral transactivator and a sequence derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF) that mediates selective dhfr mRNA degradation. These vectors have the advantage of rapidly obtaining stable cell lines without methotrexate amplification. The highest secretion obtained by these vectors was of approximately 10 μg hPRU10 6 cells/day. The other vector (pEDdc-hPRL) is based on a dicistronic expression system, containing an internal ribosome entry site isolated from the encephalomyocarditis (EMC) virus. This vector before amplification provided secretion levels at least 10 fold lower than that obtained with the other two vectors. However, after three steps of methotrexate amplification, it provided some clones able to secrete up to 30 μg hPRU10 6 cells/day. This is the first report describing the production and purification of rec-hPRL from CHO cells, obtaining secretion levels with both vectors higher than those reported so far for this hormone in other eukaryotic systems. CHO-derived rec-hPRL contained approximately 10 % of the glycosylated form, a value that is consistent with results reported for hPRL purified from the pituitary or from transformed murine C-127 cells. CHO-derived rec-hPRL was purified with good yield, obtaining also a good resolution between non-glycosylated and glycosylated prolactin. The latter, when its potency was determined via an in vitro bioassay, presented a 47 % lower bioactivity. A qualitative and quantitative analysis of these forms was also possible thanks to the setting up of a reversed

  8. White matter NAA/Cho and Cho/Cr ratios at MR spectroscopy are predictive of motor outcome in preterm infants.

    Kendall, Giles S; Melbourne, Andrew; Johnson, Samantha; Price, David; Bainbridge, Alan; Gunny, Roxanna; Huertas-Ceballos, Angela; Cady, Ernest B; Ourselin, Sebastian; Marlow, Neil; Robertson, Nicola J

    2014-04-01

    To determine (a) whether diffuse white matter injury of prematurity is associated with an increased choline (Cho)-to-creatine (Cr) ratio and a reduced N-acetylaspartate (NAA)-to-Cho ratio and whether these measures can be used as biomarkers of outcome and (b) if changes in peak area metabolite ratios at magnetic resonance (MR) spectroscopy are associated with changes in T2 and fractional anisotropy (FA) at MR imaging. The local ethics committee approved this study, and informed parental consent was obtained for each infant. At term-equivalent age, 43 infants born at less than 32 weeks gestation underwent conventional and quantitative diffusion-tensor and T2-weighted MR imaging. Single-voxel point-resolved proton (hydrogen 1) MR spectroscopy was performed from a 2-cm(3) voxel centered in the posterior periventricular white matter. Outcome was evaluated by using Bayley scales at a corrected age of 1 year. Associations were investigated with Pearson product moment or Spearman rank order correlation. Differences in ratios in infants with and infants without impairment were tested by using t tests. NAA/Cho and Cho/Cr ratios correlated with the scaled gross motor score and the composite motor score, independent of gestational age (P NAA/Cho ratio (P NAA/Cho ratio (P NAA/Cho ratio predicted impaired motor outcome at a corrected age of 1 year with a sensitivity of 0.80 (95% confidence interval [CI]: 0.57, 0.94) and a specificity of 0.80 (95% CI: 0.66, 0.88). The combination of Cho/Cr and NAA/Cho ratios measured in the posterior periventricular white matter at term-equivalent age is predictive of motor outcome at 1 year in infants born at less than 32 weeks gestation. RSNA, 2013

  9. Organisation of Dietary Control for Nutrition-Training Intervention Involving Periodized Carbohydrate (CHO) Availability and Ketogenic Low CHO High Fat (LCHF) Diet.

    Mirtschin, Joanne G; Forbes, Sara F; Cato, Louise E; Heikura, Ida A; Strobel, Nicki; Hall, Rebecca; Burke, Louise M

    2018-02-12

    We describe the implementation of a 3-week dietary intervention in elite race walkers at the Australian Institute of Sport, with a focus on the resources and strategies needed to accomplish a complex study of this scale. Interventions involved: traditional guidelines of high carbohydrate (CHO) availability for all training sessions (HCHO); a periodized CHO diet which integrated sessions with low CHO and high CHO availability within the same total CHO intake, and a ketogenic low-CHO high-fat diet (LCHF). 7-day menus and recipes were constructed for a communal eating setting to meet nutritional goals as well as individualized food preferences and special needs. Menus also included nutrition support pre, during and post-exercise. Daily monitoring, via observation and food checklists, showed that energy and macronutrient targets were achieved: diets were matched for energy (~14.8 MJ/d) and protein (~2.1 g.kg/d), and achieved desired differences for fat and CHO: HCHO and PCHO: CHO = 8.5 g/kg/d, 60% energy; fat = 20% of energy; LCHF: 0.5 g/kg/d CHO, fat = 78% energy. There were no differences in micronutrient intakes or density between HCHO and PCHO diets; however, the micronutrient density of LCHF was significantly lower. Daily food costs per athlete were similar for each diet (~AUDS$27 ± 10). Successful implementation and monitoring of dietary interventions in sports nutrition research of the scale of the present study require meticulous planning and the expertise of chefs and sports dietitians. Different approaches to sports nutrition support raise practical challenges around cost, micronutrient density, accommodation of special needs and sustainability.

  10. Cutset Quantification Error Evaluation for Shin-Kori 1 and 2 PSA model

    Choi, Jong Soo

    2009-01-01

    Probabilistic safety assessments (PSA) for nuclear power plants (NPPs) are based on the minimal cut set (MCS) quantification method. In PSAs, the risk and importance measures are computed from a cutset equation mainly by using approximations. The conservatism of the approximations is also a source of quantification uncertainty. In this paper, exact MCS quantification methods which are based on the 'sum of disjoint products (SDP)' logic and Inclusion-exclusion formula are applied and the conservatism of the MCS quantification results in Shin-Kori 1 and 2 PSA is evaluated

  11. Model for cadmium transport and distribution in CHO cells

    Hayden, T.L.; Turner, J.E.; Williams, M.W.; Cook, J.S.; Hsie, A.W.

    1982-01-01

    A compartmental model is developed to study the transport and distribution of cadmium in Chinese hamster ovary (CHO) cells. Of central importance to the model is the role played by sequestering components which bind free Cd/sup 2 +/ ions. The most important of these is a low-molecular-weight protein, metallothionein, which is produced by the cells in response to an increase in the cellular concentration of Cd/sup 2 +/. Monte Carlo techniques are used to generate a stochastic model based on existing experimental data describing the intracellular transport of cadmium between different compartments. This approach provides an alternative to the usual numerical solution of differential-delay equations that arise in deterministic models. Our model suggests subcellular structures which may be responsible for the accumulation of cadmium and, hence, could account for cadmium detoxification. 4 figures, 1 table.

  12. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

  13. Network reconstruction of the mouse secretory pathway applied on CHO cell transcriptome data

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Brandl, Julian

    2017-01-01

    , counting 801 different components in mouse. By employing our mouse RECON to the CHO-K1 genome in a comparative genomic approach, we could reconstruct the protein secretory pathway of CHO cells counting 764 CHO components. This RECON furthermore facilitated the development of three alternative methods...... to study protein secretion through graphical visualizations of omics data. We have demonstrated the use of these methods to identify potential new and known targets for engineering improved growth and IgG production, as well as the general observation that CHO cells seem to have less strict transcriptional...... regulation of protein secretion than healthy mouse cells.  Conclusions: The RECON of the secretory pathway represents a strong tool for interpretation of data related to protein secretion as illustrated with transcriptomic data of Chinese Hamster Ovary (CHO) cells, the main platform for mammalian protein...

  14. Development of hyper osmotic resistant CHO host cells for enhanced antibody production.

    Kamachi, Yasuharu; Omasa, Takeshi

    2018-04-01

    Cell culture platform processes are generally employed to shorten the duration of new product development. A fed-batch process with continuous feeding is a conventional platform process for monoclonal antibody production using Chinese hamster ovary (CHO) cells. To establish a simplified platform process, the feeding method can be changed from continuous feed to bolus feed. However, this change induces a rapid increase of osmolality by the bolus addition of nutrients. The increased osmolality suppresses cell culture growth, and the final product concentration is decreased. In this study, osmotic resistant CHO host cells were developed to attain a high product concentration. To establish hyper osmotic resistant CHO host cells, CHO-S host cells were passaged long-term in a hyper osmotic basal medium. There were marked differences in cell growth of the original and established host cells under iso- (328 mOsm/kg) or hyper-osmolality (over 450 mOsm/kg) conditions. Cell growth of the original CHO host cells was markedly decreased by the induction of osmotic stress, whereas cell growth of the hyper osmotic resistant CHO host cells was not affected. The maximum viable cell concentration of hyper osmotic resistant CHO host cells was 132% of CHO-S host cells after the induction of osmotic stress. Moreover, the hyper osmotic resistant characteristic of established CHO host cells was maintained even after seven passages in iso-osmolality basal medium. The use of hyper osmotic resistance CHO host cells to create a monoclonal antibody production cell line might be a new approach to increase final antibody concentrations with a fed-batch process. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  15. Application of a Virtual Ovation System to the ShinKori-3 Simulator

    Hong, Jin Hyuk; Lee, Myeong Soo; Chung, Kyung Hun

    2011-01-01

    The Ovation system for the Shin-Kori Unit 3 Simulator is essentially a non-redundant, repackaged subset of the actual plant I and C equipment, with additional interface computers (SimStations). This system also present in the simulator to provide communication between the plant model computer and the stimulated Ovation equipment. The stimulated Ovation equipment in the simulator system includes Operator HMI (Human- Machine Interface) equipment and Ovation virtual controllers hosted by Virtual Controller Host workstations, which are not present in the actual plant DCS system. The simulator for the Shin-Kori Unit 3 and 4 is being developed by Korea Hydro and Nuclear Power's Central Research Institute (KHNP CRI). One of the features of the simulator is its application of a virtual Ovation system capable of simulated functionalities such as run, freeze, snapshot, backtrack and others required by ANSI/ANS-3.5 in addition to the original functionality for the actual Ovation system applied at the plant. This is the first application of a virtual Ovation system to a full-scope simulator for a nuclear power plant in Korea. The purpose of this paper is to provide the overall architecture of the communication system between the virtual system and the simulator model and to describe the current situation of the development of the system and recent relevant studies

  16. The Life and Works of Han Shin Gwang : a Midwife and Nurse of Korean Modern Times

    YI Ggodme

    2006-06-01

    Full Text Available Han Shin Gwang, born in an early Christian family in Korea in 1902, could get western education different from the ordinary Korean girls in that period. She participated in the 1919 Samil Independence Movement in her teens, and got nursing and midwifery education in a missionary hospital. She got a midwife license and worked as a member in an early mother-and-child health center. She organized 'Korean Nurses' Association' in 1924 and focused on public health movement as the chairwoman. She actively participated in women's movement organizations, and Gwangjoo Student's Movement. She was known to be a representative of leading working women, and wrote articles on woman's right, the needs and works of nurses and midwives. From late Japanese colonial period, she opened her own clinic and devoted herself to midwifery. After the Korean Liberation in 1945, she began political movement and went in for a senate election. During the Korean War, she founded a shelter for mothers and children in help. After the War, she reopened a midwifery clinic and devoted to the works of Korean Midwives' Association. Han Shin Gwang's life and works belong to the first generation of Korean working women in modern times. She actively participated in women's movement, nurses' and midwives professional movement, Korea liberation movement, and mother-and-child health movement for 60 years. Her life is truly exemplary as one of the first generation of working women in modern Korea, distinguished of devotion and calling.

  17. CHO glyco-engineering using CRISPR/Cas9 multiplexing for protein production with homogeneous N-glycan profiles

    Amann, Thomas; Hansen, Anders Holmgaard; Pristovsek, Nusa

    Combining the chinese hamster ovary (CHO) - K1 draft genome1,2, identified CHO glycosyltransferases3 and the power of multiplexing gene knock-outs with CRISPR/Cas94 via co-transfection of Cas9 and one single guiding RNA (sgRNA) per target, we generated 20 Rituximab expressing CHO-S cell lines...

  18. Complete genome sequence of Vibrio anguillarum phage CHOED successfully used for phage therapy in aquaculture

    Romero, Jaime; Higuera, Gastón; Gajardo, Felipe

    2014-01-01

    Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V....... anguillarum phage CHOED....

  19. The emerging CHO systems biology era: harnessing the ‘omics revolution for biotechnology

    Kildegaard, Helene Faustrup; Baycin-Hizal, Deniz; Lewis, Nathan

    2013-01-01

    into mathematical models that describe CHO phenotypes will provide crucial biotechnology insights. As ‘omics technologies and computational systems biology mature, genome-scale approaches will lead to major innovations in cell line development and metabolic engineering, thereby improving protein production......Chinese hamster ovary (CHO) cells are the primary factories for biopharmaceuticals because of their capacity to correctly fold and post-translationally modify recombinant proteins compatible with humans. New opportunities are arising to enhance these cell factories, especially since the CHO-K1 cell...

  20. RNA-Seq Highlights High Clonal Variation in Monoclonal Antibody Producing CHO Cells

    Orellana, Camila A.; Marcellin, Esteban; Palfreyman, Robin W.

    2018-01-01

    The development of next-generation sequencing technologies has opened new opportunities to better characterize complex eukaryotic cells. Chinese hamster ovary (CHO) cells play a primary role in therapeutic protein production, with currently five of the top ten blockbuster drugs produced in CHO......-regulation of genes encoding secreted glycoproteins is found to be the most significant change. The large number of significant differences even between subclones challenges the notion of identifying and manipulating a few key genes to generate high production CHO cell lines....

  1. RNA-seq based expression analysis of the CHO cell protein secretion pathway

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Kildegaard, Helene Faustrup

    The Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant therapeutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies...... of the cell biology of the CHO cell and the potential of cell line engineering. To elucidate the poorly understood cellular processes that control and limit recombinant protein production and secretion, a system-wide study was initiated to identify possible engineering targets relevant for therapeutic protein...

  2. Cho decomposition of electrically charged one-half monopole

    Ng, Ban-Loong; Teh, Rosy; Wong, Khai-Ming [School of Physics, Universiti Sains Malaysia, 11800 USM Penang (Malaysia)

    2014-03-05

    Recently we have carried out some work on the Cho decomposition of the electrically neutral, finite energy one-half monopole solution of the SU(2) Yang-Mills-Higgs field theory. In this paper, we performed the decomposition of the electrically charged solution using the same numerical procedure. The gauge potential of the one-half dyon solution is decomposed into Abelian and non-Abelian components. The semi-infinite string singularity in the gauge potential is a contribution of the Higgs field and hence topological in nature. The string singularity cannot be cancelled by the non-Abelian components of the gauge potential. However, the string singularity is integrable and the energy of the solution is finite. By decomposing the magnetic fields and covariant derivatives of the Higgs field into three isospin space directions, we are able to provide conclusive evidence that the constructed one-half dyon is certainly a non-BPS solution even in the limit of vanishing Higgs self-coupling constant and electric charge. Furthermore, we found that the time component of gauge function is parallel to the Higgs field in isospace only at large distances, elsewhere they are non-parallel.

  3. Protein synthesis and sublethal damage repair in synchronized CHO cells

    Yezzi, M.J.; Tobias, C.A.; Blakely, E.A.

    1984-01-01

    The authors have previously reported that the split dose survival response to x-rays of asynchronous CHO-TSH1 cells is reduced if the cells are held at 40 0 C,a temperature that inhibits protein synthesis, for 2 hours before the first dose and during a 2-hour interval between doses. In conjunction with the survival experiments on asynchronous cells, the authors also examined the DNA rejoining ability in split dose studies with and without inhibition of protein synthesis. The results of these experiments suggest that inhibition of protein synthesis affects a pool of proteins that are necessary for the correct expression of the DNA, although they do not appear to be involved in rejoining DNA breaks. They have extended this work to the study of cells synchronized in G1 phase (2 hour post-mitosis) and S phase (10 hour post-mitosis). Autoradiographic analyses, using 3H-TdR pulse labeling, demonstrated that a delay in the progression of each synchronized cell population occurs after inhibition of protein synthesis. Data are reported on the effects of inhibition of protein synthesis on the ability of G1 and S phase cells to repair sublethal damage

  4. Analysis of Hydrogen Concentration Distribution during an SBO Accident for Shin-Ulchin APR1400

    Kim, Jongtae; Hong, Seong Wan [Korea Atomic energy Research Institute, Daejeon (Korea, Republic of)

    2013-10-15

    To prohibit the accumulation of hydrogen, the containment volume is considered to reduce the hydrogen concentration, or hydrogen mitigation devices such as PARs or igniters are installed in the containment. In the case of the Fukushima NPPs, the applied strategy for the hydrogen safety is the use of a containment venting system (CVS). In this way, the hydrogen accumulated in the containment vessel is vented into the environment. One of the causes of the hydrogen explosions occurring in the containment buildings of the Fukushima NPPs is expected to be the failure of the venting system. The hydrogen was therefore easily accumulated in the containment building. It is uncertain what the ignition source for the hydrogen combustion was during the accident. However, it is not too conservative to assume that an ignition source exists at any time and any place in a containment during a core-melt accident. Shin-Ulchin 1 and 2, which are construction plants of an APR 1400, are two of the newest NPPs in Korea. They have many features to enhance the safety margin during a design-based and beyond-design-based accident. One of them is the in-containment refueling water storage tank (IRWST) located inside the containment. It is used as a sink/source for feed-bleed operation. When the core is damaged along an accident progression, the hydrogen generated in the RPV can be released into the IRWST of the APR1400 with steam and water. From a previous study, it was found that a highly concentrated hydrogen/air mixture can be developed if the hydrogen is released into the IRWST. In the case of Shin-Ulchin 1 and 2, the hydrogen mitigation strategy during a high-pressure accident such as a station blackout (SBO) is changed by installing a 3-way valve. When a severe accident management (SAM) for the plant is initiated, the flow path from a pressurizer to the IRWST is changed into a steam-generator (S/G) compartment by turning the 3-wat valve actively (pilot operated). By doing so, it is

  5. CHOmine: an integrated data warehouse for CHO systems biology and modeling.

    Gerstl, Matthias P; Hanscho, Michael; Ruckerbauer, David E; Zanghellini, Jürgen; Borth, Nicole

    2017-01-01

    The last decade has seen a surge in published genome-scale information for Chinese hamster ovary (CHO) cells, which are the main production vehicles for therapeutic proteins. While a single access point is available at www.CHOgenome.org, the primary data is distributed over several databases at different institutions. Currently research is frequently hampered by a plethora of gene names and IDs that vary between published draft genomes and databases making systems biology analyses cumbersome and elaborate. Here we present CHOmine, an integrative data warehouse connecting data from various databases and links to other ones. Furthermore, we introduce CHOmodel, a web based resource that provides access to recently published CHO cell line specific metabolic reconstructions. Both resources allow to query CHO relevant data, find interconnections between different types of data and thus provides a simple, standardized entry point to the world of CHO systems biology. http://www.chogenome.org. © The Author(s) 2017. Published by Oxford University Press.

  6. A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing

    Khandeparker, R.; Verma, P.; Deobagkar, D.

    A novel halotolerant xylanase from marine bacterium Bacillus subtilis cho40 isolated from Chorao island of Mandovi estuary Goa, India has been reported. Extracellular xylanase was produced by using agricultural residue such as wheat bran as carbon...

  7. CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E.

    2015-01-01

    repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid,easy and efficient engineering of mammalian genomes. It has a wide range of applications frommodification of individual genes to genome-wide screening or regulation of genes. Facile genomeediting using CRISPR/Cas9 empowers...... researchers in the CHO community to elucidate the mechanisticbasis behind high level production of proteins and product quality attributes of interest. Inthis review, we describe the basis of CRISPR/Cas9-mediated genome editing and its applicationfor development of next generation CHO cell factories while...... highlighting both future perspectivesand challenges. As one of the main drivers for the CHO systems biology era, genome engineeringwith CRISPR/Cas9 will pave the way for rational design of CHO cell factories....

  8. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most....... Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance...... property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production....

  9. Metabolite profiling of recombinant CHO cells: Designing tailored feeding regimes that enhance recombinant antibody production.

    Sellick, C.A.; Croxford, A.S.; Maqsood, A.R.; Stephens, G.; Westerhoff, H.V.; Goodacre, R.; Dickson, A.J.

    2011-01-01

    Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the

  10. Metabolite profiling of recombinant CHO cells: designing tailored feeding regimes that enhance recombinant antibody production.

    Sellick, C.A.; Croxford, A.S.; Maqsood, A.R.; Stephens, G.; Westerhoff, H.V.; Goodacre, R.; Dickson, A.J.

    2011-01-01

    Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the

  11. THE METHOD OF PNEUMATIC IMITATION OF WEIGHT-BEARING IN CHILDREN WITH SHIN FRACTURES

    N. Yu. Serova

    2012-01-01

    Full Text Available The article contains a comparative analysis of shin fracture treatment in 102 children at the age of 4–18 years old. The main group consisted of 51 (50% children, who received postoperative treatment according to the method of pneumatic imitation of weightbearing both on health and injured limbs with «Korvit» apparatus. The control group consisted of 51 (50% patients who did not received the afore-mentioned method of postoperative treatment. The groups were comparable on age, trauma mechanism and localization, surgical operation and complex postoperative therapy. Children of the first group were found to have shorter period of time needed to achievement of pain relief, soft tissue swelling decrease, restoration of the movements in adjacent joints and somatometry values normalization, which allowed earlier verticalization and shorter period of hospitalization.

  12. The soleus syndrome. A cause of medial tibial stress (shin splints).

    Michael, R H; Holder, L E

    1985-01-01

    Radionuclide bone scans have demonstrated linear uptake along the posterior medial border of the tibia in patients with shin splints. This area was investigated by anatomical dissection (14 human cadavers), electromyographic (EMG) and muscle stimulation studies (10 patients), and open biopsy (1 patient). Histologically, the increased metabolic activity manifested on the radionuclide scan is due to a periostitis with new bone formation. The soleus muscle and its investing fascia are anatomically and biomechanically implicated in the production of these stress changes, particularly when the heel is in the pronated position. The soleus muscle and fascia form a tough "soleus bridge" over the deep compartment which is thought to be important in patients requiring surgical decompression.

  13. Bone tissue density modification in treatment of shin pseudoarthrosis by transosseous compressive osteosynthesis

    Tishkov N.V.

    2011-12-01

    Full Text Available Objective is to detect bone mineral density along the shin according to «Esperanto» levels by Hounsfield's scale. Materials and methods. The analysis of density modification in 25 patients with pseudoarthrosis of tibia with predominant localization in a lower one-third of bone has been carried out. Results. By means of computed tomography it has been revealed that the bone tissue density of the tibia in the process of false joint union when using the compressive variant of combined transosseous osteosynthesis has changed according to the regularity reproducing phase character of the accumulation of mineral substances in the bone. Conclution. The growth of mineral density of the bone tissue during treatment spreads in the directions from proximal and distal metaepiphyses to the zone of pseudoarthrosis knitting

  14. PORNOGRAFI DALAM SERIAL ANIME ANAK (ANALISIS SEMIOTIKA DALAM SERIAL CRAYON SHIN CHAN

    - Sangidun

    2017-01-01

    Full Text Available Crayon Shin Chan, a Japanese two-dimension animation series broadcast in one of private Indonesian TVs, is categorized into child’s program since it is broadcast at child’s prime time, Sunday 08.30 a.m. In spite of its broadcast time, this series consist of symbols directed not for children, such as some acts that are not appropriate to be done by children, especially in Indonesia. Moreover, adult symbols of sex are also found in the program. For this reason it will be interesting to analyze it using semiotic analysis. Semiotics is the study of symbol and its meaning which its principle concept is that both signifier and signified consist of symbols and are related to denotation and connotation.   Crayon Shin Chan merupakan serial animasi dua dimensi yang tayang di salah satu stasiun televisi swasta di Indonesia. Ini merupakan produk animasi 2 dimensi yang diimpor dari Jepang. Di Indonesia, serial ini masuk dalam kategori acara anak. Hal ini dapat dilihat dari jam penayangannya yang merupakan waktu prime time bagi anak, yakni pada hari minggu pukul 08.30. Akan tetapi, pada serial ini banyak simbol-simbol yang mengarah pada tayangan yang bukan untuk anak-anak, yakni adeganadegan yang tidak pantas dilakukan oleh anak khususnya di Indonesia. Serta adanya pula simbol-simbol yang mengarah pada tayangan berbau dewasa. Tentu akan menarik jika tayangan ini diteliti menggunakan analisis semiotika. Semiotika sendiri merupakan kajian ilmu mengenai tanda dan makna. Yang pada prinsipnya, konsep penting seperti penanda (signifier dan petanda (signified sama-sama terdiri dari tanda dan terkait dengan denotasi dan konotasi.

  15. Widespread extrahippocampal NAA/(Cr+Cho) abnormalities in TLE with and without mesial temporal sclerosis.

    Mueller, Susanne G; Ebel, Andreas; Barakos, Jerome; Scanlon, Cathy; Cheong, Ian; Finlay, Daniel; Garcia, Paul; Weiner, Michael W; Laxer, Kenneth D

    2011-04-01

    MR spectroscopy has demonstrated extrahippocampal NAA/(Cr+Cho) reductions in medial temporal lobe epilepsy with (TLE-MTS) and without (TLE-no) mesial temporal sclerosis. Because of the limited brain coverage of those previous studies, it was, however, not possible to assess differences in the distribution and extent of these abnormalities between TLE-MTS and TLE-no. This study used a 3D whole brain echoplanar spectroscopic imaging (EPSI) sequence to address the following questions: (1) Do TLE-MTS and TLE-no differ regarding severity and distribution of extrahippocampal NAA/(Cr+Cho) reductions? (2) Do extrahippocampal NAA/(Cr+Cho) reductions provide additional information for focus lateralization? Forty-three subjects (12 TLE-MTS, 13 TLE-no, 18 controls) were studied with 3D EPSI. Statistical parametric mapping (SPM2) was used to identify regions of significantly decreased NAA/(Cr+Cho) in TLE groups and in individual patients. TLE-MTS and TLE-no had widespread extrahippocampal NAA/(Cr+Cho) reductions. NAA/(Cr+Cho) reductions had a bilateral fronto-temporal distribution in TLE-MTS and a more diffuse, less well defined distribution in TLE-no. Extrahippocampal NAA/(Cr+Cho) decreases in the single subject analysis showed a large inter-individual variability and did not provide additional focus lateralizing information. Extrahippocampal NAA/(Cr+Cho) reductions in TLE-MTS and TLE-no are neither focal nor homogeneous. This reduces their value for focus lateralization and suggests a heterogeneous etiology of extrahippocampal spectroscopic metabolic abnormalities in TLE.

  16. Metaphase chromosome and nucleoid differences between CHO-K1 and its radiosensitive derivative xrs-5

    Schwartz, J.L.; Stephens, J.; Vaughan, A.T.M.

    1992-05-01

    The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. Chromatin structure also appears altered in xrs-5 cells compared to the parental CHO-K1 cells. Metaphase chromosomes from xrs-5 are more condensed in appearance than CHO-K1 chromosomes. The overcondensed look is not the result of colcemid sensitivity. Electron microscopy studies suggest that xrs-5 metaphase chromosomes have larger loops of chromatin extending out from the chromosome core. There are also differences between CHO-K1 and xrs-5 cells in the size and fluorescence pattern of ethidium bromide-stained nucleoid preparations. These results suggest that there is a fundamental difference between CHO-K1 and xrs-5 in either the organization of the supercoiled loops of DNA attached to the nuclear matrix or in the nature of the proteins that attach the DNA to the matrix. These alterations in chromosome structure may underlie, in part, the radiation sensitivity of xrs-5 cells

  17. Metaphase chromosome and nucleoid differences between CHO-K1 and its radiosensitive derivative xrs-5

    Schwartz, J.L.; Stephens, J.; Vaughan, A.T.M.

    1993-01-01

    The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiosensitivity is associated with a defect in DNA double-strand break rejoining. Chromatin structure also appears altered in xrs-5 cells compared with the parental CHO-K1 cells. Metaphase chromosomes form xrs-5 are more condensed in appearance than CHO-K1 chromosomes. The overcondensed look is not the result of colcemid sensitivity. Electron microscopy studies suggest that xrs-5 metaphase chromosomes have larger loops of chromatin extending out from the chromosome core. There are also differences between CHO-K1 and xrs-5 cells in the size and fluorescence pattern of ethidium bromide-stained nucleoid preparations. These results suggest that there is a fundamental difference between CHO-K1 and xrs-5 in either the organization of the supercoiled loops of DNA attached to the nuclear matrix or in the nature of the proteins that attach the DNA to the matrix. These alterations in chromosome structure may underlie, in part the radiation sensitivity of xrs-5 cells. (Author)

  18. Evaluation of SPACE code for simulation of inadvertent opening of spray valve in Shin Kori unit 1

    Kim, Seyun; Youn, Bumsoo

    2013-01-01

    SPACE code is expected to be applied to the safety analysis for LOCA (Loss of Coolant Accident) and Non-LOCA scenarios. SPACE code solves two-fluid, three-field governing equations and programmed with C++ computer language using object-oriented concepts. To evaluate the analysis capability for the transient phenomena in the actual nuclear power plant, an inadvertent opening of spray valve in startup test phase of Shin Kori unit 1 was simulated with SPACE code. To evaluate the analysis capability for the transient phenomena in the actual nuclear power plant, an inadvertent opening of spray valve in startup test phase of Shin Kori unit 1 was simulated with SPACE code

  19. Report on the project for working out the new energy vision of the Kamoto Town area. Feasibility study; 2001 nendo Kamoto machi chiiki shin energy vision sakutei tou jigyo fijibiriti hokokusho

    NONE

    2002-02-01

    For the realization of 'the introduction of biomass of animal manure/garbage,' an important item in 'the new energy vision of the Kamoto Town area (Kumamoto Prefecture),' a feasibility study was conducted. For the resource retrieval of animal manure, etc., only composting is generally conducted. In this project, however, two projects are combinedly conducted: project for producing biogas (main component: methane gas) and liquid fertilizer and project for producing compost. The project pursues the reduction in running cost by self-sufficiency of energy and the environmental preservation by recovery of methane gas. The plant for producing biogas and liquid fertilizer consists of garbage crusher, storage tank, anaerobic fermentation tank and generator. The plant for producing compost consists of solid liquid separator, fermentation tank, agitator and smell remover. In the feasibility study, the following were carried out: study of the establishment of specifications/selection of the necessary conditions for a plant tentatively named the Kamoto Town Biomass Center, study of the proposed site for plant, survey of the financial plan, survey of the operational system, study of the project promotion system, etc. (NEDO)

  20. Report on the initial-stage survey of the new energy vision of the Izumi city area; 2001 nendo Izumi shi chiiki shin energy vision shoki dankai chosa hokokusho

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Izumi City, Osaka Prefecture, an investigational study was conducted of the structure of energy consumption, existence amount of new energy, subjects in utilization of new energy, etc. The energy consumption amount of the city is increasing mostly in electric power in the residential sector, but is decreasing in the business sector and industrial sector. It is leveling off in the transportation sector, but only the consumption amount of gasoline is markedly increasing. In the potential amount of new energy utilization in the city, the rate of solar energy/clean energy vehicles was high. As a result of the study, the following were made clear: It is effective to introduce new energy mostly used for citizen such as the household use photovoltaic power system/solar heat use hot water supplier/hybrid private car. Of waste energy, wind energy, biomass energy and temperature difference energy, it is effective for city to take the initiative in introducing them. It is effective for city/proprietor to introduce the natural gas cogeneration. (NEDO)

  1. Survey report of FY 1996 on the questionnaires related to new energies for local governments in Kinki district; 1996 nendo chosa hokokusho (shin energy ni kansuru Kinki chiiki jichitai anketo chosa)

    NONE

    1997-03-01

    The photovoltaic (PV) power generation system having a lot of merits for environment and energy has been put into a diffusion stage from the viewpoint of its technology due to its progressive technology innovation. However, it is not widely diffused in the whole society due to its high cost. Systematic investigation for the future PV power generation was conducted through the verification of factors obstructing the diffusion promotion and the investigation of the problems. Then, questionnaires were conducted for local governments in the Kinki district. Proportion of the local governments positively promoting the introduction of PV power generation was 3.4%. Among new energies, 45.7% of the governments were interested in solar heat utilization, and 43.1% were interested in PV power generation. Proportion of the local governments understanding the subsidy system of MITI was 57.7%. For the explanation meeting of new energy introduction promotion, 55.4% of the local governments wanted to participate. Among the interesting themes, 61.0% were interested in the introduction examples, 52.4% were in the introduction support, 52.1% were in the introduction of fundamental knowledge, and 30.0% were in the latest technology trends and in the guide for introduction

  2. Regional new energy vision for Kamaishi City. Energy to create community's future out of natural resources; 2001 nendo Kamaishi shi chiiki shin energy vision. Megumi kara machi no mirai wo tsukuru energy

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Kamaishi City, Iwate Prefecture, surveys and studies were conducted involving the amount of energy needed by the city, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The city consumed 5,334 times 10{sup 12}J in fiscal 1999, which amount occupied 0.034% of the nation-wide consumption. Concerning the total consumed by the city, the manufacturing sector was responsible for 36% and the transportation sector for 24%. Oil provided 60% of the energy, and electric power 24%. These percentages were similar to those found across the nation. The amount of carbon dioxide emissions was estimated to be equivalent to 463-million kg-CO2. Projects for new energy introduction were studied, which included photovoltaic power generation for primary and junior high school buildings, the new city office, road signs along the municipal roads, and the harbor area; hybrid photovoltaic/wind power generation for a newly built fish market; solar heat utilization for heating frozen roads; wind power generation for the Wayama and Naranoki stock farms and for the harbor area; and a business for utilizing waste heat discharged by Kamaishi City's Eco-Town project. (NEDO)

  3. New energy visions for the Town of Naie district. Human-friendly Town of Naie City by environment-friendly energies; Naiemachi chiiki shin energy vision. Kankyo ni yasashii energy ga tsukuru hito ni yasashii machi Naie

    NONE

    2001-03-01

    The new energy visions are drawn for Town of Naie, Sorachi-gun in Hokkaido, where the industrial sector (manufacturing industries) consumes more than 60% of the total energy consumption. By energy type, oil and others account for 80%. When arranged in the order of expected recoverable quantity, the new energies for the town include utilization of solar energy, photovoltaic power generation, wind power generation, and utilization of wastes, biomass energy (agricultural products) and snow energy. The basic guidelines for introduction of new energies are for health and welfare, clearing and utilization of snow, environmental education and bringing up talented persons, supporting the related entrepreneurs, and integration of new energy with promotion of energy-saving movement. The priority projects the town plans to promote include utilization of biogas and temperature-differential energy for the town's water purification center; foundation of the Naie eco-school; and drawing conceptions for utilization of underground heat, utilization of low-temperature and snow/ice energy, co-energy around the spa of Naie, and mega-solar and eco-park at the Sorachi housing complex. (NEDO)

  4. Report on regional new energy vision for Kawara town. Toward the creation of eco-symbiotic community (simplified version); Kawaracho chiiki shin energy vision gaiyoban. Kankyo kyoseigata shakai no sozo ni mukete

    NONE

    2001-02-01

    A regional new energy vision has been formulated for Kawara town, Fukuoka Prefecture, in the hope that the industry, administration, and citizenry will be concerned with each other for the realization of an eco-symbiotic community. The fruits of endeavors exerted for this purpose are described in seven chapters, which are (1) the outline of surveys, (2) local characteristics of Kawara town, (3) policy toward introducing new energy, (4) measures for introducing new energy, (5) study of model projects, (6) introduction of new energy into Kawara town, and (7) efforts to realize the introduction. Described in chapter (5) are the introduction of photovoltaic power generation into primary and junior high schools, heat supply business utilizing waste heat from a plant, energy conservation at administration buildings, development of an activities program for people to learn energy related matters, and the results of studies conducted on the respective model projects. Concerning the activities program, several applications are considered, dependent upon who are to implement the program: volunteer students, local community leaders, experts, or administration officials. For the initial stage of learning under the program, a system is built under which experts, local community leaders, primary and junior highschool children, and their teachers cooperate with each other under the leadership of a project coordinator. (NEDO)

  5. Building a network of flowers, green, and ocean. Investigation report on establishment of new energy vision in Wada Town area (Chiba Prefecture); Hana midori umi no network zukuri. Wadacho chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2001-02-01

    With the idea of living together with well-blessed nature placed as the base of building the town, new energy utilization promoting measures were established spanning over ten years starting fiscal 2000. Specifically, the vision was established with the following points as the substance: introduction of new energy technologies into the agricultural and fishery industries as the basic district industry, initiative introduction into public facilities in consideration of repercussion effects brought about by the introduction, provision of energy related information by improving publicity magazines and information technologies, and expanded provision of environmental information collaborated with schools. Specific measures discussed include photovoltaic power generation, utilization of bio-mass energies made available from livestock excreta, and algae, utilization of hydro-energy through the improvement project of the Komukai dam, utilization of wind power energy and ocean wave power energy, and utilization of rape-seed oil as automotive fuel. Such educational activities will also be promoted actively as the setting of economy indexes, assistance to autonomous activities, and holding training meetings, seminars, and experience education class rooms. (NEDO)

  6. New energy visions for the Town of Naie district. Human-friendly Town of Naie City by environment-friendly energies; Naiemachi chiiki shin energy vision. Kankyo ni yasashii energy ga tsukuru hito ni yasashii machi Naie

    NONE

    2001-03-01

    The new energy visions are drawn for Town of Naie, Sorachi-gun in Hokkaido, where the industrial sector (manufacturing industries) consumes more than 60% of the total energy consumption. By energy type, oil and others account for 80%. When arranged in the order of expected recoverable quantity, the new energies for the town include utilization of solar energy, photovoltaic power generation, wind power generation, and utilization of wastes, biomass energy (agricultural products) and snow energy. The basic guidelines for introduction of new energies are for health and welfare, clearing and utilization of snow, environmental education and bringing up talented persons, supporting the related entrepreneurs, and integration of new energy with promotion of energy-saving movement. The priority projects the town plans to promote include utilization of biogas and temperature-differential energy for the town's water purification center; foundation of the Naie eco-school; and drawing conceptions for utilization of underground heat, utilization of low-temperature and snow/ice energy, co-energy around the spa of Naie, and mega-solar and eco-park at the Sorachi housing complex. (NEDO)

  7. Report on the survey on the project for working out the new energy vision of Oyamada Village; 2001 nendo Oyamada mura chiiki shin energy vision sakutei tou jigyo chosa hokokusho

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Oyamada Village, Mie Prefecture, an investigational study was conducted of the energy demand amount, existence amount of new energy and project for new energy introduction, and a vision was worked out. The energy demand amount of the village was 672,289 x 10{sup 3} MJ/y. The rate of energy source was about 27% of electric power and 65% of petroleum-base such as gasoline, light oil and kerosene. The amount of CO2 emitted from these was estimated at 50,333 t-CO2. As viewed by sector, the energy demand amount consisted of 46.7% in the transportation sector, 36.3% in the industrial sector, 9.6% in the residential sector and 7.3% in the business sector. As the project for new energy introduction, the following were studied: Aoyama Heights wind farm (wind power generation: 700kW x 12 turbines); wind power generation project jointly with citizen; introduction of photovoltaic power generation/solar heat utilization to public facilities; greenhouse of Western orchard using waste heat from hot spring, stockbreeding waste utilization project, recycle project for thinnings, introduction of clean energy vehicle, etc. (NEDO)

  8. Report on the survey for working out the new energy vision of the Sen'nan city area; 2001 nendo Sen'nan shi chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Sen'nan City, Osaka Prefecture, an investigational study was conducted of the structure of energy demand, existence amount of new energy, project for new energy introduction, etc. The energy consumption amount of the city in FY 1999 was estimated at 4,971TJ. It consisted of 40.6% in the industrial sector, 29.7% in the transportation sector and 29.6% in the commercial/residential sector. The rate of energy source was 48.8% of petroleum, 32.1% of electric power and 19.1% of gas. As the project for new energy introduction, the following were studied: introduction of photovoltaic power generation and solar heat utilization facilities to the city office/elementary school/junior high school/municipal dwelling houses; street light combinedly using photovoltaic power generation and wind power generation; introduction of clean energy vehicle to official vehicle; preferential treatment for introduction of low-emission car for citizen; introduction of natural gas cogeneration and fuel cell to the city office, etc.; introduction of small-size wind power generation/small- and medium-size hydraulic power generation to public facilities, park, etc.; construction of the waste energy utilization system. (NEDO)

  9. Report on regional new energy vision for Kawara town. Toward the creation of eco-symbiotic community; Kawaracho chiiki shin energy vision hokokusho. Kankyo kyoseigata shakai no sozo ni mukete

    NONE

    2001-02-01

    A regional new energy vision has been formulated for Kawara town, Fukuoka Prefecture, in the hope that the industry, administration, and citizenry will be concerned with each other for the realization of an eco-symbiotic community. The fruits of endeavors exerted for this purpose are described in seven chapters, which are (1) the outline of surveys, (2) local characteristics of Kawara town, (3) policy toward introducing new energy, (4) measures for introducing new energy, (5) study of model projects, (6) introduction of new energy into Kawara town, and (7) efforts to realize the introduction. Described in chapter (5) are the introduction of photovoltaic power generation into primary and junior high schools, heat supply business utilizing waste heat from a plant, energy conservation at administration buildings, and the development of an activities program for people to learn energy related matters. Several applications are considered concerning the activities program, dependent upon who are to implement the program: volunteer students, local community leaders, experts, or administration officials. In relation to heat supply business, studies are conducted about the supply of waste heat from a cement plant to public facilities such as the town hall and administration buildings in the form of medium pressure steam or hot water. (NEDO)

  10. FY 2001 report on the initial-stage survey of the project for working out the new energy vision of the Okinawa City area; 2001 nendo Okinawa shi chiiki shin energy vision sakutei chosa. shoki dankai chosa hokokusho

    NONE

    2002-02-01

    For working out a new energy vision in Okinawa City, an investigational survey was conducted, as the initial-stage survey, of the state of energy demand of the city, estimated amount of existence of new energy, proposed project (draft) on new energy introduction, etc. The energy demand amount of Okinawa City was estimated at 6,865 x 10{sup 6} MJ/y. By sector, it consisted of 41.6% in the transportation sector, 35.6% in the commercial/residential sector and 22.8% in the industrial sector. The rate of energy source was 50.5% of petroleum-base fuel, 31.0% of electric power and 9.5% of LP gas. The expected minable amount of new energy was estimated at 318,513 x 10{sup 3} MJ/y, which is equivalent to 17 x the energy consumption amount/day of the city. As the proposed project (draft) for new energy introduction, study was made of the introduction of photovoltaic power system/solar heat utilization system to the children future zone park/elementary school/junior high school/kindergarten/public hall, introduction of clean energy vehicle to official vehicle of the city, refuse incineration power generation/utilization of incineration heat at Kurahama sanitary facilities, and others. (NEDO)

  11. New energy vision of the Tagawa city area. Aiming at a town for symbiosis of humans and the environment; 2001 nendo Tagawa shi chiiki shin energy vision. Hito to kankyo ga kyoseisuru machi wo mezashite

    NONE

    2002-03-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Tagawa City, Fukuoka Prefecture, an investigational study was conducted of the energy supply/demand of the city, existence amount of new energy, project for new energy introduction, etc., and a vision was worked out. The energy consumption amount of the city was estimated at 6,678,932 GJ/y. It consisted of 42% in the industrial sector, 35% in the transportation sector and 23% in the commercial/residential sector. The rate of energy source was 75% of petroleum-base energy and 25% of electric power. As the project for new energy introduction, the following were studied: power generation/thermal exchange/composting by fermentation of methane from stockbreeding waste; environmental preservation type agriculture such as large-scale hothouse using exhaust heat from cement plant; utilization of ground mine water at coal goaf for space cooling and as water source; introduction of photovoltaic power generation/solar energy use hot water supply system to public facilities; introduction of clean energy car to vehicle in welfare/education fields; preparation of a program for energy environment education; preparation of bases for energy environment education. (NEDO)

  12. Report on the survey for working out the new energy vision of the core city/town/village area in the Honjo region; 2001 nendo Honjo chiho kyoten toshi chiiki shin energy vision sakutei chosa hokokusho

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in the core city/town/village area in the Honjo region, Saitama Prefecture, an investigational study was conducted of the energy consumption amount, existence amount of new energy and project for new energy introduction, and a vision was worked out. The energy consumption of the area in FY 1999 was 9.8 x 10{sup 15}J in amount and was broken down into 47% of petroleum, 27% of electric power, 9% of town gas and 9% of LPG. As viewed by sector, the energy consumption consisted of 39% in the industrial sector, 29% in the transportation sector, 23% in the residential sector and 9% in the commercial sector. As the project for new energy introduction, the following were studied: introduction of wind power generation/photovoltaic power generation to municipal office/public facilities/traffic facilities/research facilities/street light/water filtration plant/disaster prevention facilities/residential houses; utilization of biomass like garbage/animal feces/residue from food plant/ligneous waste/thinnings and utilization of fuel cell by biogas; introduction of clean energy vehicle. (NEDO)

  13. Kickoff into the future - Community full of light and balmy wind, Naraha. Regional new energy vision for Naraha Town; 2001 nendo Naraha machi chiiki shin energy vision. Mirai he no kick off - Hikari to kaze no machi Naraha

    NONE

    2002-02-01

    For promoting the introduction of new energy and for enhancing people's consciousness of such at Naraha Town, Fukushima Prefecture, surveys and studies were conducted involving the trend of energy consumption of the town, the amount of new energy resources in existence, and new energy introduction projects, and a vision was formulated. The annual energy consumption of the town is 1,134,300 GJ, with industries consuming 41%, transportation 36%, households 12%, and commerce 11%. The energy comprised 76% from oil based fuels and 24% from electric power. Some new energy introduction projects were discussed, which covered the exhibition, public relations activities, and education through the use of wind power generation and photovoltaic power generation or solar heat utilization for Tenjin Point Sports Park, natural park surrounding the Kidogawa dam, on-the-road station Naraha Michinoeki, solar cars, and hybrid type street lamps; photovoltaic power generation, solar heat utilization, and clean energy vehicles for a special nursing home for elderly people, nursery schools, kindergartens, and a processing center for agriculture, forestry, and fishery products; and disseminating and enlightening activities conducted for people in general and business operators. (NEDO)

  14. Investigation on initial stage of new energy vision in Izumi-otsu City in Osaka Prefecture. Report for fiscal 2000; Izumiotsushi chiiki shin energy vision shoki dankai chosa. 2000 nendo hokokusho

    NONE

    2001-02-01

    With objectives to achieve promotion of new energy introduction with the environment creating city as the basic concept, and to depict the future image of the city energies by clearly showing the way to suppressing environmental load, a new energy vision is being established. Fiscal 2000 has investigated the energy demand and supply conditions and existence quantity of new energies. The investigation results were put into order by the following seven items: 1) summary of the investigation, 2) district characteristics of Izumi-otsu City, 3) investigation on quantity of new energies available in the city, 4) investigation on the energy demand quantity, 5) putting the problems into order, 6) the Citizens' Energy Conference, and 7) summary of the investigation on examples of new energy utilization. Item 3 is composed of the new energies utilizable in Izumi-otsu City, and calculations of existence quantity of new energies that are to be utilized. The utilizable quantity of new energies is 9.9 times 10{sup 5} Gcal/year, which corresponds to about 60% of the energy used in fiscal 1990. (NEDO)

  15. Report on new energy vision in Utazu Town area (aiming at building town where people and nature live together); Utazucho chiiki shin energy vision hokokusho. Ningen to shizen no kyoseisuru machizukuri wo mezashite

    NONE

    2001-02-01

    A new energy vision was established in Utazu Town in Miyagi Prefecture. The population of the town is decreasing year after year to 5,905 persons in fiscal 1996. The industrial structure has the ratio of primary, secondary, and tertiary industries at 43:26:31 in 1995, wherein the marine products industry is the basic industry. The annual energy consumption of the town in fiscal 1998 is 69,950 Gcal, and is accounted for by the transportation department at 48.8%, the consumer and household department at 29.9%, the consumer business department at 12.4%, and the industrial department at 8.7%. Evaluation on utilizable quantity of new energies revealed 9,925 MWh annually by wind power generation, 1,000 MWh annually by small to medium hydropower generation, 33,033 GJ annually by bio-mass, and 11,565 GJ annually by refuses. The new energy introducing project includes utilization of photovoltaic power generation at the Road Station, the town office, the public hall, primary and middle schools, fishing boats, fishery cultivation and fishing industry, introduction of wind power generation into the Tazukayama sightseeing road, introduction of small to medium hydropower generation into the Harai River dam, introduction of clean energy type community buses, and introduction of cogeneration system to the meal supply center. (NEDO)

  16. New energy vision at Sabae City. Toward realization of 'environmental international city'; 2001 nendo Sabae shi chiiki shin energy vision. Kankyo kokusai toshi Sabae no jitsugen ni mukete

    NONE

    2002-03-01

    With an object of accelerating introduction of and uplifting consciousness on new energies at Sabae City in Fukui Prefecture, a new energy vision was put into order based on the result of the initial stage investigation having been performed in the previous fiscal year. Solar light and solar heat energies will be introduced positively into public facilities, and at the same time works will be implemented to accelerate the proliferation thereof into general households. The target of introduction by the year 2010y is set to 2,600 kW by photovoltaic power generation, and the introduction thereof is planned to schools, nursery schools, municipality operated houses, and unattended facilities. With regard to solar heat water warmer, the present proliferation rate of about 7.3% will be raised to 15%. Regarding wastes generated from industrial areas, efficient energy extraction by thermal recycling is intended on the assumption of suppression of wastes generation, their re-use, and recycling. Systems may include the bio-gas system, cogeneration, and recovery of energies from plastics wastes. For acceleration of proliferation of new energies in general, promotion of introduction will be achieved on mini-wind power generation, small hydroelectric power generation, and fuel cell automobiles. (NEDO)

  17. Report on the project for working out the new energy vision of the Chinen Village area in FY 2001; 2001 nendo Chinen son chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Chinen Village, Okinawa Prefecture, an investigational study was conducted of the trend of energy consumption of the village, existence amount of new energy, case study for new energy introduction, etc., and a vision was worked out. The energy consumption amount of Chinen Village in FY 1999 was estimated at 591,935 x 10{sup 3} MJ/y. By sector, the consumption amount in the transportation sector was the highest, approximately 40%, followed in order by the household sector, business sector and industrial sector. The rate of energy source was 49.2% of petroleum, 42.3% of electric power and 8.5% of gas. As the case study for new energy introduction, the following were studied: introduction of photovoltaic power generation to the village office/Azama Sun Beach/hydroponics/JA branch office/prawn farm, introduction of photovoltaic power generation/wind power generation to the village gymnasium/village drainage pump/refrigerating facilities of the fisheries cooperative association, etc. Further, as the new project, the following were studied: introduction of photovoltaic power generation/wind power generation to the agricultural irrigation project/comprehensive sports park, introduction of photovoltaic power generation to the new building of Chinen Elementary School/maker of processed medicinal herbs. (NEDO)

  18. System and Software Design for the Plant Protection System for Shin-Hanul Nuclear Power Plant Units 1 and 2

    Hwang, In Seok; Kim, Young Geul; Choi, Woong Seock; Sohn, Se Do

    2015-01-01

    The Reactor Protection System(RPS) protects the core fuel design limits and reactor coolant system pressure boundary for Anticipated Operational Occurrences (AOOs), and provides assistance in mitigating the consequences of Postulated Accidents (PAs). The ESFAS sends the initiation signals to Engineered Safety Feature - Component Control System (ESF-CCS) to mitigate consequences of design basis events. The Common Q platform Programmable Logic Controller (PLC) was used for Shin-Wolsung Nuclear Power Plant Units 1 and 2 and Shin-Kori Nuclear Power Plant Units 1, 2, 3 and 4 since Digital Plant Protection System (DPPS) based on Common Q PLC was applied for Ulchin Nuclear Power Plant Units 5 and 6. The PPS for Shin-Hanul Nuclear Power Plant Units 1 and 2 (SHN 1 and 2) was developed using POSAFE-Q PLC for the first time for the PPS. The SHN1 and 2 PPS was delivered to the sites after completion of Man Machine Interface System Integrated System Test (MMIS-IST). The SHN1 and 2 PPS was developed to have the redundancy in each channel and to use the benefits of POSAFE-Q PLC, such as diagnostic and data communication. The PPS application software was developed using ISODE to minimize development time and human errors, and to improve software quality, productivity, and reusability

  19. System and Software Design for the Plant Protection System for Shin-Hanul Nuclear Power Plant Units 1 and 2

    Hwang, In Seok; Kim, Young Geul; Choi, Woong Seock; Sohn, Se Do [KEPCO EnC, Daejeon (Korea, Republic of)

    2015-10-15

    The Reactor Protection System(RPS) protects the core fuel design limits and reactor coolant system pressure boundary for Anticipated Operational Occurrences (AOOs), and provides assistance in mitigating the consequences of Postulated Accidents (PAs). The ESFAS sends the initiation signals to Engineered Safety Feature - Component Control System (ESF-CCS) to mitigate consequences of design basis events. The Common Q platform Programmable Logic Controller (PLC) was used for Shin-Wolsung Nuclear Power Plant Units 1 and 2 and Shin-Kori Nuclear Power Plant Units 1, 2, 3 and 4 since Digital Plant Protection System (DPPS) based on Common Q PLC was applied for Ulchin Nuclear Power Plant Units 5 and 6. The PPS for Shin-Hanul Nuclear Power Plant Units 1 and 2 (SHN 1 and 2) was developed using POSAFE-Q PLC for the first time for the PPS. The SHN1 and 2 PPS was delivered to the sites after completion of Man Machine Interface System Integrated System Test (MMIS-IST). The SHN1 and 2 PPS was developed to have the redundancy in each channel and to use the benefits of POSAFE-Q PLC, such as diagnostic and data communication. The PPS application software was developed using ISODE to minimize development time and human errors, and to improve software quality, productivity, and reusability.

  20. Couinaud's classification v.s. Cho's classification. Their feasibility in the right hepatic lobe

    Shioyama, Yasukazu; Ikeda, Hiroaki; Sato, Motohito; Yoshimi, Fuyo; Kishi, Kazushi; Sato, Morio; Kimura, Masashi

    2008-01-01

    The objective of this study was to investigate if the new classification system proposed by Cho is feasible to clinical usage comparing with the classical Couinaud's one. One hundred consecutive cases of abdominal CT were studied using a 64 or an 8 slice multislice CT and created three dimensional portal vein images for analysis by the Workstation. We applied both Cho's classification and the classical Couinaud's one for each cases according to their definitions. Three diagnostic radiologists assessed their feasibility as category one (unable to classify) to five (clear to classify with total suit with the original classification criteria). And in each cases, we tried to judge whether Cho's or the classical Couinaud' classification could more easily transmit anatomical information. Analyzers could classified portal veins clearly (category 5) in 77 to 80% of cases and clearly (category 5) or almost clearly (category 4) in 86-93% along with both classifications. In the feasibility of classification, there was no statistically significant difference between two classifications. In 15 cases we felt that using Couinaud's classification is more convenient for us to transmit anatomical information to physicians than using Cho's one, because in these cases we noticed two large portal veins ramify from right main portal vein cranialy and caudaly and then we could not classify P5 as a branch of antero-ventral segment (AVS). Conversely in 17 cases we felt Cho's classification is more convenient because we could not divide right posterior branch as P6 and P7 and in these cases the right posterior portal vein ramified to several small branches. The anterior fissure vein was clearly noticed in only 60 cases. Comparing the classical Couinaud's classification and Cho's one in feasility of classification, there was no statistically significant difference. We propose we routinely report hepatic anatomy with the classical Couinauds classification and in the preoperative cases we

  1. The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

    Zhang, Yang; Halim, Adnan; Narimatsu, Yoshiki

    2014-01-01

    The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its “human-like” glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors......, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis...... of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially...

  2. Sequencing the CHO DXB11 genome reveals regional variations in genomic stability and haploidy

    Kaas, Christian Schrøder; Kristensen, Claus; Betenbaugh, Michael J.

    2015-01-01

    Background: The DHFR negative CHO DXB11 cell line (also known as DUX-B11 and DUKX) was historically the first CHO cell line to be used for large scale production of heterologous proteins and is still used for production of a number of complex proteins.  Results: Here we present the genomic sequence...... of the CHO DXB11 genome sequenced to a depth of 33x. Overall a significant genomic drift was seen favoring GC -> AT point mutations in line with the chemical mutagenesis strategy used for generation of the cell line. The sequencing depth for each gene in the genome revealed distinct peaks at sequencing...... in eight additional analyzed CHO genomes (15-20% haploidy) but not in the genome of the Chinese hamster. The dhfr gene is confirmed to be haploid in CHO DXB11; transcriptionally active and the remaining allele contains a G410C point mutation causing a Thr137Arg missense mutation. We find similar to 2...

  3. Enhancement of Human Prolactin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

    Herbert Rodrigues Goulart

    2010-01-01

    Full Text Available Sodium butyrate (NaBu has been used as a productivity enhancer for the synthesis of recombinant proteins in Chinese hamster ovary (CHO cells. Thus, the influence of NaBu on the production of recombinant human prolactin (hPRL from CHO cells was investigated for the first time. CHO cell cultures were submitted to a treatment with different concentrations of NaBu (0.25 to 4 mM. Quantitative and qualitative analyses by reverse-phase high-performance liquid chromatography (RP-HPLC and Western blot or SDS-PAGE, carried out directly on CHO-conditioned medium, showed that the highest hPRL expression was obtained with 1 mM NaBu. In vitro biological assays based on noble rat lymphoma (Nb2 and mouse pro-B lymphoma (Ba/F3-LLP cells were carried out on purified hPRL. Its bioactivity in the presence of NaBu was not apparently different from that of the First International Reference Reagent of recombinant hPRL (WHO 97/714. Our results show that NaBu increased the synthesis of recombinant hPRL in CHO cells, apparently without compromising either its structure or function.

  4. The art of CHO cell engineering: A comprehensive retrospect and future perspectives.

    Fischer, Simon; Handrick, René; Otte, Kerstin

    2015-12-01

    Chinese hamster ovary (CHO) cells represent the most frequently applied host cell system for industrial manufacturing of recombinant protein therapeutics. CHO cells are capable of producing high quality biologics exhibiting human-like post-translational modifications in gram quantities. However, production processes for biopharmaceuticals using mammalian cells still suffer from cellular limitations such as limited growth, low productivity and stress resistance as well as higher expenses compared to bacterial or yeast based expression systems. Besides bioprocess, media and vector optimizations, advances in host cell engineering technologies comprising introduction, knock-out or post-transcriptional silencing of engineering genes have paved the way for remarkable achievements in CHO cell line development. Furthermore, thorough analysis of cellular pathways and mechanisms important for bioprocessing steadily unravels novel target molecules which might be addressed by functional genomic tools in order to establish superior production cell factories. This review provides a comprehensive summary of the most fundamental achievements in CHO cell engineering over the past three decades. Finally, the authors discuss the potential of novel and innovative methodologies that might contribute to further enhancement of existing CHO based production platforms for biopharmaceutical manufacturing in the future. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Strong CH/O interactions between polycyclic aromatic hydrocarbons and water: Influence of aromatic system size.

    Veljković, Dušan Ž

    2018-03-01

    Energies of CH/O interactions between water molecule and polycyclic aromatic hydrocarbons with a different number of aromatic rings were calculated using ab initio calculations at MP2/cc-PVTZ level. Results show that an additional aromatic ring in structure of polycyclic aromatic hydrocarbons significantly strengthens CH/O interactions. Calculated interaction energies in optimized structures of the most stable tetracene/water complex is -2.27 kcal/mol, anthracene/water is -2.13 kcal/mol and naphthalene/water is -1.97 kcal/mol. These interactions are stronger than CH/O contacts in benzene/water complex (-1.44 kcal/mol) while CH/O contacts in tetracene/water complex are even stronger than CH/O contacts in pyridine/water complexes (-2.21 kcal/mol). Electrostatic potential maps for different polycyclic aromatic hydrocarbons were calculated and used to explain trends in the energies of interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Thermal radiosensitization in heat- and radiation-sensitive mutants of CHO cells

    Kampinga, H.H.; Kanon, B.; Konings, A.W.T.; Stackhouse, M.A.; Bedford, J.S.

    1993-01-01

    In the current study, the extent of hyperthermic radiosensitization in a new γ-radiation-sensitive cell line, irs-20, recently isolated by Stackhouse and Bedford (1991) and a heat-sensitive mutant hs-36 (Harvey and Bedford 1988) was compared with the radiosensitization of their mutual parent CHO 10B12 cell line. The irs-20 and CHO 10B12 cells have comparable heat (43.5 o C) sensitivities, whereas hs-36 and CHO 10B12 show a similar sensitivity to γ- and X-rays. Radiosensitization due to pre-exposure to 43.5 o C heating of plateau phase cultures was found for all three cell lines, even after relatively mild heat treatment killing <20% of cells. Experiments using CHEF electrophoresis confirmed the dsb repair deficiency of the irs-20 cells (Stackhouse and Bedford 1992) and showed that heat inhibited dsb repair in all three cell lines. (Author)

  7. N-Glycosylation optimization of recombinant antibodies in CHO cell through process and metabolic engineering

    Fan, Yuzhou

    , analysis, control and optimization of N-glycosylation were thoroughly reviewed. In particular, how to control and optimize N-glycosylation in CHO cells was exclusively studied. The main focus of this PhD project is to find effective approaches of modulating N-glycosylation of CHO-derived recombinant...... galactose as feed additives, changing process parameters such as seeding density and cultivation duration are all demonstrated to be effective. The causal explanation of their impact on glycosylation can be various, including product, metabolism, proteome and physiology-associated mechanism. In the middle...... part of the thesis, both literature reviews and experimental applications were provided to demonstrate how to use omics data and implement systems biology to understand biological activities, especially N-glycosylation in CHO cells. In the last part of the thesis, the second strategy that apply genetic...

  8. Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools.

    Rajendra, Yashas; Balasubramanian, Sowmya; Peery, Robert B; Swartling, James R; McCracken, Neil A; Norris, Dawn L; Frye, Christopher C; Barnard, Gavin C

    2017-03-01

    Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534-540, 2017. © 2017 American Institute of Chemical Engineers.

  9. Book Review ~ Advancing Online Learning in Asia. Editors: David Murphy, Namin Shin, and Weiyuan Zhang

    Insung Jung

    2004-08-01

    Full Text Available The Internet, high-speed electronic communications, and computers have transformed the way we teach and learn. With the development of these new information and communication technologies, the idea of online education has been adopted in many developed, and more recently in developing countries, to bring wider opportunities to people in the form of increased access to flexible and interactive, open and distance learning systems. As stated in the Introduction of “Advancing Online Learning in Asia” edited by Murphy, Shin, and Zhang, online education is now everywhere and it “is changing the ways in which educational institutions interact with their students, for both traditional and distance education universities.” By examining recent developments of online education in Asia from multiple perspectives, this book has a potential to be an invaluable resource to educators. Taking cases from the Asian region in which online learning was introduced, implemented, and experienced, this book presents the cases from a number of perspectives, especially from student perspectives, and addresses pedagogical and technical issues faced by online educators. The breadth of the articles in this book provides a wide range of online learning cases and varied perspectives, which should clearly appeal to educators, researchers, administrators, and policy makers in online education.

  10. K-Ar chronological study of the quaternary volcanic activity in Shin-etsu Highland

    Kaneko, Takayuki; Shimizu, Satoshi; Itaya, Tetsumaru.

    1989-01-01

    In order to clarify the temporal and spatial patterns in arc volcanism, 55 K-Ar ages of volcanic rocks from 17 volcanoes in Shin-etsu Highland, central Japan were determined. In addition, life spans, volume of erupted materials and eruption rates of each volcano were estimated. Graphical analysis demonstrates that volume of ejecta varies proportionately with both life span and eruption rate, and that there is no significant correlation between eruption rate and distance from the volcanic front. The life span of each volcano in this Highland is less than 0.6 m.y. In the central Shiga and southern Asama area, the volcanism started at 1 Ma and is still active. However the former had a peak in the activity at around 0.5 Ma, while the latter is apparently most intense at present. Northern Kenashi area has the volcanism without peak in 1.7 - 0.2 Ma, though the activity within a volcanic cluster or chain in central Japan lasts generally for 1 m.y. or less with a peak. (author)

  11. Introduction of new energies that gives vitality to town of Mano, industries, and people. Report on establishment of new energy vision in Mano Town area (Niigata Prefecture); Mano no machi, sangyo, hito ni katsuryoku wo ataeru shin energy no donyu. Manocho chiiki shin energy vision hokokusho

    NONE

    2001-03-01

    Based on assurance of energy availability in Sado Island, and age advancing of the population, an energy vision was established with the target of building 'Mano Town that seem real to everyone to find it good to live therein'. The population of Mano Town is about 6000 persons, and the energy consumption in total is 22,350 kl/year as converted into petroleum (industries and public sector at 12,162, households at 2,720, and transportation at 7,433). The new energy introduction is divided into three steps of the initial stage, middle stage, and long term stage. The initial stage will execute introduction of photovoltaic power generation into primary schools, promotion of utilization of solar energy at homes, promotion of introducing firewood and chaff boilers, and introduction of cogeneration into Yutoripia Mano. For the middle stage, discussions were given on introduction of photovoltaic power generation into the city office building, manufacture of light oil substituting fuel through rape seed and soy bean cultivation, introduction of hybrid automobiles, and manufacture of pellet fuel by utilizing wood wastes. For the long term stage, discussions were given on wind power generation, and ethanol fuel manufacture. (NEDO)

  12. New energy vision in Sawauchi Village area. Toward establishment of Sawauchi Village having unaided power based on 'new energies, lessons learned from past'; Sawauchimura chiiki shin energy vison. 'Onkochi shin energy' ni yoru jiriki aru Sawauchimura no kakuritsu ni mukete

    NONE

    2001-02-01

    A new energy vision was established in the Sawauchi Village area in Iwate Prefecture. As the energy species to be discussed for introduction, Sawauchi Village has assumed 'solar (beam and heat)', 'forest bio-mass', 'stock breeding bio-mass', 'small hydro-power' and 'snow'. The new energy introduction project included the proliferation and enlightenment activities using the Snow Country Culture Research Institute as the core, new energy introduction into the Sawauchi Baden, introduction of small hydropower generation and micro hydropower generation, new energy introduction into snow melting systems, new energy introduction into agricultural greenhouses, introduction of snow room-cooling systems into public facilities, development of new business targeted at promotion of forestry, and proliferation and enlightenment activities coordinated with relevant organizations. Particularly in the development of new business targeted at promotion of forestry, tackling will be moved forward to sale and manufacture of pellet stoves, manufacture of wood pellets, research, development, and commercialization of bio-mass power plant and new housings incorporating snow country cultures, with an aim of promotion of utilization of woods and thinned woods available in the village. (NEDO)

  13. New energy vision of Rokunohe Town. Maple green plan (Town creation using the riches of Nature and industries of the area); 2001 nendo Rokunohe machi chiiki shin energy vision. Meipuru Green keikaku - Yutaka na shizen to chiiki no sangyo wo ikashita machidukuri

    NONE

    2002-02-01

    For the purpose of promoting the introduction of new energy and enhancing the awareness of it in Rokunohe Town, Aomori Prefecture, an investigational study was conducted of the state of energy supply/demand, existence amount of new energy, introduction of new energy, etc., and a vision was worked out. The population of Rokunohe Town was 10,481 according to the results of the national census taken in 2000, which is slightly decreasing. The energy consumption amount of the town is 16,063.6t in oil conversion, and the consumption depends on fossil fuels such as electric power, gasoline, light oil, heavy oil and LPG gas. For the action plan for introduction of new energy, the following were cited: field study of the photovoltaic power generation, preparation of the shopping street/school street which are safe and unfrozen (snow melting of sidewalk), potential study of the agricultural development of snow/ice energy, use of the agricultural waste such as animal feces and waste vegetable (study of introduction of small-scale bio-system), promotion of recycle of cooking oil (study of the use of clean energy vehicles for official vehicles and use of diesel substituting fuel), study of use of energy crops for the idle field, various education projects, etc. (NEDO)

  14. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup

    2015-01-01

    gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following...

  15. Gas phase UV and IR absorption spectra of CxF2x+1CHO (x=1-4)

    Hashikawa, Y; Kawasaki, M; Waterland, RL

    2004-01-01

    The UV and IR spectra of CxF2x+1 CHO (x = 1-4) were investigated using computational and experimental techniques. CxF2x+1CHO (x = 1-4) have broad UV absorption features centered at 300-310 nm. The maximum absorption cross-section increases significantly and shifts slightly to the red with increased...

  16. Silurian to Devonian magmatism, molybdenite mineralization, regional exhumation and brittle strike-slip deformation along the Loch Shin Line, NW Scotland

    Holdsworth, R.; Dempsey, E.; Selby, D.; Darling, James Richard; Feely, M.; Costanzo, A.; Strachan, Robin A; Waters, P.; Finlay, A.J.

    2015-01-01

    The Loch Shin Line is a geological–geophysical lineament associated with a zone of mantle-derived appinites, granites and strike-slip faulting that runs NW–SE across the Moine Nappe, northern Scotland. U–Pb zircon and Re–Os molybdenite dating of the Loch Shin and Grudie plutons, which lie immediately SW of the NW–SE Loch Shin–Strath Fleet fault system, yield c. 427–430 Ma ages that overlap within error. They also coincide with previously obtained U–Pb zircon ages for the Rogart pluton, which ...

  17. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies

    Noh, Soo Min; Shin, Seunghyeon; Min Lee, Gyun

    2018-01-01

    To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1...... and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated...... in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation...

  18. Selection of chemically defined media for CHO cell fed-batch culture processes

    Pan, X.; Streefland, M.; Dalm, C.; Wijffels, R.H.; Martens, D.E.

    2017-01-01

    Two CHO cell clones derived from the same parental CHOBC cell line and producing the same monoclonal antibody (BC-G, a low producing clone; BC-P, a high producing clone) were tested in four basal media in all possible combinations with three feeds (=12 conditions) in fed-batch cultures.
    Higher

  19. Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells

    Grav, Lise Marie; Julie la Cour Karottki, Karen; Lee, Jae Seong

    2017-01-01

    and yields. In this chapter, we present our protocol on how to use the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knockout engineering target genes in CHO cells. As an example, we refer to the glutamine synthetase (GS...

  20. Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

    van Es, H. H.; Renkema, H.; Aerts, H.; Schurr, E.

    1994-01-01

    Expression of the pfmdr1-encoded Pgh1 protein of Plasmodium falciparum in CHO cells confers a phenotype of increased sensitivity to chloroquine due to an increased Pgh1-mediated accumulation of this antimalarial. Pgh1 carrying amino acid substitutions associated with chloroquine resistance in P.

  1. C-terminal KDEL-modified cystatin C is retained in transfected CHO cells

    Johansen, Teit Eliot; Vogel, Charlotte Katrine; Schwartz, Thue W.

    1990-01-01

    The significance of a C-terminal tetrapeptide, Lys-Asp-Glu-Leu (KDEL), as a retention signal for the endoplasmatic reticulum was studied using cystatin C, a general thiol protease inhibitor, as the reporter protein. Clones of CHO cells were analyzed after stable transfection with eukaryotic...

  2. Cholesterol oxidase (ChoE) is not important in the virulence of Rhodococcus equi.

    Pei, Yanlong; Dupont, Chris; Sydor, Tobias; Haas, Albert; Prescott, John F

    2006-12-20

    To analyze further the role in virulence of the prominent cholesterol oxidase (ChoE) of Rhodococcus equi, an allelic exchange choE mutant from strain 103+ was constructed and assessed for virulence in macrophages, in mice, and in foals. There was no difference between the mutant and parent strain in cytotoxic activity for macrophages or in intra-macrophage multiplication. No evidence of attenuation was obtained in macrophages and in mice, but there was slight attenuation apparent in four intra-bronchially infected foals compared to infection of four foals with the virulent parent strain, based on a delayed rise in temperature of the choE-mutant infected foals. However, bacterial colony counts in the lung 2 weeks after infection were not significantly different, although there was a slight but non-significant (P=0.12) difference in lung:body weight ratio of the choE mutant versus virulent parent infected foals (mean 2.67+/-0.25% compared to 4.58+/-0.96%). We conclude that the cholesterol oxidase is not important for the virulence of R. equi.

  3. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

    2012-04-01

    Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

  4. A proteomic study of cMyc improvement of CHO culture

    Dunn Michael J

    2010-03-01

    Full Text Available Abstract Background The biopharmaceutical industry requires cell lines to have an optimal proliferation rate and a high integral viable cell number resulting in a maximum volumetric recombinant protein product titre. Nutrient feeding has been shown to boost cell number and productivity in fed-batch culture, but cell line engineering is another route one may take to increase these parameters in the bioreactor. The use of CHO-K1 cells with a c-myc plasmid allowing for over-expressing c-Myc (designated cMycCHO gives a higher integral viable cell number. In this study the differential protein expression in cMycCHO is investigated using two-dimensional gel electrophoresis (2-DE followed by image analysis to determine the extent of the effect c-Myc has on the cell and the proteins involved to give the new phenotype. Results Over 100 proteins that were differentially expressed in cMycCHO cells were detected with high statistical confidence, of which 41 were subsequently identified by tandem mass spectrometry (LC-MS/MS. Further analysis revealed proteins involved in a variety of pathways. Some examples of changes in protein expression include: an increase in nucleolin, involved in proliferation and known to aid in stabilising anti-apoptotic protein mRNA levels, the cytoskeleton and mitochondrial morphology (vimentin, protein biosysnthesis (eIF6 and energy metabolism (ATP synthetase, and a decreased regulation of all proteins, indentified, involved in matrix and cell to cell adhesion. Conclusion These results indicate several proteins involved in proliferation and adhesion that could be useful for future approaches to improve proliferation and decrease adhesion of CHO cell lines which are difficult to adapt to suspension culture.

  5. Single-dose Toxicity of ShinYangHur Herbal Acupuncture

    Eunhye Cha

    2015-06-01

    Full Text Available Objectives: This study was carried out to analyze the single-dose toxicity of ShinYangHur (SYH herbal acupuncture injected into the muscles of Sprague-Dawley (SD rats. Methods: The SYH herbal acupuncture was made in a clean room at the Korean Pharmacopuncture Institute (KPI, Korea-Good Manufacturing Practice, K-GMP. After the mixing process with sterile distilled water, the pH was controlled to between 7.0 and 7.5. Then, NaCl was added to make a 0.9% isotonic solution by using sterilized equipment. All experiments were conducted at Biotoxtech, an institution authorized to perform non clinical studies under the regulations of Good Laboratory Practice (GLP. SD rats were chosen for the pilot study. Doses of SYH herbal acupuncture, 0.25, 0.5, and 1.0 mL, were administered to the experimental groups, and a dose of normal saline solution, 1.0 mL, was administered to the control group. This study was conducted under the approval of the Institutional Animal Ethics Committee. Results: No deaths or abnormalities occurred in any of the four groups. No significant changes in weight, hematological parameters or clinical chemistry between the control group and the experimental groups were observed. To check for abnormalities in organs and tissues, we used microscopy was used to examine representative histological sections of each specified organ; the results showed no significant differences in any of the organs or tissues. Conclusion: The above outcomes suggest that treatment with SYH herbal acupuncture is relatively safe. Further studies on this subject are needed to yield more concrete evidence.

  6. Comparison of the Radionuclides Dispersion at the UAE Barakah Site with that at the ROK Shin-Kori Site - Comparison of the radionuclides dispersion in Barakah site with that in Shin-Kori site

    Kim, Sung-yeop; Lee, Kun Jai; Chang, Soon Heung [Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of); Beeley, Philip A. [Khalifa University of Science, Technology and Research, P.O. Box 127788, Abu Dhabi (United Arab Emirates)

    2014-07-01

    In order to understand the characteristics of atmospheric dispersion of radionuclides in the desert environment of Barakah site in UAE, comparison research with the results of other environments could be an appropriate way to facilitate it. Shin-Kori site is the proper comparison target because same reactor type of APR1400 with that in Barakah site is under construction. Hypothetical accident scenario was considered and accident source term which had been developed in previous research has been applied as releasing source. After reviewing several computation codes, ADMS5 has been selected as an atmospheric dispersion modeling tool which is installing advanced Gaussian plum model and plentiful options. The climate data of both Barakah and Shin-Kori were acquired and the environments of both sites have been simulated considering wind speed, wind direction, temperature, humidity, ground surface roughness and etc. Near field final human doses on the maps have been schematised regarding statistical meteorological data of both sites and dose conversion factors from the publications of ICRP and federal guidance report of EPA. The results of this research are expected to enhance the understanding about differences between two environments which have same reactor type and to improve the comprehension of desert environment of Barakah site as well. Applying different dose conversion factors to Barakah site considering the desert biosphere could be further study to obtain more accurate results. (authors)

  7. A study on Requirements of Data Base Translator for APR1400 Computerized Procedure System at Shin-Hanul unit 1 and 2

    Seong, Nokyu; Lee, Sungjin

    2015-01-01

    The CPS is one of the Man Machine Interface (MMI) resources and the CPS can directly display plant graphic objects which are in the Digital Control System (DCS). And the CPS can send a request to DCS to provide DCS screen which is called step support display through DCS link button on a computerized procedure. The procedure writers can insert DCS graphic information to computerized procedure through data base which is provided by CPS Editing System (CPSES). The data base which is provided by CPSES conforms to the naming rule of DCS graphic objects. The naming rule of DCS graphic objects is defined by vendor thus status of DCS graphic objects which are in computerized procedure at Shin-Kori plant cannot be displayed on CPS at Shin-Hanul plant. To use computerized procedure which is written by other plant procedure writer, DCS graphic objects shall be translated by its plant data base. This paper introduces requirements of data base translator to reduce translation and re-inserting graphic objects burden. This paper introduces the requirements of data base translator of CPSES for APR1400 CPS at Shin-Hanul unit 1 and 2. The translator algorithms shall be tested to update data base of CPSES effectively. The prototype of translator is implemented and is being tested using real plant DB. This translator can be applied to Shin- Hanul unit1 and 2 through software V and V

  8. A study on Requirements of Data Base Translator for APR1400 Computerized Procedure System at Shin-Hanul unit 1 and 2

    Seong, Nokyu; Lee, Sungjin [KHNP, Daejeon (Korea, Republic of)

    2015-05-15

    The CPS is one of the Man Machine Interface (MMI) resources and the CPS can directly display plant graphic objects which are in the Digital Control System (DCS). And the CPS can send a request to DCS to provide DCS screen which is called step support display through DCS link button on a computerized procedure. The procedure writers can insert DCS graphic information to computerized procedure through data base which is provided by CPS Editing System (CPSES). The data base which is provided by CPSES conforms to the naming rule of DCS graphic objects. The naming rule of DCS graphic objects is defined by vendor thus status of DCS graphic objects which are in computerized procedure at Shin-Kori plant cannot be displayed on CPS at Shin-Hanul plant. To use computerized procedure which is written by other plant procedure writer, DCS graphic objects shall be translated by its plant data base. This paper introduces requirements of data base translator to reduce translation and re-inserting graphic objects burden. This paper introduces the requirements of data base translator of CPSES for APR1400 CPS at Shin-Hanul unit 1 and 2. The translator algorithms shall be tested to update data base of CPSES effectively. The prototype of translator is implemented and is being tested using real plant DB. This translator can be applied to Shin- Hanul unit1 and 2 through software V and V.

  9. Improving the representation of modal choice into bottom-up optimization energy system models - The MoCho-TIMES model

    Tattini, Jacopo; Ramea, Kalai; Gargiulo, Maurizio

    2018-01-01

    and mathematical expressions required to develop the approach. This study develops MoCho-TIMES in the standalone transportation sector of TIMES-DK, the integrated energy system model for Denmark. The model is tested for the Business as Usual scenario and for four alternative scenarios that imply diverse......This study presents MoCho-TIMES, an original methodology for incorporating modal choice into energy-economy-environment-engineering (E4) system models. MoCho-TIMES addresses the scarce ability of E4 models to realistically depict behaviour in transport and allows for modal shift towards transit...

  10. Kinetics of the Br2-CH3CHO Photochemical Chain Reaction

    Nicovich, J. M.; Shackelford, C. J.; Wine, P. H.

    1997-01-01

    Time-resolved resonance fluorescence spectroscopy was employed in conjunction with laser flash photolysis of Br2 to study the kinetics of the two elementary steps in the photochemical chain reaction nBr2 + nCH3CHO + hv yields nCH3CBrO + nHBr. In the temperature range 255-400 K, the rate coefficient for the reaction Br((sup 2)P(sub 3/2)) + CH3CHO yields CH3CO + HBr is given by the Arrhenius expression k(sub 6)(T) = (1.51 +/- 0.20) x 10(exp -11) exp(-(364 +/- 41)/T)cu cm/(molecule.s). At 298 K, the reaction CH3CO + Br2 yields CH3CBrO + Br proceeds at a near gas kinetic rate, k(sub 7)(298 K) = (1.08 +/- 0.38) x 10(exp -10)cu cm/(molecule.s).

  11. Quantitative intracellular flux modeling and applications in biotherapeutic development and production using CHO cell cultures.

    Huang, Zhuangrong; Lee, Dong-Yup; Yoon, Seongkyu

    2017-12-01

    Chinese hamster ovary (CHO) cells have been widely used for producing many recombinant therapeutic proteins. Constraint-based modeling, such as flux balance analysis (FBA) and metabolic flux analysis (MFA), has been developing rapidly for the quantification of intracellular metabolic flux distribution at a systematic level. Such methods would produce detailed maps of flows through metabolic networks, which contribute significantly to better understanding of metabolism in cells. Although these approaches have been extensively established in microbial systems, their application to mammalian cells is sparse. This review brings together the recent development of constraint-based models and their applications in CHO cells. The further development of constraint-based modeling approaches driven by multi-omics datasets is discussed, and a framework of potential modeling application in cell culture engineering is proposed. Improved cell culture system understanding will enable robust developments in cell line and bioprocess engineering thus accelerating consistent process quality control in biopharmaceutical manufacturing. © 2017 Wiley Periodicals, Inc.

  12. Effects of heavy water on ultrastructural and functional status of Hep 2 and CHO cells lysosomes

    Buzgariu, Wanda; Caloianu, Maria; Zarnescu, Otilia; Cimpean, Anisoara; Titescu, Gh.; Stefanescu, I.

    2002-01-01

    The heavy water effects on the ultrastructure and function of Hep 2 and CHO lysosomal cell compartment were investigated using electron microscopy and enzymatic studies. The cell viability, measured by neutral red uptake assay, and the total protein content determination, have shown a dose dependent decrease in cell growth for both studied cell types. The electron microscopy study has revealed a progressive increase in number and size of lysosomes and autophagosomes after 96 h exposure to different deuterium concentration media in a dose dependent manner. The enzymatic determination in the lysosomal pellet revealed an increased acid phosphatase activity in both cell types (15% and 33% for Hep 2 and 24% and 52% for CHO, respectively) exposed to media with high (65%, 90%) D 2 O content. (authors)

  13. CHO On A Detox: Removing By-Product Formation Through Cell Engineering

    Pereira, Sara; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    Chinese Hamster Ovary (CHO) cells are the preferred hosts for the production of therapeutic glycoproteins. However, there is a need for improvement of the bioprocesses towards increased cell growth and higher productivities without compromising the product quality. Efforts to obtain tailor-made p......-made products with the desired properties that meet the requirements of regulatory authorities are continuously being made. Of equal relevance is to develop methods to engineer cell lines with improved by-product metabolism....

  14. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

  15. Improving lactate metabolism in an intensified CHO culture process: productivity and product quality considerations.

    Xu, Sen; Hoshan, Linda; Chen, Hao

    2016-11-01

    In this study, we discussed the development and optimization of an intensified CHO culture process, highlighting medium and control strategies to improve lactate metabolism. A few strategies, including supplementing glucose with other sugars (fructose, maltose, and galactose), controlling glucose level at Productivity and product quality attributes differences between batch, fed-batch, and concentrated fed-batch cultures were discussed. The importance of process and cell metabolism understanding when adapting the existing process to a new operational mode was demonstrated in the study.

  16. Fed-batch CHO cell culture for lab-scale antibody production

    Fan, Yuzhou; Ley, Daniel; Andersen, Mikael Rørdam

    2017-01-01

    Fed-batch culture is the most commonly used upstream process in industry today for recombinant monoclonal antibody production using Chinese hamster ovary cells. Developing and optimizing this process in the lab is crucial for establishing process knowledge, which enable rapid and predictable tech......-transfer to manufacturing scale. In this chapter, we will describe stepwise how to carry out fed-batch CHO cell culture for lab-scale antibody production....

  17. Isolation of uv-sensitive variants of CHO-Kl by nylon cloth replicaplating

    Stamato, T.D.; Waldren, C.A.

    1977-01-01

    Techniques are described which permit the identification and isolation of uv-sensitive variants from mutagenized populations of Chinese hamster ovary (CHO) cells. Identification is based on the observation that within two days after receiving a dose of approximately 240 ergs/mm 2 of uv irradiation, most of the cells in a colony of CHO detach from the surface of a plastic tissue culture dish. At a lower dose of uv, which does not kill or detach a significant number of parental cells, uv-sensitive colonies are killed and become detached. Thus a clear plaque is produced in a lawn of unirradiated parental cells, marking the site occupied by a sensitive colony. Live cells from such sensitive colonies have been recovered from a nylon cloth replica prepared prior to irradiation and characterized. One uv-sensitive variant (CHO-UV-1) is indistinguishable from parental cells in x-ray resistance, chromosome number, generation time, and duration of the phases of the cell cycle

  18. Precision control of recombinant gene transcription for CHO cell synthetic biology.

    Brown, Adam J; James, David C

    2016-01-01

    The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology. Copyright © 2015. Published by Elsevier Inc.

  19. Thermotolerance and thermosensitization in CHO and R1H cells: a comparative study

    Dikomey, E.; Eickhoff, J.; Jung, H.

    1984-01-01

    In CHO and R1H cells thermotolerance was induced by a pre-incubation at 40 0 C, by an acute heat shock at 43 0 C followed by a time interval at 37 0 C, and during continuous heating at 42 0 C. Thermotolerance, which was tested at 43 0 , primarily causes an increase in D 0 of the heat-response curve. The degree of maximum thermotolerance was found to be generally more pronounced in CHO than in R1H cells, but the time interval at 37 0 C, as well as at 40 0 C, to reach this maximum level was the same in both cell lines. CHO and R1H cells could be sensitized to 40 0 C by a pre-treatment at 43 0 C. When compared for the same survival rate after pre-treatment at 43 0 C alone the degree of thermosensitization was about the same in both cell lines. In either cell line thermosensitization was found to be suppressed when cells were made thermotolerant by a previous incubation at 40 0 C for 16 hours. (author)

  20. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    Awasthi, Kumud Kant [University of Rajasthan, Department of Zoology (India); Awasthi, Anjali; Kumar, Narender; Roy, Partha [Indian Institute of Technology Roorkee, Department of Biotechnology (India); Awasthi, Kamlendra, E-mail: kamlendra.awasthi@gmail.com [Malaviya National Institute of Technology, Department of Physics (India); John, P. J., E-mail: placheriljohn@yahoo.com [University of Rajasthan, Department of Zoology (India)

    2013-09-15

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV-Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408-410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 {+-} 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC{sub 50}) for CHO cells is 68.0 {+-} 2.65 {mu}g/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 {mu}g/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity.

  1. Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

    Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2014-03-01

    Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. Copyright © 2013 Elsevier GmbH. All rights reserved.

  2. Multi-omic profiling of EPO-producing CHO cell panel reveals metabolic adaptation to heterologous protein production

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we...... discovered indications of metabolic adaptation of the amino acid catabolism in favor of heterologous protein production. We established a panel of stably EPO expressing CHO-K1 clones spanning a 25-fold productivity range and characterized the clones in batch and chemostat cultures. For this, we employed...... a multi-omic physiological characterization including metabolic foot printing of amino acids, metabolite fingerprinting of glycolytic intermediates, NAD(P)H-/NAD(P)+ and adenosine nucleotide phosphates. We used qPCR, qRT-PCR, western blots and Affymetrix CHO microarrays to assess EPO gene copy numbers...

  3. One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

    Grav, Lise Marie; Lee, Jae Seong; Thomsen, Signe Gerling

    2015-01-01

    The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously....... Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further....

  4. Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies.

    Noh, Soo Min; Shin, Seunghyeon; Lee, Gyun Min

    2018-03-29

    To characterize a glutamine synthetase (GS)-based selection system, monoclonal antibody (mAb) producing recombinant CHO cell clones were generated by a single round of selection at various methionine sulfoximine (MSX) concentrations (0, 25, and 50 μM) using two different host cell lines (CHO-K1 and GS-knockout CHO). Regardless of the host cell lines used, the clones selected at 50 μM MSX had the lowest average specific growth rate and the highest average specific production rates of toxic metabolic wastes, lactate and ammonia. Unlike CHO-K1, high producing clones could be generated in the absence of MSX using GS-knockout CHO with an improved selection stringency. Regardless of the host cell lines used, the clones selected at various MSX concentrations showed no significant difference in the GS, heavy chain, and light chain gene copies (P > 0.05). Furthermore, there was no correlation between the specific mAb productivity and these three gene copies (R 2  ≤ 0.012). Taken together, GS-mediated gene amplification does not occur in a single round of selection at a MSX concentration up to 50 μM. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.

  5. Safety Evaluation of Full Digital Plant Protection System of Shin-Kori 3 and 4 in Korea

    Koh, J. S.; Kim, D. I.; Jeong, C. H.; Park, H. S.; Ji, S. H.; Kang, Y. D.; Park, G. Y.

    2009-01-01

    Keeping pace with the emerging trend of digital computer technologies, KHNP has utilized full digital plant protection system into the design of I and C systems at SKN 3 and 4. This paper presents safety review activities and results related to digital plant protection systems during the licensing of construction permit for the Shin-Kori 3 and 4(SKN 3 and 4) in Korea. The major licensing issues regarding the digital systems were software quality and cyber security during planning stage, system integrity with fail-safe design, EMI equipment qualification of digital systems, FPGA qualification and communication independence between safety and non-safety System. This paper addresses our approach to evaluate full digital protection systems with revised safety review guidelines and the resulting discussion to resolve the licensing issues

  6. Project Experience of MMIS for Shin-Hanul units 1 and 2 (Component Design, Manufacturing and Testing)

    Koo, Seo-ryong; Kim, Kook-hun [Doosan Heavy Industries and Construction Co. Ltd., Hwaseong (Korea, Republic of)

    2015-10-15

    Doosan is uniquely positioned in the field of nuclear power plant in the world-wide. Doosan has secured the first step to nuclear technology field through the development of the nuclear power plant (NPP) man machine interface system (MMIS), which is one of the three major core technologies of non-independence. In the nuclear power plant, MMIS takes a function as the brain and nerve system in the human body. Korean MMIS was selected for the Shin-Hanul units 1 and 2 for the first time and Doosan is charge of a component design and equipment supply. At the end of successful completion of MMIS for Shin-Hanul units 1 and 2, Doosan will be a total provider of the MMIS from development, validation, and manufacturing to delivery in Korea. Man Machine Interface System (MMIS) is equivalent to the brain and nerve system of a nuclear power plant. It monitors and controls the operating conditions to prevent accidents proactively. Doosan achieved a complete development of MMIS which is core part of nuclear technology that was relied on oversea's companies in the past by an accomplishment of RnD in cooperation with domestic utility, KHNP and research institutions. Doosan's MMIS is very user-friendly, easy to understand, transparent to audits, and guarantees maximum reliability, availability, maintainability, and safety. Doosan proved safety and reliability through test and inspection of integrated function of nuclear safety and non-safety control system. Doosan MMIS was evaluated a one of the best system through IAEA IERIC's review via safety guide from IAEA specialist's group in 2010. Doosan also achieved the improvements of quality through the KINS audits in 2014.

  7. 11C-CHO PET in optimization of target volume delineation and treatment regimens in postoperative radiotherapy for brain gliomas

    Li Fangming; Nie Qing; Wang Ruimin; Chang, Susan M.; Zhao Wenrui; Zhu Qi; Liang Yingkui; Yang Ping; Zhang Jun; Jia Haiwei; Fang Henghu

    2012-01-01

    Objective: We explored the clinical values of 11 C-choline ( 11 C-CHO) PET in optimization of target volume delineation and treatment regimens in postoperative radiotherapy for brain gliomas. Methods: Sixteen patients with the pathological confirmation of the diagnosis of gliomas prior to receiving radiotherapy (postoperative) were included, and on whom both MRI and CHO PET scans were performed at the same position for comparison of residual tumors with the two techniques. 11 C-CHO was used as the tracer in the PET scan. A plain T1-weighted, T2-weighted and contrast-enhanced T1-weighted imaging scans were performed in the MRI scan sequence. The gliomas' residual tumor volume was defined as the area with CHO-PET high-affinity uptake and metabolism (V CHO ) and one with MRI T1-weighted imaging high signal intensity (V Gd ), and was determined by a group of experienced professionals and clinicians. Results: (1) In CHO-PET images, the tumor target volume, i.e., the highly metabolic area with a high concentration of isotopes (SUV 1.016–4.21) and the corresponding contralateral normal brain tissues (SUV0.1–0.62), was well contrasted, and the boundary between lesions and surrounding normal brain tissues was better defined compared with MRI and 18 F-FDG PET images. (2) For patients with brain gliomas of WHO Grade II, the SUV was 1.016–2.5; for those with WHO Grades III and IV, SUVs were >26–4.2. (3) Both CHO PET and MRI were positive for 10 patients and negative for 2 patients. The residual tumor consistency between these two studies was 75%. Four of the 10 CHO-PET-positive patients were negative on MRI scans. The maximum distance between V Gd and V CHO margins was 1.8 cm. (4) The gross tumor volumes (GTVs) and the ensuing treatment regimens were changed for 31.3% (5/16) of patients based on the CHO-PET high-affinity uptake and metabolism, in which the change rate was 80% (4/5), 14.3 % (1/7) and 0% (0/4) for patients with WHO Grade II III, and IV gliomas

  8. Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis.

    Han, Gil-Soo; Carman, George M

    2017-08-11

    The PAH1 -encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae , exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1 -encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1 Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1 Δ mutant is induced through the inositol-sensitive upstream activation sequence (UAS INO ), a cis -acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UAS INO mutation suppressed pah1 Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1 -encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1 Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. miR-2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality.

    Fischer, Simon; Paul, Albert Jesuran; Wagner, Andreas; Mathias, Sven; Geiss, Melanie; Schandock, Franziska; Domnowski, Martin; Zimmermann, Jörg; Handrick, René; Hesse, Friedemann; Otte, Kerstin

    2015-10-01

    Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells. © 2015 Wiley Periodicals, Inc.

  10. The relationship between Cho/NAA and glioma metabolism: implementation for margin delineation of cerebral gliomas.

    Guo, Jun; Yao, Chengjun; Chen, Hong; Zhuang, Dongxiao; Tang, Weijun; Ren, Guang; Wang, Yin; Wu, Jinsong; Huang, Fengping; Zhou, Liangfu

    2012-08-01

    The marginal delineation of gliomas cannot be defined by conventional imaging due to their infiltrative growth pattern. Here we investigate the relationship between changes in glioma metabolism by proton magnetic resonance spectroscopic imaging ((1)H-MRSI) and histopathological findings in order to determine an optimal threshold value of choline/N-acetyl-aspartate (Cho/NAA) that can be used to define the extent of glioma spread. Eighteen patients with different grades of glioma were examined using (1)H-MRSI. Needle biopsies were performed under the guidance of neuronavigation prior to craniotomy. Intraoperative magnetic resonance imaging (MRI) was performed to evaluate the accuracy of sampling. Haematoxylin and eosin, and immunohistochemical staining with IDH1, MIB-1, p53, CD34 and glial fibrillary acidic protein (GFAP) antibodies were performed on all samples. Logistic regression analysis was used to determine the relationship between Cho/NAA and MIB-1, p53, CD34, and the degree of tumour infiltration. The clinical threshold ratio distinguishing tumour tissue in high-grade (grades III and IV) glioma (HGG) and low-grade (grade II) glioma (LGG) was calculated. In HGG, higher Cho/NAA ratios were associated with a greater probability of higher MIB-1 counts, stronger CD34 expression, and tumour infiltration. Ratio threshold values of 0.5, 1.0, 1.5 and 2.0 appeared to predict the specimens containing the tumour with respective probabilities of 0.38, 0.60, 0.79, 0.90 in HGG and 0.16, 0.39, 0.67, 0.87 in LGG. HGG and LGG exhibit different spectroscopic patterns. Using (1)H-MRSI to guide the extent of resection has the potential to improve the clinical outcome of glioma surgery.

  11. Minimizing transfusion requirements for children undergoing craniosynostosis repair: the CHoR protocol.

    Vega, Rafael A; Lyon, Camila; Kierce, Jeannette F; Tye, Gary W; Ritter, Ann M; Rhodes, Jennifer L

    2014-08-01

    Children with craniosynostosis may require cranial vault remodeling to prevent or relieve elevated intracranial pressure and to correct the underlying craniofacial abnormalities. The procedure is typically associated with significant blood loss and high transfusion rates. The risks associated with transfusions are well documented and include transmission of infectious agents, bacterial contamination, acute hemolytic reactions, transfusion-related lung injury, and transfusion-related immune modulation. This study presents the Children's Hospital of Richmond (CHoR) protocol, which was developed to reduce the rate of blood transfusion in infants undergoing primary craniosynostosis repair. A retrospective chart review of pediatric patients treated between January 2003 and Febuary 2012 was performed. The CHoR protocol was instituted in November 2008, with the following 3 components; 1) the use of preoperative erythropoietin and iron therapy, 2) the use of an intraoperative blood recycling device, and 3) acceptance of a lower level of hemoglobin as a trigger for transfusion (protocol implementation served as controls. A total of 60 children were included in the study, 32 of whom were treated with the CHoR protocol. The control (C) and protocol (P) groups were comparable with respect to patient age (7 vs 8.4 months, p = 0.145). Recombinant erythropoietin effectively raised the mean preoperative hemoglobin level in the P group (12 vs 9.7 g/dl, p protocol that includes preoperative administration of recombinant erythropoietin, intraoperative autologous blood recycling, and accepting a lower transfusion trigger significantly decreased transfusion utilization (p < 0.001). A decreased length of stay (p < 0.001) was seen, although the authors did not investigate whether composite transfusion complication reductions led to better outcomes.

  12. Rate Coefficients for the OH + (CHO)2 (Glyoxal) Reaction Between 240 and 400 K

    Feierabend, K. J.; Talukdar, R. K.; Zhu, L.; Ravishankara, A. R.; Burkholder, J. B.

    2006-12-01

    Glyoxal (CHO)2, the simplest dialdehyde, is an end product formed in the atmospheric oxidation of biogenic hydrocarbons, for example, isoprene. As such, glyoxal plays a role in regional air quality and ozone production in certain locations. Glyoxal is lost in the atmosphere via UV photolysis and reaction with OH. However, the currently available rate coefficient data for the OH + glyoxal reaction is limited to a single room- temperature measurement made using the relative rate method. A determination of the rate coefficient temperature dependence is therefore needed for a more complete interpretation of the atmospheric processing of glyoxal. This study reports the rate coefficient for the OH + (CHO)2 reaction measured under pseudo- first-order conditions in OH ([(CHO)2] > 1000 [OH]0). OH radicals were produced using 248 nm pulsed laser photolysis of H2O2 or HNO3 and detected by pulsed laser induced fluorescence. The concentration of glyoxal in the reactor was determined using three independent techniques; gas flow rates as well as in situ UV and IR absorption. The total pressure in the reactor was varied from 40 to 300 Torr (He), and the rate coefficient was found to be independent of pressure over the temperature range studied. The rate coefficient exhibits a negative temperature dependence between 240 and 400 K consistent with the dependence previously observed for many other aldehydes. Our room-temperature rate coefficient is smaller than the relative rate value that is currently recommended for use in atmospheric model calculations. Our measured rate coefficients are discussed with respect to those for other aldehydes. The atmospheric implications of our work will also be discussed.

  13. Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.

    de Wit, C; Fautz, C; Xu, Y

    2000-09-01

    Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell

  14. Genetically modified CHO cells for studying the genotoxicity of heterocyclic amines from cooked foods

    Thompson, L.H.; Wu, R.W.; Felton, J.S.

    1995-07-01

    We have developed metabolically competent CHO cells to evaluate the genotoxicity associated with heterocyclic amines, such as those that are present in cooked foods. Into repair-deficient UV5 cells we introduced cDNAs for expressing cytochrome P450IA2 and acetyltransferases. We then genetically reverted these transformed lines to obtain matched metabolically competent repair-deficient/proficient lines. For a high mutagenic response, we find a requirement for acetyltransferase with IQ but not with PhIP. This system allows for both quantifying mutagenesis and analyzing the mutational spectra produced by heterocyclic amines

  15. Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

    Jones, Meredith B.; Tomiya, Noboru; Betenbaugh, Michael J.; Krag, Sharon S.

    2010-01-01

    Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man 5 GlcNAc 2 -P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man 9 GlcNAc 2 -P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc 3 Man 9 GlcNAc 2 -P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man 5 GlcNAc 2 -PP-Dol through Glc 1 Man 9 GlcNAc 2 -PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc 3 Man 9 GlcNAc 2 -P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

  16. Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

    Jones, Meredith B., E-mail: mbauman7@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Tomiya, Noboru, E-mail: ntomiya1@jhu.edu [Department of Biology, Johns Hopkins University, 3400 North Charles Street, Mudd Hall 104A, Baltimore, MD 21218 (United States); Betenbaugh, Michael J., E-mail: beten@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Krag, Sharon S., E-mail: skrag@jhsph.edu [Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 (United States)

    2010-04-23

    Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

  17. Influence of catechins on bystander responses in CHO cells induced by alpha-particle irradiation

    Law, Y.L.; Wong, T.P.W. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong); Yu, K.N. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong (Hong Kong)], E-mail: peter.yu@cityu.edu.hk

    2010-04-15

    In this work, we studied alpha-particle induced and medium-mediated bystander effects in Chinese hamster ovary (CHO) cells through micronucleus (MN) assay. We showed that signal transduction from irradiated cells to bystander cells occur within a short time after irradiation. We then studied the effects of ROS (reactive oxygen species)-scavenging catechins in the medium before irradiation. We observed decreases in the percentage of bystander cells with MN formation and thus proved the protection effect of catechins on bystander cells from radiation.

  18. Induction of micronuclei and binucleated cells by treatment with radiation and cisplatin in CHO cells

    Rodilla, V.; Seymour, C.B.; Mothersill, C.; Pertusa, J.; Pellicer, J.A.

    1991-01-01

    The frequencies of CHO cells with micronuclei in the cisplatin-treated cultures showed an increase reaching a maximum 48 hours after treatment. Within the next 48 hours a slight decrease in the frequencies was observed. In γ-irradiated cultures (1.2 Gy/min at 80 cm source-skin distance) the maximum in micronuclei-induction was reached at 24 hours post-irradiation, decreasing thereafter. Cultures receiving both treatments showed a similar curve, with a peak at 24 hours, decreasing thereafter. (UK)

  19. "Shin splint" syndrome and tibial stress fracture in the same patient diagnosed by means of (99m)Tc-HMDP SPECT/CT.

    Vicente, Justo Serrano; Grande, Maria Luz Domínguez; Torre, Jose Rafael Infante; Madrid, Juan Ignacio Rayo; Barquero, Carmen Durán; Bernardo, Lucía García; Sánchez, Román Sánchez

    2013-04-01

    We show a patient who presented leg pain triggered by intense exercise. The most likely diagnosis was a possible tibial stress fracture or a "shin splint" syndrome (soleus enthesopathy). We performed a bone scintigraphy including SPECT/CT that revealed the presence of the two concomitant pathologies. SPECT/CT identified the hot spot superimposed with bone lesion in the tibial stress fracture and only remodeling activity without evidence of cortical lesions in the enthesopathy processes.

  20. Cross-cultural adaptation of the CHO-KLAT for boys with hemophilia in rural and urban china

    Wu Runhui

    2012-09-01

    Full Text Available Abstract Background Quality of life (QoL is increasingly recognized as an important outcome measure in clinical trials. The Canadian Hemophilia Outcomes-Kids Life Assessment Tool (CHO-KLAT shows promise for use in China. Objective To adapt the CHO-KLAT version 2.0 for use in clinical trials in China. Methods Forward and back translations of the CHO-KLAT2.0 were completed in 2008. Between October 2009 and June 2010, a series of 3 focus groups were held with 20 boys and 31 parents in rural and urban China to elicit additional concepts, important to their QoL, for the Chinese CHO-KLAT2.0. All of the items identified by boys and parents were reviewed by a group of experts, resulting in a Chinese version of the CHO-KLAT2.0. This version underwent a detailed cognitive debriefing process between October 2010 and June 2011. Thirteen patient-parent pairs participated in this cognitive debriefing process until a stable and clearly understood Chinese version of the CHO-KLAT2.0 was obtained. Results The initial back translation of the Chinese CHO-KLAT2.0 was slightly discrepant from the original English version on 12 items. These were all successfully adjudicated. The focus groups identified 9 new items that formed an add-on Socio-Economic Context (SEC module for China. Linguistic improvements were made after the 2nd, 5th, 7th and 13th cognitive debriefings pairs and affected a total of 18 items. The result was a 35 item CHO-KLAT2.0 and a SEC module in Simplified Chinese, both of which have good content validity. Conclusion This detailed process proved to be extremely valuable in ensuring the items were accurately interpreted by Chinese boys with hemophilia ages ≤18 years. The need for the additional SEC module highlighted the different context that currently exists in China with regard to hemophilia care as compared to many Western countries, and will be important in tracking progress within both rural and urban China over time. Changes based on the

  1. Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

    Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

    2017-08-10

    To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  2. Effect of 5-aminolevulinic acid on kinetics of protoporphyrin IX production in CHO cells.

    W Warchoł

    2004-07-01

    Full Text Available 5-aminolevulinic acid (ALA is utilized in a photodynamic therapy as a compound capable of augmenting intracellular pool of protoporphyrin IX (PpIX, which exhibits properties of a photosensitizer. The studies were aimed at monitoring accumulation of endogenous protoporphyrin IX in CHO cells under effect of various concentrations of ALA in culture medium and following removal of the compound from the culture medium. Cell content of PpIX was determined following incubation of the cells for 72 h in a culture medium containing different concentration of ALA. Moreover, the cells were preincubated for 2 h in ALA at various concentrations and separated from the compound by medium change and their PpIX content was monitored following incubation. PpIX content was defined by a fluorescent technique under the confocal microscope. In the course of continuous incubation of cells with ALA, biphasic alterations were noted in cellular PpIX concentration. Removal of ALA from the incubation medium resulted at first in a decrease in PpIX content in cells, which was followed by an evidently augmented accumulation of the compound in the cells. The results suggested that in the case of CHO cells, exogenous ALA was not an exclusive source of PpIX synthesis and that alterations in enzyme activities were responsible for production of PpIX.

  3. Mutant spectra of irradiated CHO AL cells determined with multiple markers analyzed by flow cytometry

    Ross, Carley D.; French, C. Tenley; Keysar, Stephen B.; Fox, Michael H.

    2007-01-01

    We have previously developed a sensitive and rapid mammalian cell mutation assay which is based on a Chinese hamster ovary cell line that stably incorporates human chromosome 11 (CHO A L ) and uses flow cytometry to measure mutations in CD59. We now show that multiparameter flow cytometry may be used to simultaneously analyze irradiated CHO A L cells for mutations in five CD genes along chromosome 11 (CD59, CD44, CD90, CD98, CD151) and also a GPI-anchor gene. Using this approach, 19 different mutant clones derived from individual sorted mutant cells were analyzed to determine the mutant spectrum induced by ionizing radiation. All clones analyzed were negative for CD59 expression and PCR confirmed that at least CD59 exon 4 was also absent. As expected, ionizing radiation frequently caused large deletions along chromosome 11. This technology can readily be used to rapidly analyze the mutant yield as well as the spectrum of mutations caused by a variety of genotoxic agents and provide greater insight into the mechanisms of mutagenesis

  4. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  5. Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells.

    Gácsi, Mariann; Antal, Otilia; Vasas, Gábor; Máthé, Csaba; Borbély, György; Saker, Martin L; Gyori, János; Farkas, Anna; Vehovszky, Agnes; Bánfalvi, Gáspár

    2009-06-01

    In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

  6. Recovery of CHO cells from hyperthermic potentiation to x rays: repair of DNA and chromatin

    Clark, E.P.; Dewey, W.C.; Lett, J.T.

    1981-01-01

    Above the critical temperature, ca. 42.5 0 C, hyperthermic potentiation of Chinese hamster ovary (CHO) cells to x irradiation was accompanied by increased binding of nonhistone proteins to DNA and by reduced rates of rejoining of DNA strand breaks. These biochemical changes were reversed as the cells recovered from the hyperthermic exposures at 37 0 C. If the hyperthermically treated cells were incubated at 37 0 C before x irradiation, the ratio of nonhistone protein to DNA returned to normal in 12 h but the depressed rate of rejoining of DNA strand breaks and increased cell radiosensitivity remained unaltered. Cell radiosensitivity began to decrease after 12 h and recovery from hyperthermia-potentiated radiosensitivity was complete by 48 h. In the same interval, the rate of rejoining of DNA strand breaks also returned to normal. From this behavior, we conclude that the reduction in the rate of rejoining of DNA strand breaks involved changes in DNA structure which were restored only after the thermal enhancement of protein binding was reversed. These experiments provide support for the viewpoint that critical hyperthermic potentiation (i.e., above 42.5 0 C for CHO cells) may have logistical advantages over subcritical hyperthermic potentiation (i.e., below 42.5 0 C) in clinical situations

  7. Effective suppression of bystander effects by DMSO treatment of irradiated CHO cells

    Kashino, Genro; Prise, K.M.; Suzuki, Keiji

    2007-01-01

    Evidence is accumulating that irradiated cells produce some signals which interact with non-exposed cells in the same population via a bystander effect. Here, we examined whether dimethyl sulfoxide (DMSO) is effective in suppressing radiation induced bystander effects in Chinese hamster ovary (CHO) and repair deficient xrs5 cells. When 1 Gy-irradiated CHO cells were treated with 0.5% DMSO for 1 hr before irradiation, the induction of micronuclei in irradiated cells was suppressed to 80% of that in non-treated irradiated cells. The suppressive effect of DMSO on the formation of bystander signals was examined and the results demonstrated that 0.5% DMSO treatment of irradiated cells completely suppressed the induction of micronuclei by the bystander effect in non-irradiated cells. It is suggested that irradiated cells ceased signal formation for bystander effects by the action of DMSO. To determine the involvement of reactive oxygen species on the formation of bystander signals, we examined oxidative stress levels using the 2',7'-dichlorofluorescein (DCFH) staining method in irradiated populations. The results showed that the treatment of irradiated cells with 0.5% DMSO did not suppress oxidative stress levels. These results suggest that the prevention of oxidative stress is independent of the suppressive effect of DMSO on the formation of the bystander signal in irradiated cells. It is suggested that increased reactive oxygen species (ROS) in irradiated cells is not a substantial trigger of a bystander signal. (author)

  8. Adhesion and migration of CHO cells on micropatterned single layer graphene

    Keshavan, S.; Oropesa-Nuñez, R.; Diaspro, A.; Canale, C.; Dante, S.

    2017-06-01

    Cell patterning technology on single layer graphene (SLG) is a fairly new field that can find applications in tissue engineering and biomaterial/biosensors development. Recently, we have developed a simple and effective approach for the fabrication of patterned SLG substrates by laser micromachining, and we have successfully applied it for the obtainment of geometrically ordered neural networks. Here, we exploit the same approach to investigate the generalization of the cell response to the surface cues of the fabricated substrates and, contextually, to quantify cell adhesion on the different areas of the patterns. To attain this goal, we tested Chinese hamster ovary (CHO) cells on PDL-coated micropatterned SLG substrates and quantified the adhesion by using single cell force spectroscopy (SCFS). Our results indicate higher cell adhesion on PDL-SLG, and, consequently, an initial CHO cell accumulation on the graphene areas, confirming the neuronal behaviour observed previously; interestingly, at later time point in culture, cell migration was observed towards the adjacent SLG ablated regions, which resulted more favourable for cell proliferation. Therefore, our findings indicate that the mechanism of interaction with the surface cues offered by the micropatterned substrates is strictly cell-type dependent.

  9. Glycoprofiling effects of media additives on IgG produced by CHO cells in fed-batch bioreactors

    Kildegaard, Helene Faustrup; Fan, Yuzhou; Wagtberg Sen, Jette

    2016-01-01

    Therapeutic monoclonal antibodies (mAbs) are mainly produced by heterogonous expression in Chinese hamster ovary (CHO) cells. The glycosylation profile of the mAbs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production. In this......Therapeutic monoclonal antibodies (mAbs) are mainly produced by heterogonous expression in Chinese hamster ovary (CHO) cells. The glycosylation profile of the mAbs has major impact on the efficacy and safety of the drug and is therefore an important parameter to control during production....... In this study, the effect on IgG N-glycosylation from feeding CHO cells with eight glycosylation precursors during cultivation was investigated. The study was conducted in fed-batch mode in bioreactors with biological replicates to obtain highly controlled and comparable conditions. We assessed charge...

  10. Monitoring of BHT-quinone and BHT-CHO in the gas of capsules of Asclepias physocarpa.

    Ma, Bing-Ji; Peng, Hua; Liu, Ji-Kai

    2006-01-01

    Three volatile components, namely benzoic acid ethyl ester (1), 2,6-di-tert-butyl-p-benzoquinone (BHT-quinone) (2), and 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO) (3), were detected from the gas in the capsules of Asclepias physocarpa by means of GC/MS analysis. BHT-quinone and BHT-CHO as organic pollutants are the degradation products of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT). Ground water, lake water and/or rain water are a source of BHT metabolites in the plant Asclepias physocarpa.

  11. Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N......-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell...

  12. Gene mutation, quantitative mutagenesis, and mutagen screening in mammalian cells: study with the CHO/HGPRT system

    Hsie, A.W.

    1980-01-01

    We have employed CHO cells to develop and define a set of stringent conditions for studying mutation induction to TG resistance. Several lines of evidence support the CHO/HGPRT system as a specific-locus mutational assay. The system permits quantification of mutation at the HGPRT locus induced by various physical and chemical mutagens. The quantitative nature of the system provides a basis for the study of structure-function relationships of various classes of chemical mutagens. The intra- and interlaboratory reproducibility of this system suggests its potential for screening environmental agents for mutagenic activity

  13. Benchmarking of commercially available CHO cell culture media for antibody production.

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-06-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable

  14. Software V and V of PPS for Shin-Hanul Nuclear Power Plant Units 1 and 2

    Park, Cheollak; Kang, Dongpa; Choe, Changhui; Sohn, Sedo; Beak, Seungmin

    2013-01-01

    Software V and V processes determine whether the development products of a given activity conform to the requirements of that activity and whether the software satisfies its intended use and user needs. This paper introduces the software V and V activities and tasks performed during the software development life cycle performed during the software development life cycle of the Plant Protection System (PPS) for Shin-Hanul Nuclear Power Plant Units 1 and 2 (SHN 1 and 2). The PPS generates signals to actuate Reactor Trip (RT) and Engineered Safety Features (ESF) whenever monitored processes exceed predetermined limits, and the PPS software is classified safety critical and an independent V and V is thus required according to regulations, code and standards. The software V and V efforts, sufficiently disciplined and rigorous, are quite essential to demonstrate that the software development process is of a high quality. The software V and V of PPS for SHN 1 and 2 has been accomplished successfully with systematic V and V procedures and methods established until test phase in compliance with related code and standards. In particular, the use of automated tools such as LDRA and DOORS greatly has contributed to an improvement of a software quality, and a reduction of a verification time and human errors

  15. Cell survival and chromosomal aberrations in CHO-K1 cells irradiated by carbon ions

    Czub, J. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Banas, D. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Holycross Cancer Center, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Blaszczyk, A. [Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University, Grudziadzka 5, 87-100 Torun (Poland); Braziewicz, J. [Institute of Physics, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Holycross Cancer Center, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Buraczewska, I. [Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw (Poland); Choinski, J. [Heavy Ion Laboratory, Warsaw University, ul. Pasteura 5A, 02-093 Warsaw (Poland); Gorak, U. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland); Jaskola, M.; Korman, A. [Andrzej Soltan Institute for Nuclear Studies, 05-400 Otwock-Swierk (Poland); Lankoff, A.; Lisowska, H. [Institute of Biology, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland); Lukaszek, A. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland); Main School of Fire Service, ul. Slowackiego 52/54, 01-629 Warsaw (Poland); Szeflinski, Z. [Institute of Experimental Physics, Warsaw University, ul. Hoza 69, 00-681 Warsaw (Poland)], E-mail: szef@fuw.edu.pl; Wojcik, A. [Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw (Poland); Institute of Biology, Swietokrzyska Academy, ul. Swietokrzyska 15, 25-406 Kielce (Poland)

    2009-03-15

    Chinese hamster ovary CHO-K1 cells were exposed to high LET {sup 12}C-beam (LET: 830 keV/{mu}m) in the dose range of 0-6 Gy and to {sup 60}Co irradiation and the RBE value was obtained. Effects of {sup 12}C-beam exposure on cell survival and chromosomal aberrations were calculated. The chromosomal aberration data were fitted with linear equation. The distribution of aberration in cells was examined with a standard u-test and used to evaluate the data according to Poisson probabilities. The variance to the mean ratio {sigma}{sup 2}/Y and the dispersion index (u) were determined. Overdispersion was significant (p<0.05) when the value of u exceeded 1.96.

  16. Relationship of radiation sensitivity and aberrant DNA synthesis in repair deficient CHO cells

    Newman, C.N.; Hagler, H.; Miller, J.H.

    1986-11-01

    Comparison of alkaline sucrose gradient profiles of pulse-labeled DNA from a normal CHO cell line and its radiation-sensitive mutant, xrs-5, reveals significant differences in the replicon elongation/maturation process in these two cells. During a one hr period of growth subsequent to labeling, the molecular weight of pulse-labeled DNA from the mutant cell increases considerably more rapidly than that of the parent cell. For xrs-5, the presence of 2 mM deoxycytidine (CdR) in the culture medium reduces the replication rate to one approaching that of the parent cell growing in the standard medium. Corresponding uv resistance of the mutant likewise increases to nearly that of the parent cell line. These results suggest that the locus conferring radiation sensitivity to xrs-5 affects the DNA replisome complex and that replicative activity and radiation sensitivity are jointly modulated by CdR. 19 refs., 4 figs

  17. Mutagenicity of silver nanoparticles in CHO cells dependent on particle surface functionalization and metabolic activation

    Guigas, Claudia; Walz, Elke; Gräf, Volker; Heller, Knut J.; Greiner, Ralf

    2017-06-01

    The potential of engineered nanomaterials to induce genotoxic effects is an important aspect of hazard identification. In this study, cytotoxicity and mutagenicity as a function of metabolic activation of three silver nanoparticle (AgNP) preparations differing in surface coating were determined in Chinese hamster ovary (CHO) subclone K1 cells. Three silver nanoparticle preparations ( x 90,0 culture medium containing 10% fetal calf serum (FCS) than in medium without FCS. The HPRT test without metabolic activation system S9 revealed that compared to the other AgNP formulations, citrate-coated Ag showed a lower genotoxic effect. However, addition of S9 increased the mutation frequency of all AgNPs and especially influenced the genotoxicity of Citrate-Ag. The results showed that exogenous metabolic activation of nanosilver is crucial even if interactions of the metabolic activation system, nanosilver, and cells are not really understood up to now.

  18. The effect of purine phosphonomethoxyalkyl derivatives on DNA synthesis in Cho Chinese hamster cells

    Stetina, R [Institute of Experimental Medicine, Laboratory of Developmental Toxicology, Academy of Sciences of Czech Republic, 51783 Olesnice v Orlickych horach (Czech Republic); Votruba, I; Holy, A; Merta, A [Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic (Czech Republic)

    1994-12-31

    The inhibition of incorporation of {sup 3}H-thymidine and the changes of the rate of nascent DNA chain elongation were investigated in Cho Chinese hamster cells treated with (S)-(3-hydroxy-2-phosphonomethoxypropyl) (HPMP) and N-(2-phosphonomethoxyethyl) (PME) derivatives of adenine (A), guanine (G) and 2,6-diaminopurine (DAP). No direct correlation was observed in PME and HPMP derivatives between cytotoxicity, inhibition of {sup 3}H-thymidine incorporation and inhibition of nascent DNA chain elongation. The highest cytotoxicity and inhibition of DNA synthesis were caused by PMEG. The limited extent of inhibition of DNA elongation was encountered in the case of HPMPG and HPMPA. With PMEA, weak inhibition of elongation of DNA was observed only after a prolonged exposure (6 h). None of the investigated drugs induced DNA breaks. (author) 4 figs., 23 refs.

  19. Heating of low-density CHO-foam layers by means of soft X-rays

    Rosmej, O.N., E-mail: o.rosmej@gsi.de [GSI Helmholtzzentrum fuer Schwerionenforschung, Planckstrasse 1, 164291 Darmstadt (Germany); Bagnoud, V.; Eisenbarth, U. [GSI Helmholtzzentrum fuer Schwerionenforschung, Planckstrasse 1, 164291 Darmstadt (Germany); Vatulin, V.; Zhidkov, N.; Suslov, N.; Kunin, A.; Pinegin, A. [All Russian Scientific Research Institute of Experimental Physics, RFNC-VNIIEF, Mira St. 37, Sarov (Russian Federation); Schaefer, D.; Nisius, Th.; Wilhein, Th. [RheinAhrCampus Remagen, Institute for X-optics, Suedallee 2, 53424 Remagen (Germany); Rienecker, T.; Wiechula, J.; Jacoby, J. [Goethe University, Frankfurt am Main (Germany); Zhao, Y. [Institute of Modern Physics, CAS, Nanchang Road 509, 730000 Lanzhou (China); Vergunova, G.; Borisenko, N. [Lebedev Physical Institute, Leninskii Prospekt, 65 Moscow (Russian Federation); Orlov, N. [Joint Institute for High Temperatures RAS, Institute for High Energy Density, Izhorskaya. 13, building 2, 125412 Moscow (Russian Federation)

    2011-10-11

    Interaction of soft X-ray thermal radiation with polymer foam layers has been studied experimentally. Indirectly heated CHO-foams were used to create a plasma target for applications in combined heavy ion beam-laser experiments that are aimed at investigation of the heavy ion energy loss in ionized matter. In this work, we report experimental results on heating of low Z foams by means of the Planckian radiation generated in gold hohlraums. The experimental goal was to study the hohlraum radiation field, duration of the soft X-ray pulse, the conversion efficiency of the laser energy into soft X-rays, measurements of the absorption properties of foam layers and parameters of the foam targets heated by the Plankian radiation.

  20. Heating of low-density CHO-foam layers by means of soft X-rays

    Rosmej, O.N.; Bagnoud, V.; Eisenbarth, U.; Vatulin, V.; Zhidkov, N.; Suslov, N.; Kunin, A.; Pinegin, A.; Schaefer, D.; Nisius, Th.; Wilhein, Th.; Rienecker, T.; Wiechula, J.; Jacoby, J.; Zhao, Y.; Vergunova, G.; Borisenko, N.; Orlov, N.

    2011-01-01

    Interaction of soft X-ray thermal radiation with polymer foam layers has been studied experimentally. Indirectly heated CHO-foams were used to create a plasma target for applications in combined heavy ion beam-laser experiments that are aimed at investigation of the heavy ion energy loss in ionized matter. In this work, we report experimental results on heating of low Z foams by means of the Planckian radiation generated in gold hohlraums. The experimental goal was to study the hohlraum radiation field, duration of the soft X-ray pulse, the conversion efficiency of the laser energy into soft X-rays, measurements of the absorption properties of foam layers and parameters of the foam targets heated by the Plankian radiation.

  1. Quantitative mammalian cell mutagenesis and mutagen screening: study with CHO cells

    Hsie, A.W.; O'Neill, J.P.; San Sebastian, J.R.; Brimer, P.A.

    1979-01-01

    The CHO/HGPRT system has been developed and defined for quantifying mutation induced by various physical and chemical agents at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells. In all direct-acting chemical mutagens studied, mutation induction increases linearly as a function of the concentration, with no apparent threshold. Some chemicals induce mutation at non-cytotoxic concentrations. The mutagenicity of ethyl methanesulfonate has been quantified as a function of exposure concentration x treatment time. The sensitive and quantitative nature of the system enables studies of the structure-activity (mutagenicity) relationships of various classes of chemicals, including alkylating agents, heterocyclic nitrogen mustards, and platinum compounds. When rat liver S 9 -mediated metabolic activation is present, procarcinogens such as benzo(a)pyrene, 2-acetylaminofluorene, and dimethylnitrosamine are mutagenic, whereas their noncarcinogenic structural analogues pyrene, fluorene, and dimethylamine are not. The system has been shown to be useful in determining the interactive effects between physical and chemical agents, and in screening for mutagenicity of fractionated organic mixtures and industrial chemicals in both liquid and gaseous state. For the system to be used successfully in routine screening, further studies should be directed toward the development of a metabolic activation system suitable for a broad spectrum of chemicals, a sensitive and reliable statistical method, and an experimental design to determine compounds with low mutagenicity. The system has been expanded for determination of mutagen-induced chromosome aberration, sister-chromatid exchange, and micronucleus formation in addition to gene mutation and cytotoxicity; it can also be used to study inhibition of DNA synthesis

  2. Effects of copper on CHO cells: cellular requirements and product quality considerations.

    Yuk, Inn H; Russell, Stephen; Tang, Yun; Hsu, Wei-Ting; Mauger, Jacob B; Aulakh, Rigzen P S; Luo, Jun; Gawlitzek, Martin; Joly, John C

    2015-01-01

    Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes. © 2014 American Institute of Chemical Engineers.

  3. Impact of sodium butyrate and mild hypothermia on metabolic and physiological behaviour of CHO TF 70R cells

    Veronica Avello

    2017-05-01

    Conclusions: The combination of NaBu addition and mild hypothermic condition causes an impact on physiological and metabolic state of CHO TF 70R cells, decreasing cell growth rate and improving glucose consumption efficiency. These results therefore provide a promising strategy to increase specific productivity of rh-tPA.

  4. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay*

    DALBY, TINE; SØRENSEN, CHARLOTTE; PETERSEN, JESPER WESTPHAL; KROGFELT, KAREN ANGELIKI

    2010-01-01

    Dalby T, Sørensen C, Petersen JW, Krogfelt KA. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay. APMIS 2010; 118: 968–72. Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT. PMID:21091778

  5. Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

    Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

    2013-12-16

    Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

  6. Identification of potential molecular markers of ionizing radiation-induced mutations at the hprt locus in CHO cells

    Schwartz, J.L.; Sun, J.; Porter, R.C.

    1995-01-01

    Using multiplex polymerase chain reaction-based exon deletion analysis, we have analyzed mutations at the hprt locus from independent CHO cell mutants isolated from untreated, 60 Co x-ray-, and 212 Bi-exposed CHO-K1 cello and its radiation-sensitive derivative, xrs-5. In the 71 spontaneous CHO-K1 mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of 1-8 exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of γ rays (10% survival) produced 45% of the 20 mutants analyzed showing partial deletion, and 30% showing total deletion. Exposure to an equitoxic dose of a radiation from 212 Bi, a 220 Rn daughter, resulted in a spectrum similar to the γ-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The α-radiation, however, tended to produce larger intragenic deletions that γ radiation. Of the 87 spontaneous xrs-5 mutants analyzed for deletions 44% showed partial deletion, and 14% showed total deletion. Exposure to α radiation (10% survival) resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed no change in exon number or size, 16% showed partial deletion, and 41% showed total deletion. While the defect in xrs-5 has a profound effect on spontaneous mutation spectra, it does not appear to affect α-induced mutation spectra

  7. Design and implementation of an advanced protection system for the Shin-Kori 3 and 4 nuclear power plant

    Kim, Yonghak; Choi, Woongseock; Kwon, Jongsoo; Wilkosz, Stephen J.; Ridolfo, Charles F.; Yanosy, Paul L.

    2008-01-01

    The Nuclear Power Industry is currently embracing modern digital technology for upgrades to existing Instrumentation and Control (I and C) infrastructures as well as for incorporation into the next generation of new plants which will be coming 'on-line' during the next decade. This technology is being fully exploited for the next generation of advanced plant protection systems which will be initially deployed on the Shin-Kori 3 and 4 Nuclear Power Plant in the Republic of Korea. The system design for this plant protection system is being performed by the Korea Power Engineering Company (KOPEC) and builds upon the past generation of digital safety systems which were initially implemented at Ulchin 5 and 6. The advanced protection system is an evolution of this existing design and includes a number of improved operating attributes including: · Integration of Reactor Protection, Engineered Safety Features Actuation, and Qualified Indication and Alarm functions which were previously implemented by separate systems in the past. · Use of a 'soft control' interface which provides convenient accessibility to the safety systems from 'operator workstations' · Implementation of a Large Display Panel (LDP) which provides a consistent and constant representation of the overall plant state and of the plant safety status. The equipment for the advanced plant protection system is being provided by Westinghouse Electric Company (WEC) and utilizes the Westinghouse 'Common Q' Standardized qualified platform (where 'Q' denotes 'qualified'). The 'Common Q' platform is comprised of commercially dedicated Programmable Logic Controllers (PLC's), color-graphic Flat Panel Displays (FPD's) with integral touch screens, and high speed data communication links. It is a mature product that is in wide use for a number of safety-related applications. Among its key attributes are: · High overall system availability, which is achieved via use of a multiple channel configuration that is tolerant

  8. Tibial stress reaction presenting as bilateral shin pain in a man taking denosumab for giant cell tumor of the bone.

    Lim, Sian Yik; Rastalsky, Naina; Choy, Edwin; Bolster, Marcy B

    2015-12-01

    Prolonged bisphosphonate use has been associated with increased risk of atypical femoral fractures. Very few cases of atypical femoral fractures have been reported with denosumab. We report a case of bilateral tibial stress reactions in a 60-year-old man with no history of osteoporosis who was on prolonged high-dose denosumab for the treatment of giant cell tumor of bone. He presented with a 3-month history of pain in his bilateral shins worsening with activity and improving with rest. Although initial radiographs were unremarkable, he was found to have changes consistent with a stress reaction on magnetic resonance imaging of the distal tibia. To our knowledge, bilateral tibial stress reactions have not been previously reported with anti-resorptive therapies (neither bisphosphonates nor denosumab). Our case is intriguing in terms of the development of stress reactions as a precursor to stress fractures which may also relate to atypical fractures. Our case suggests a possible association between denosumab use and stress reactions. Of note the indication for denosumab in our case was for the treatment of giant cell tumor of bone where the Food and Drug Administration (FDA) approved dose is substantially higher than the FDA approved dose for osteoporosis treatment. Although rare, clinicians should consider the possibility of stress fractures in patients on anti-resorptive medications such as denosumab, especially when a patient presents with new onset thigh pain, hip pain or pain over an area affecting the long bones. Evaluation by imaging of affected areas should be pursued to enable early detection and intervention, as well as prevention of morbidity and associated ongoing risk to the patient. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

    Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

    2015-09-01

    Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

    Nakamura, Tsuyoshi; Omasa, Takeshi

    2015-09-01

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Engineering CHO cells with an oncogenic KIT improves cells growth, resilience to stress, and productivity.

    Mahameed, Mohamed; Tirosh, Boaz

    2017-11-01

    An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form. Here, we investigated the effect of oncogenic D816V KIT on the performance of CHO-K1 cells under conventional tissue culture growth settings and when adapted, to shaking conditions. The onco-KIT promoted global protein synthesis, elevated the expression of a secretable transgene, enhanced proliferation, and improved the overall titers of a model glycoprotein. Moreover, the expression of the onco-KIT endowed the cells with a remarkable resistance to various stress conditions. Our data suggest that the introduction of onco-KIT can serve as a strategy for improving glycoprotein biomanufacturing. Biotechnol. Bioeng. 2017;114: 2560-2570. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. The relationship of metabolic burden to productivity levels in CHO cell lines.

    Zou, Wu; Edros, Raihana; Al-Rubeai, Mohamed

    2018-03-01

    The growing demand for recombinant therapeutics has driven biotechnologists to develop new production strategies. One such strategy for increasing the expression of heterologous proteins has focused on enhancing cell-specific productivity through environmental perturbations. In this work, the effects of hypothermia, hyperosmolarity, high shear stress, and sodium butyrate treatment on growth and productivity were studied using three (low, medium, and high producing) CHO cell lines that differed in their specific productivities of monoclonal antibody. In all three cell lines, the inhibitory effect of these parameters on proliferation was demonstrated. Additionally, compared to the control, specific productivity was enhanced under all conditions and exhibited a consistent cell line specific pattern, with maximum increases (50-290%) in the low producer, and minimum increases (7-20%) in the high producer. Thus, the high-producing cell line was less responsive to environmental perturbations than the low-producing cell line. We hypothesize that this difference is most likely due to the bottleneck associated with a higher metabolic burden caused by higher antibody expression. Increased recombinant mRNA levels and pyruvate carboxylase activities due to low temperature and hyperosmotic stress were found to be positively associated with the metabolic burden. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  13. Ultrastructural study of mitochondrial damage in CHO cells exposed to hyperthermia.

    Cole, A; Armour, E P

    1988-09-01

    A unique direct-view stereo electron microscope technique was used to visualize the structure and three-dimensional distributions of mitochondria in CHO cells in situ following hyperthermic treatments. Aberrations induced by various heating regimens were recorded. The protocol included a trypsin digestion that may have enhanced the expression of the initial heat damage. The developed damage was observed as increasing levels of mitochondrial distortion, swelling, and dissociation. Minimal damage was induced at 42 degrees C for exposures of up to 4 h, while significant damage was induced at 43 degrees C for exposures of more than 30 min and at 45 degrees C for exposures of more than 10 min. For moderate exposures, a partial recovery of mitochondrial integrity was observed when the heat treatment was followed by incubation at 37 degrees C for 24 h. Mitochondrial damage was related to the heat dose in that increasing treatment temperature resulted in greater damage, but when compared to cell survival the damage did not parallel cell killing under all time-temperature conditions.

  14. Conditional Knockdown of Endogenous MicroRNAs in CHO Cells Using TET-ON-SanDI Sponge Vectors.

    Costello, Alan; Lao, Nga; Clynes, Martin; Barron, Niall

    2017-01-01

    MicroRNAs (miRNAs) are small, noncoding RNAs of about 22 nucleotides in length and have proven to be useful targets for genetic modifications for desirable phenotype in the biotech industry. The use of constitutively expressed "miRNA sponge" vectors in which multiple, tandem miRNA binding sites containing transcripts are transcriptionally regulated by a constitutive promoter for down regulating the levels of endogenous microRNAs in Chinese hamster ovary (CHO) cells has shown to be more advantageous than using synthetic antisense oligonucleotides. The application of miRNA sponges in biotechnological processes, however, could be more effective, if expression of miRNA sponges could be tuned. In this chapter, we present a method for the generation of stable CHO cell lines expressing a TET-ON-SanDI-miRNA-sponge that is in theory expressed only in the presence of an inducer.

  15. Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients

    Pereira, Sara; Kildegaard, Helene F.; Andersen, Mikael R.

    2018-01-01

    and process optimization and monitoring to perform efficiently. One of the main reasons for this is the production and accumulation of toxic and growth-inhibiting metabolites during culture. Lactate and ammonium are the most known, but many more have been identified. In this review, we present an overview...... of metabolites that deplete and accumulate throughout the course of cultivations with toxic and growth inhibitory effects to the cells. We further provide an overview of the CHO metabolism with emphasis to metabolic pathways of amino acids, glutathione (GSH), and related compounds which have growth...... of resources that describe the cellular mechanisms of CHO and are available on-line. Finally, we discuss the application of this knowledge for bioprocess and medium development and cell line engineering....

  16. Engineer Medium and Feed for Modulating N-Glycosylation of Recombinant Protein Production in CHO Cell Culture.

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell. In this chapter, we will focus on how to carry out experiments for N-glycosylation modulation through medium and feed optimization. The workflow and typical methods involved in the experiment process will be presented.

  17. Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Hansen, Henning Gram

    2014-01-01

    of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved...... mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27...

  18. Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

    Böhm, Ernst; Seyfried, Birgit K; Dockal, Michael; Graninger, Michael; Hasslacher, Meinhard; Neurath, Marianne; Konetschny, Christian; Matthiessen, Peter; Mitterer, Artur; Scheiflinger, Friedrich

    2015-09-18

    BACKGROUND & Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

  19. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    Golubnitchaya-Labudova, O.; Hoefer, M.; Portele, A.; Vacata, V.; Rink, H.; Lubec, G.

    1997-01-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer the question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the inserts of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9. (author)

  20. Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2

    Vita, N.; Magazin, M.; Marchese, E.; Lupker, J.; Ferrara, P.

    1990-01-01

    We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and the structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2

  1. Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

    Kim, Che Lin; Kwang Ha, Tae; Min Lee, Gyun

    2016-01-01

    Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (qp)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), Li......Cl was added to the CHO-NK and human embryonic kidney 293E (HEK293E) cell cultures before and/or after transfection with polyethylenimine as a transfection reagent. The effect of this addition on transfection efficiency (pre-treatment) and qp enhancement during TGE (post-treatment) was examined. For the TGE...... of monoclonal antibody (mAb) in CHO-NK cells, pretreatment alone with 10 mM LiCl and post-treatment alone with 5 mM LiCl resulted in 1.2- and 3.4-fold increase of maximum mAb concentration (MMC), respectively, compared with the TGE without LiCl treatment. Furthermore, combinatorial treatment with LiCl (10 m...

  2. Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM

    Kim, Hyonchol; Arakawa, Hideo; Hatae, Noriyuki; Sugimoto, Yukihiko; Matsumoto, Osamu; Osada, Toshiya; Ichikawa, Atsushi; Ikai, Atsushi

    2006-01-01

    The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5x10 -18 J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1x10 4 under the assumption that the area of the cell surface was about 5000 μm 2 . These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM

  3. Randomized Controlled Trial of Hospital-Based Hygiene and Water Treatment Intervention (CHoBI7) to Reduce Cholera.

    George, Christine Marie; Monira, Shirajum; Sack, David A; Rashid, Mahamud-ur; Saif-Ur-Rahman, K M; Mahmud, Toslim; Rahman, Zillur; Mustafiz, Munshi; Bhuyian, Sazzadul Islam; Winch, Peter J; Leontsini, Elli; Perin, Jamie; Begum, Farzana; Zohura, Fatema; Biswas, Shwapon; Parvin, Tahmina; Zhang, Xiaotong; Jung, Danielle; Sack, R Bradley; Alam, Munirul

    2016-02-01

    The risk for cholera infection is >100 times higher for household contacts of cholera patients during the week after the index patient seeks hospital care than it is for the general population. To initiate a standard of care for this high-risk population, we developed Cholera-Hospital-Based-Intervention-for-7-Days (CHoBI7), which promotes hand washing with soap and treatment of water. To test CHoBI7, we conducted a randomized controlled trial among 219 intervention household contacts of 82 cholera patients and 220 control contacts of 83 cholera patients in Dhaka, Bangladesh, during 2013-2014. Intervention contacts had significantly fewer symptomatic Vibrio cholerae infections than did control contacts and 47% fewer overall V. cholerae infections. Intervention households had no stored drinking water with V. cholerae and 14 times higher odds of hand washing with soap at key events during structured observation on surveillance days 5, 6, or 7. CHoBI7 presents a promising approach for controlling cholera among highly susceptible household contacts of cholera patients.

  4. DNA and chromosome breaks induced by 123I-estrogen in CHO cells

    Schwartz, J.L.

    1997-01-01

    The effects of the Auger electron-emitting isotope I-123, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen positive Chinese hamster ovary (CHO-ER) cells. Exposure to the 123 I-estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.2. The corresponding ratio with 60 Co gamma rays was 15.6. The dose-response was biphasic suggesting that either receptor sites are saturated at high does, or that there is a nonrandom distribution of breaks induced by the 123 I-estrogen. The 123 I-estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1,000 disintegrations per cell. This corresponds to the mean lethal dose of 123 I-estrogen for these cells suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that 123 I-estrogen-induced chromosome breaks are rejoined. The F-ratio, the ratio of dicentrics to centric rings, was 5.8 ± 1.7, which is similar to that seen with high LET radiations. Their results suggest that I-123 bound to estrogen is an efficient clastogenic agent, that the cytotoxic damage produced by I-123 bound to estrogen is very like high LET-induced damage, and the I-123 in the estrogen-receptor-DNA complex is probably in close proximity to the sugar-phosphate backbone of the DNA

  5. Infrared spectra of cyanoacetaldehyde (NCCH2CHO): a potential prebiotic compound of astrochemical interest.

    Benidar, Abdessamad; Georges, Robert; Guillemin, Jean-Claude; Mó, Otilia; Yáñez, Manuel

    2013-08-26

    Cyanoacetaldehyde (NC-CH2CH=O) and its isomer, cyanovinylalcohol (NC-CH=CH-OH), as possible components of the interstellar medium, comets, or planetary atmospheres, exist in equilibrium in the gas phase, although the latter compound is very much in the minority (2%). The recording and analysis of the gas-phase infrared spectrum of the former compound within the 4000-500 cm(-1) spectroscopic range and the potential presence of the latter isomer, which could be vital for their detection in these media, are reported. CCSD(T) and G4 high-level ab initio methods, as well as density functional theory calculations, predict the existence of two stable rotamers of cyanoacetaldehyde. The global minimum has a structure with an unusual O-C-C-C dihedral angle (150°) that falls between the antiperiplanar (180°) and anticlinal forms (120°). The second rotamer, which is about 4.0 kJ mol(-1) less stable in terms of free energy, has a planar structure that corresponds to the synperiplanar form (O-C-C-C dihedral angle: 0°). The absorption vibrational bands of the two aldehyde rotamers that are present in the mixture lead to a spectrum with a very complex structure in the region of deformation movements, in which several low-intensity bands overlap. A complete and unambiguous assignment of the experimental spectrum has been achieved by using the calculated harmonic and anharmonic vibrational frequencies. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. FANCG knockout CHO cells display sensitivity to diverse DNA damaging agents and possible genomic instability

    Hinz, J.M.; Tebbs, R.S.; Yamada, N.A.; Salazar, E.P.; Kopf, V.L.; Thompson, L.H.

    2003-01-01

    Full text: The function of the proteins encoded by the genes responsible for the disease Fanconi anemia (FA) have not been elucidated. Several of these proteins (FancA, C, E, F, and G) form a complex in the nucleus, and cells deficient in any one of these proteins are sensitive to crosslinking agents, suggesting a possible role for these proteins in some aspect of DNA repair, chromosomal maintenance, or replication. We constructed a FancG knockout mutant (FGKO40) in CHO AA8 cells, as well as FancG-corrected FGKO40 cells (KO40BP6, which is a pool of six BAC-clone transformants). FGKO40 cells are sensitive to a wide variety of DNA damaging agents. Sensitivity to the cross-linking agents MMC (3x) and chloroethyl-nitrosourea (3x) does not exceed that of methyl methanesulfonate (MMS) (4x), methyl-nitrosourea (4x), or ethyl-nitrosourea (3x), or the purine analog 6-thioguanine (5x). Other agents show mild sensitivity in FGKO40 cells: ionizing radiation (1.2x), UV-C (1.5x), hydroxyurea (1.2x), camptothecin (1.2x), and excess thymidine (normal). The length of S phase was carefully measured by monitoring the progression of highly synchronous G1 cells obtained by centrifugal elutriation. FGKO40 cells traversed S phase normally but had a slightly longer G2 phase than parental cells. Treatment of synchronized G1 cells with MMS did not increase S phase in parental or mutant cells, but again G2 was slightly longer for FGKO40. Mutation rates were measured at the hrpt and aprt loci, where gene inactivation confers resistance to 6-thioguanine or 8-azaadenine, respectively. FGKO40 had a slightly reduced mutation rate for hprt mutants, suggesting reduced recovery of large deletions at this locus. Moreover, the rate of methotrexate resistance was elevated 2.5-fold in mutant cells compared to controls. Resistance to this drug is generally associated with amplification of the dhfr locus, suggesting FancG plays a role in this aspect of genome stability. We suggest that the defect in FGKO

  7. Task-based detectability comparison of exponential transformation of free-response operating characteristic (EFROC) curve and channelized Hotelling observer (CHO)

    Khobragade, P.; Fan, Jiahua; Rupcich, Franco; Crotty, Dominic J.; Gilat Schmidt, Taly

    2016-03-01

    This study quantitatively evaluated the performance of the exponential transformation of the free-response operating characteristic curve (EFROC) metric, with the Channelized Hotelling Observer (CHO) as a reference. The CHO has been used for image quality assessment of reconstruction algorithms and imaging systems and often it is applied to study the signal-location-known cases. The CHO also requires a large set of images to estimate the covariance matrix. In terms of clinical applications, this assumption and requirement may be unrealistic. The newly developed location-unknown EFROC detectability metric is estimated from the confidence scores reported by a model observer. Unlike the CHO, EFROC does not require a channelization step and is a non-parametric detectability metric. There are few quantitative studies available on application of the EFROC metric, most of which are based on simulation data. This study investigated the EFROC metric using experimental CT data. A phantom with four low contrast objects: 3mm (14 HU), 5mm (7HU), 7mm (5 HU) and 10 mm (3 HU) was scanned at dose levels ranging from 25 mAs to 270 mAs and reconstructed using filtered backprojection. The area under the curve values for CHO (AUC) and EFROC (AFE) were plotted with respect to different dose levels. The number of images required to estimate the non-parametric AFE metric was calculated for varying tasks and found to be less than the number of images required for parametric CHO estimation. The AFE metric was found to be more sensitive to changes in dose than the CHO metric. This increased sensitivity and the assumption of unknown signal location may be useful for investigating and optimizing CT imaging methods. Future work is required to validate the AFE metric against human observers.

  8. Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

    Ana de la Cueva

    Full Text Available Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

  9. Transferability study of CHO cell clustering assays for monitoring of pertussis toxin activity in acellular pertussis vaccines.

    Isbrucker, R; Daas, A; Wagner, L; Costanzo, A

    2016-01-01

    Current regulations for acellular pertussis (aP) vaccines require that they are tested for the presence of residual or reversion-derived pertussis toxin (PTx) activity using the mouse histamine sensitisation test (HIST). Although a CHO cell clustering assay can be used by manufacturers to verify if sufficient inactivation of the substance has occurred in-process, this assay cannot be used at present for the final product due to the presence of aluminium adjuvants which interfere with mammalian cell cultures. Recently, 2 modified CHO cell clustering assays which accommodate for the adjuvant effects have been proposed as alternatives to the HIST. These modified assays eliminate the adjuvant-induced cytotoxicity either through dilution of the vaccine (called the Direct Method) or by introducing a porous barrier between the adjuvant and the cells (the Indirect Method). Transferability and suitability of these methods for testing of products present on the European market were investigated during a collaborative study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). Thirteen laboratories participated in this study which included 4 aP-containing vaccines spiked by addition of PTx. This study also assessed the transferability of a standardised CHO cell clustering assay protocol for use with non-adjuvanted PTx preparations. Results showed that the majority of laboratories were able to detect the PTx spike in all 4 vaccines at concentrations of 4 IU/mL or lower using the Indirect Method. This sensitivity is in the range of the theoretical sensitivity of the HIST. The Direct Method however did not show the expected results and would need additional development work.

  10. Protective effect of propolis on radiation-induced chromosomal damage on Chinese hamster ovary cells (CHO-K1)

    Spigoti, Geyza; Bartolini, Paolo; Okazaki, Kayo [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: kokazaki@ipen.br; Tsutsumi, Shiguetoshi [Amazon Food Ltd., Tokyo (Japan)], e-mail: fwip5138@mb.infoweb.ne.jp

    2009-07-01

    In the last years, particular interest has been given to investigations concerning natural, effective and nontoxic compounds with radioprotective capacity in concert with increasing utilization of different types of ionizing radiation for various applications. Among them, propolis, a resinous mixture of substances collected by honey bees (Apis mellifera) has been considered promising since it presents several advantageous characteristics, i.e., antiinflammatory, anticarcinogenic, antimicrobial and free radical scavenging action. It is, therefore, a direct antioxidant that protects cells and organisms from the adverse effects of ionizing radiation. These relevant biological activities are mainly mediated by the flavonoids, present at relatively high concentrations in the propolis. Considering that the chemical composition and, consequently, the biological activity of propolis is variable according to the environmental plant ecology, the present study was conducted in order to evaluate the radioprotective capacity of Brazilian propolis, collected in the State of Rio Grande do Sul, against genotoxic damages induced by {sup 60}Co {gamma}-radiation in Chinese hamster ovary cells (CHO-K1). for this purpose, micronucleus induction was analyzed concerning irreparable damage, specifically related to DNA double-strand breaks, that are potentially carcinogenic. CHO-K1 cells were submitted to different concentrations of propolis (3 - 33 {mu}g/ml), 1 h before irradiation, with 1 Gy of {gamma} radiation (0.722 Gy/min). The data obtained showed a decreasing tendency in the quantity of radioinduced damage on cells previously treated with propolis. The radioprotective effect was more prominent at higher propolis concentration. The treatment with propolis alone did not induce genotoxic effects on CHO-K1 cells. Beside that, the treatment with propolis, associated or not with radiation, did not influence the kinetics of cellular proliferation. (author)

  11. Protective effect of propolis on radiation-induced chromosomal damage on Chinese hamster ovary cells (CHO-K1)

    Spigoti, Geyza; Bartolini, Paolo; Okazaki, Kayo; Tsutsumi, Shiguetoshi

    2009-01-01

    In the last years, particular interest has been given to investigations concerning natural, effective and nontoxic compounds with radioprotective capacity in concert with increasing utilization of different types of ionizing radiation for various applications. Among them, propolis, a resinous mixture of substances collected by honey bees (Apis mellifera) has been considered promising since it presents several advantageous characteristics, i.e., antiinflammatory, anticarcinogenic, antimicrobial and free radical scavenging action. It is, therefore, a direct antioxidant that protects cells and organisms from the adverse effects of ionizing radiation. These relevant biological activities are mainly mediated by the flavonoids, present at relatively high concentrations in the propolis. Considering that the chemical composition and, consequently, the biological activity of propolis is variable according to the environmental plant ecology, the present study was conducted in order to evaluate the radioprotective capacity of Brazilian propolis, collected in the State of Rio Grande do Sul, against genotoxic damages induced by 60 Co γ-radiation in Chinese hamster ovary cells (CHO-K1). for this purpose, micronucleus induction was analyzed concerning irreparable damage, specifically related to DNA double-strand breaks, that are potentially carcinogenic. CHO-K1 cells were submitted to different concentrations of propolis (3 - 33 μg/ml), 1 h before irradiation, with 1 Gy of γ radiation (0.722 Gy/min). The data obtained showed a decreasing tendency in the quantity of radioinduced damage on cells previously treated with propolis. The radioprotective effect was more prominent at higher propolis concentration. The treatment with propolis alone did not induce genotoxic effects on CHO-K1 cells. Beside that, the treatment with propolis, associated or not with radiation, did not influence the kinetics of cellular proliferation. (author)

  12. Cytotoxicity Evaluation of Anatase and Rutile TiO₂ Thin Films on CHO-K1 Cells in Vitro.

    Cervantes, Blanca; López-Huerta, Francisco; Vega, Rosario; Hernández-Torres, Julián; García-González, Leandro; Salceda, Emilio; Herrera-May, Agustín L; Soto, Enrique

    2016-07-26

    Cytotoxicity of titanium dioxide (TiO₂) thin films on Chinese hamster ovary (CHO-K1) cells was evaluated after 24, 48 and 72 h of culture. The TiO₂ thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C) toward the anatase to rutile phase transformation. The root-mean-square (RMS) surface roughness of TiO₂ films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM) results showed that the TiO₂ films' thickness values fell within the nanometer range (290-310 nm). Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO₂ thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO₂ thin films, the number of CHO-K1 cells on the control substrate and on all TiO₂ thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO₂ films annealed at 800 °C. These results indicate that TiO₂ thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO₂ thin films in biomedical science.

  13. Cytotoxicity Evaluation of Anatase and Rutile TiO2 Thin Films on CHO-K1 Cells in Vitro

    Blanca Cervantes

    2016-07-01

    Full Text Available Cytotoxicity of titanium dioxide (TiO2 thin films on Chinese hamster ovary (CHO-K1 cells was evaluated after 24, 48 and 72 h of culture. The TiO2 thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C toward the anatase to rutile phase transformation. The root-mean-square (RMS surface roughness of TiO2 films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM results showed that the TiO2 films’ thickness values fell within the nanometer range (290–310 nm. Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO2 thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO2 thin films, the number of CHO-K1 cells on the control substrate and on all TiO2 thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO2 films annealed at 800 °C. These results indicate that TiO2 thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO2 thin films in biomedical science.

  14. Cytotoxicity Evaluation of Anatase and Rutile TiO2 Thin Films on CHO-K1 Cells in Vitro

    Cervantes, Blanca; López-Huerta, Francisco; Vega, Rosario; Hernández-Torres, Julián; García-González, Leandro; Salceda, Emilio; Herrera-May, Agustín L.; Soto, Enrique

    2016-01-01

    Cytotoxicity of titanium dioxide (TiO2) thin films on Chinese hamster ovary (CHO-K1) cells was evaluated after 24, 48 and 72 h of culture. The TiO2 thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C) toward the anatase to rutile phase transformation. The root-mean-square (RMS) surface roughness of TiO2 films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM) results showed that the TiO2 films’ thickness values fell within the nanometer range (290–310 nm). Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO2 thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO2 thin films, the number of CHO-K1 cells on the control substrate and on all TiO2 thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO2 films annealed at 800 °C. These results indicate that TiO2 thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO2 thin films in biomedical science. PMID:28773740

  15. Photolysis of CH3CHO at 248 nm: Evidence of triple fragmentation from primary quantum yield of CH3 and HCO radicals and H atoms

    Morajkar, Pranay; Bossolasco, Adriana; Schoemaecker, Coralie; Fittschen, Christa

    2014-06-01

    Radical quantum yields have been measured following the 248 nm photolysis of acetaldehyde, CH3CHO. HCO radical and H atom yields have been quantified by time resolved continuous wave Cavity Ring Down Spectroscopy in the near infrared following their conversion to HO2 radicals by reaction with O2. The CH3 radical yield has been determined using the same technique following their conversion into CH3O2. Absolute yields have been deduced for HCO radicals and H atoms through fitting of time resolved HO2 profiles, obtained under various O2 concentrations, to a complex model, while the CH3 yield has been determined relative to the CH3 yield from 248 nm photolysis of CH3I. Time resolved HO2 profiles under very low O2 concentrations suggest that another unknown HO2 forming reaction path exists in this reaction system besides the conversion of HCO radicals and H atoms by reaction with O2. HO2 profiles can be well reproduced under a large range of experimental conditions with the following quantum yields: CH3CHO + hν248nm → CH3CHO*, CH3CHO* → CH3 + HCO ϕ1a = 0.125 ± 0.03, CH3CHO* → CH3 + H + CO ϕ1e = 0.205 ± 0.04, CH3CHO*{to 2pc{rArrfill}}limits^{o2}CH3CO + HO2 ϕ1f = 0.07 ± 0.01. The CH3O2 quantum yield has been determined in separate experiments as φ_{CH3} = 0.33 ± 0.03 and is in excellent agreement with the CH3 yields derived from the HO2 measurements considering that the triple fragmentation (R1e) is an important reaction path in the 248 nm photolysis of CH3CHO. From arithmetic considerations taking into account the HO2 and CH3 measurements we deduce a remaining quantum yield for the molecular pathway: CH3CHO* → CH4 + CO ϕ1b = 0.6. All experiments can be consistently explained with absence of the formerly considered pathway: CH3CHO* → CH3CO + H ϕ1c = 0.

  16. Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

    Gaëlle Gonzalez

    Full Text Available Cell microparticles (MPs released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5, and serotype 35 (HAdV35, respectively. We found that MPs derived from CHO cells (MP-donor cells constitutively expressing CAR (MP-CAR or CD46 (MP-CD46 were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

  17. CRISPR/Cas9-induced transgene insertion and telomere-associated truncation of a single human chromosome for chromosome engineering in CHO and A9 cells.

    Uno, Narumi; Hiramatsu, Kei; Uno, Katsuhiro; Komoto, Shinya; Kazuki, Yasuhiro; Oshimura, Mitsuo

    2017-10-06

    Chromosome engineering techniques including gene insertion, telomere-associated truncation and microcell-mediated chromosome transfer (MMCT) are powerful tools for generation of humanised model animal, containing megabase-sized genomic fragments. However, these techniques require two cell lines: homologous recombination (HR)-proficient DT40 cells for chromosome modification, and CHO cells for transfer to recipient cells. Here we show an improved technique using a combination of CRISPR/Cas9-induced HR in CHO and mouse A9 cells without DT40 cells following MMCT to recipient cells. Transgene insertion was performed in CHO cells with the insertion of enhanced green fluorescence protein (EGFP) using CRISPR/Cas9 and a circular targeting vector containing two 3 kb HR arms. Telomere-associated truncation was performed in CHO cells using CRISPR/Cas9 and a linearised truncation vector containing a single 7 kb HR arm at the 5' end, a 1 kb artificial telomere at the 3' end. At least 11% and 6% of the targeting efficiency were achieved for transgene insertion and telomere-associated truncation, respectively. The transgene insertion was also confirmed in A9 cells (29%). The modified chromosomes were transferrable to other cells. Thus, this CHO and A9 cell-mediated chromosome engineering using the CRISPR/Cas9 for direct transfer of the modified chromosome is a rapid technique that will facilitate chromosome manipulation.

  18. FY 2001 report on the new energy vision of the Ishigaki City area. Survey for working out 'the project for the introduction of biomass energy in the central urban area of Ishigaki City'; 2001 nendo Ishigaki shi chiiki shin energy vision sakutei tou jigyo hokokusho

    NONE

    2002-02-01

    Study was made of potentialities of the project of 'an energy supply system for biomass energy using business-derived raw refuse' indicated in 'the new energy vision of Ishigaki City, Okinawa Prefecture.' The amount of the recovered business-derived raw refuse was set at 5.6t at the minimum as against the discharged amount of 8t, and it was calculated that the amount of the biogas generated from the high temperature methane fermentation would be 757.7 m{sup 3}/d. The amount of the suppliable energy calculated from the above was 1,102.4 kWh/d in gas engine. Further, as to the energy demand, supposing the energy demand in large-scale hot bath facilities using salt as a resource of the area, it was calculated that the energy demand would be approximately 1MWh/d altogether with that in the biogas plant. As a result of the study of profitability of the project, the following was found out: it takes 25 years to recover the invested capital in the case of carrying out the biogas plant project alone and 18 years to recover it in the case of carrying out the hot bath facility project alone. In the case of carrying out both projects as one, it takes 18 years to recover the invested capital, the same as in the case of carrying out the hot bath facility project alone, but a great shortage from 6 years to 3 years is shown of the period required to get out of the cumulative deficit. (NEDO)

  19. Fiscal 2000 survey report. Survey of preparation of energy consumption index for Asian region and of prediction of amount of new energy to be introduced; 2000 nendo Asia chiiki no energy shohi koritsu shihyo no sakusei to shin energy donyuryo no yosoku ni kansuru chosa hokokusho

    NONE

    2001-03-01

    For Japan whose domestic reduction of greenhouse gas emissions is but limited, an approach to the problem with emission tradings and CDMs (clean development mechanisms) taken into consideration is necessary, and improvement on energy consumption in the Asian region as a whole is indispensable. From this viewpoint, countries in Asia who might be candidate importers of Japan's technical assistance were selected, and their energy conservation potential and farming village electrification potential were investigated. Covered by the survey were China, Philippines, Thailand, Vietnam, Malaysia, Indonesia, and India. Investigations were conducted into the fields named below, and the results were grouped into the same. The fields were (1) economic growth and energy supply/demand in the Asian region, (2) energy conservation in the Asian region, (3) electrification of farm villages in the Asian region, (4) cost analysis for farm village electrification in the Asian region, and (5) simulation of new energy introduction. The results show that there is a great difference between the seven countries in all the five fields. (NEDO)

  20. Fiscal 2000 regional new energy vision formulation - Financially aided project. Report on regional new energy vision formulation for Ueno village (Object of survey: Commercialization feasibility study for a stand-alone type wind power generation system at Ueno German Culture Village); 2000 nendo Uenoson chiiki shin energy vision sakutei hokokusho. Ueno Doitsu bunkamura dokuritsugata furyoku hatsuden system jigyoka feasibility study chosa

    NONE

    2001-02-01

    A survey is conducted about the profitability of installing a stand-alone type wind power system at Ueno village (Miyako Island), Okinawa Prefecture. The German Culture Village, which is an integrated resort house provided with accommodations (500 people), educational and cultural facilities, and sports facilities consumes 272 MWh/year in electric power. The results of the survey are summarized in six chapters, which are (1) the survey of the business under the project to be the object of feasibility study, (2) project to be the object of feasibility study, (3) outline of the Okinawa Electric Power Co., Inc., Miyakojima system and problems to ensue from its interconnection with wind turbines, (4) survey of stand-alone type wind turbine power generation systems, (5) technical/economic feasibility study, and (6) project management and future tasks. It is concluded that there will be profit in the presence of a subsidy that covers 65% or more of the total cost of constructing a system comprising three 600 KW wind turbines, three 400 kW class diesel generators, and storage batteries. (NEDO)

  1. New energy vision in Iide Town (Yamagata Prefecture), town of 'circulation type energy' that connects nature, people and life together; Iidecho chiiki shin energy vision. Shizen hito kurashi wo tsunageru 'junkangata energy' no machi

    NONE

    2001-02-01

    With an aim of regenerating a sustainable circulation type society, a new energy introduction plan was established that saves energy and utilizes reproducible resources. The plan lasts until 2010 with the target of the new energy introduction quantity in 2010 to reach 3.1% of the total primary energy supply quantity. The activity achievements were put into order by the following seven items: 1) summary of Iide Town, 2) types and utilization methods of new energies, 3) trends of energy demand in Iide Town, 4) possibility of utilizing new energies in Iide Town, 5) basic conception for introducing new energies, 6) systems of new energy vision, and 7) what is to be done with emphasis for the time being. In Item 3, considerations were given on the energy using modes and demand quantity. Fields of the object include public facilities, general households, and agricultural production. The using modes cover heat, electric power, and automotive use. The total demand was calculated as 145,891 Gcal/year, divided into the heat utilization at 50.7%, the electric power at 8.8%, and the automotive use at 40.6%. (NEDO)

  2. Feasibility study on plan to utilize livestock excreta for bio-gas in Miyagi Village (Gunma Prefecture). Investigations on projects including district new energy vision establishment in fiscal 2000, and on feasibility for commercialization; Miyagimura chikusan haisetsubutsu bio gas ka energy riyo keikaku feasibility study. 2000 nendo chiiki shin energy vision sakutei nado jigyo, jigyoka feasibility study

    NONE

    2001-02-01

    With objectives to utilize energy available from livestock excreta, and to properly treat the livestock excreta, a feasibility study was performed on a plan to utilize livestock excreta for bio-gas. The system to be developed is a livestock wastes treatment system to utilize gas generated by efficiently fermenting the excreta as fuel, and residual sludge solids as compost. The activity achievements were put into order by the following nine items: 1) purpose of the feasibility study, 2) method for the feasibility study, 3) the situation where the livestock industry and the hog raising industry were selected as the object of the investigation, 4) properties of hog excreta, characteristics in urine foul water treatment, technologies for urine foul water treatment (biological treatment), 5) bio-gasification of livestock wastes, 6) the basic conditions for investigating the demonstration bio-bas plants, 7) proposals and standard cases of the demonstration bio gas plants, 8) discussions and positioning of the basic system for the demonstration bio gas plants, and 9) conclusion. (NEDO)

  3. Development of a new bioprocess scheme using frozen seed train intermediates to initiate CHO cell culture manufacturing campaigns.

    Seth, Gargi; Hamilton, Robert W; Stapp, Thomas R; Zheng, Lisa; Meier, Angela; Petty, Krista; Leung, Stephenie; Chary, Srikanth

    2013-05-01

    Agility to schedule and execute cell culture manufacturing campaigns quickly in a multi-product facility will play a key role in meeting the growing demand for therapeutic proteins. In an effort to shorten campaign timelines, maximize plant flexibility and resource utilization, we investigated the initiation of cell culture manufacturing campaigns using CHO cells cryopreserved in large volume bags in place of the seed train process flows that are conventionally used in cell culture manufacturing. This approach, termed FASTEC (Frozen Accelerated Seed Train for Execution of a Campaign), involves cultivating cells to high density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags. This article reviews the development and optimization of individual steps of the FASTEC bioprocess scheme: scaling up cells to greater than 70 × 10(6) cells/mL and freezing in bags with an optimized controlled rate freezing protocol and using a customized rack configuration. Flow cytometry analysis was also employed to understand the recovery of CHO cells following cryopreservation. Extensive development data were gathered to ensure that the quantity and quality of the drug manufactured using the FASTEC bioprocess scheme was acceptable compared to the conventional seed train process flow. The result of offering comparable manufacturing options offers flexibility to the cell culture manufacturing network. Copyright © 2012 Wiley Periodicals, Inc.

  4. Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells

    Camille C. Hanot

    2015-12-01

    Full Text Available Superparamagnetic iron-oxide nanoparticles (SPIONs show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells. We evaluated the effect of particle diameter (50 and 100 nm and polyethylene glycol (PEG chain length (2k, 5k and 20k Da on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT and sulforhodamine B (SRB assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS. Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications.

  5. Stable expression of human thyrotropin (hTSH) in mammalian cells (CHO) expressing α2,6 sialyltransferase

    Damiani, Renata

    2009-01-01

    A CHO cell line, previously genetically modified by the introduction of rat α2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of α2,3- and 39% of α2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 μM methotrexate, presented a secretion level of ∼2 μg hTSH/10 6 cells/day, useful for product purification and characterization. The relative molecular masses (M r ) of the heterodimer and of the α- and β-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T 4 release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.5-fold more potent than native hTSH (p < 0.001). (author)

  6. Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro.

    Mosesso, Pasquale; Bohm, Lothar; Pepe, Gaetano; Fiore, Mario; Carpinelli, Alice; Gäde, Gerd; Nagini, Siddavaram; Ottavianelli, Alessandro; Degrassi, Francesca

    2012-09-18

    In this work we have examined the genotoxic potential of the bioinsecticide Azadirachtin A (AZA) and its influence on cell proliferation on human lymphocytes and Chinese Hamster ovary (CHO) cells. AZA genotoxicity was assessed by the analysis of chromosomal aberrations and sister chromatid exchanges (SCEs) in the absence and presence of rat liver S9 metabolism. Primary DNA damage was also investigated by means of the comet assay. The results obtained clearly indicate that AZA is not genotoxic in mammalian cells. On the other hand, AZA proved to interfere with cell cycle progression as shown by modulation of frequencies of first (M1) and second division (M2) metaphases detected by 5-Bromo-2'-deoxyuridine labeling. Accumulation of M1 metaphases were more pronounced in human lymphocytes. In the transformed CHO cell line, however, significant increases of multinucleated interphases and polyploid cells were observed at long treatment time. At higher dose-levels, the incidence of polyploidy was close to 100%. Identification of spindle structure and number of centrosomes by fluorescent immunostaining with α- and γ-tubulin antibodies revealed aberrant mitoses exhibiting multipolar spindles with several centrosomal signals. These findings suggest that AZA can act either through a stabilizing activity of microtubules or by inhibition of Aurora A, since both mechanisms are able to generate genetically unstable polyploid cells with multipolar spindles and multinucleated interphases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  7. Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement.

    Mosser, Mathilde; Kapel, Romain; Chevalot, Isabelle; Olmos, Eric; Marc, Ivan; Marc, Annie; Oriol, Eric

    2015-01-01

    Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates. © 2015 American Institute of Chemical Engineers.

  8. Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

    Stankowski, L.F. Jr.; Tindall, K.R.; Hsie, A.W.

    1983-01-01

    Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10 6 clonable cells. As with HGPRT - mutants, XGPRT - mutants can be selected in 6-thioguanine. AS52 (XGPRT + ) and wild type CHO (HGPRT + ) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT - to HGPRT - mutants induced per unit dose (J/m 2 for UV light and μg/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT - AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment

  9. Women in church and society: Report of research done by a research team at the PU vir CHO

    F.J. van Rensburg

    2002-08-01

    Full Text Available The research project “Women in Church and Society” was conducted under the auspices of one of the focus areas for research and postgraduate education at the Potchefstroomse Universiteit vir Christelike Hoër Onderwys: “Reformed Theology and the Development of the South African Society”. This focus area is based in the Faculty of Theology (PU vir CHO and is directed by Herrie van Rooy. Project 2 of this focus area is “The socio-historic context of the Bible and its implications for the development of South African Society” and is under the leadership of Fika J. van Rensburg. The first sub-project of Project 2 to be completed is “Women in Church and Society”. It commenced in 2000 and had its fourth and final workshop in September 2002. It was managed by a five-person executive committee and had the following categories of collaborators: 16 PU vir CHO researchers, 10 researchers from other South African universities, 6 international researchers, 19 masters’ and doctoral students, and 21 researchers with special expertise in relevant areas. In total 48 papers1 were read and discussed at the four workshops; and most of them have either been published or are in the process of being published as articles in accredited journals. This article is a report on the activities and outcome of the research project.

  10. Photolysis of CH₃CHO at 248 nm: evidence of triple fragmentation from primary quantum yield of CH₃ and HCO radicals and H atoms.

    Morajkar, Pranay; Bossolasco, Adriana; Schoemaecker, Coralie; Fittschen, Christa

    2014-06-07

    Radical quantum yields have been measured following the 248 nm photolysis of acetaldehyde, CH3CHO. HCO radical and H atom yields have been quantified by time resolved continuous wave Cavity Ring Down Spectroscopy in the near infrared following their conversion to HO2 radicals by reaction with O2. The CH3 radical yield has been determined using the same technique following their conversion into CH3O2. Absolute yields have been deduced for HCO radicals and H atoms through fitting of time resolved HO2 profiles, obtained under various O2 concentrations, to a complex model, while the CH3 yield has been determined relative to the CH3 yield from 248 nm photolysis of CH3I. Time resolved HO2 profiles under very low O2 concentrations suggest that another unknown HO2 forming reaction path exists in this reaction system besides the conversion of HCO radicals and H atoms by reaction with O2. HO2 profiles can be well reproduced under a large range of experimental conditions with the following quantum yields: CH3CHO + hν(248nm) → CH3CHO*, CH3CHO* → CH3 + HCO ϕ(1a) = 0.125 ± 0.03, CH3CHO* → CH3 + H + CO ϕ(1e) = 0.205 ± 0.04, CH3CHO*[Formula: see text]CH3CO + HO2 ϕ(1f) = 0.07 ± 0.01. The CH3O2 quantum yield has been determined in separate experiments as ϕ(CH₃) = 0.33 ± 0.03 and is in excellent agreement with the CH3 yields derived from the HO2 measurements considering that the triple fragmentation (R1e) is an important reaction path in the 248 nm photolysis of CH3CHO. From arithmetic considerations taking into account the HO2 and CH3 measurements we deduce a remaining quantum yield for the molecular pathway: CH3CHO* → CH4 + CO ϕ(1b) = 0.6. All experiments can be consistently explained with absence of the formerly considered pathway: CH3CHO* → CH3CO + H ϕ(1c) = 0.

  11. Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines

    Kaas, Christian Schrøder; Bolt, Gert; Hansen, Jens J

    2015-01-01

    orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII...... FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce...

  12. Evaluation of CHO Benchmarks on the Arria 10 FPGA using Intel FPGA SDK for OpenCL

    Jin, Zheming [Argonne National Lab. (ANL), Argonne, IL (United States); Yoshii, Kazutomo [Argonne National Lab. (ANL), Argonne, IL (United States); Finkel, Hal [Argonne National Lab. (ANL), Argonne, IL (United States); Cappello, Franck [Argonne National Lab. (ANL), Argonne, IL (United States)

    2017-05-23

    The OpenCL standard is an open programming model for accelerating algorithms on heterogeneous computing system. OpenCL extends the C-based programming language for developing portable codes on different platforms such as CPU, Graphics processing units (GPUs), Digital Signal Processors (DSPs) and Field Programmable Gate Arrays (FPGAs). The Intel FPGA SDK for OpenCL is a suite of tools that allows developers to abstract away the complex FPGA-based development flow for a high-level software development flow. Users can focus on the design of hardware-accelerated kernel functions in OpenCL and then direct the tools to generate the low-level FPGA implementations. The approach makes the FPGA-based development more accessible to software users as the needs for hybrid computing using CPUs and FPGAs are increasing. It can also significantly reduce the hardware development time as users can evaluate different ideas with high-level language without deep FPGA domain knowledge. Benchmarking of OpenCL-based framework is an effective way for analyzing the performance of system by studying the execution of the benchmark applications. CHO is a suite of benchmark applications that provides support for OpenCL [1]. The authors presented CHO as an OpenCL port of the CHStone benchmark. Using Altera OpenCL (AOCL) compiler to synthesize the benchmark applications, they listed the resource usage and performance of each kernel that can be successfully synthesized by the compiler. In this report, we evaluate the resource usage and performance of the CHO benchmark applications using the Intel FPGA SDK for OpenCL and Nallatech 385A FPGA board that features an Arria 10 FPGA device. The focus of the report is to have a better understanding of the resource usage and performance of the kernel implementations using Arria-10 FPGA devices compared to Stratix-5 FPGA devices. In addition, we also gain knowledge about the limitations of the current compiler when it fails to synthesize a benchmark

  13. Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.

    Shin, Jongoh; Lee, Namil; Cho, Suhyung; Cho, Byung-Kwan

    2018-01-01

    Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

  14. Sister chromatid exchanges induced in CHO cells by X-rays or 5.5 MeV neutrons

    Bocian, E.; Rosiek, O.; Sablinski, J.; Ziemba-Zoltowska, B.

    1986-01-01

    The induction of sister chromatid exchanges (SCEs) by X-rays (1-9 Gy) and 5.5 MeV neutrons (0.5-4 Gy) was studied in CHO cells. A dose-dependent increase of the frequency of SCE was found for both radiations when cells with BrdUrd substituted DNA were irradiated. The similar doubling dose, approx. 4 Gy, was found for X-rays and neutrons. The increase of the SCE frequency was not clearly dependent on the dose when cells with BrdUrd unsubstituted DNA were irradiated. In this case a dose of 4 Gy enhanced the SCE frequency only by the factor of 1.3. (author)

  15. Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian

    2015-01-01

    optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylationrelated product quality. In this work, different fed-batch processes with two chemically defined proprietary media......Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process...... and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality....

  16. Analysis of native cellular DNA after heavy ion irradiation: DNA double-strand breaks in CHO-K1 cells

    Heilmann, J.; Taucher-Scholz, G.; Kraft, G.

    1994-11-01

    A fast assay for the detection of DNA double-strand breaks was developed involving constant field gel electrophoresis (Taucher-Scholz et al., 1994) and densitometric scanning of agarose gels stained with ethidium bromide. With this technique, DSB induction was investigated after irradiation of CHO cells with carbon ions with LET values between 14 keV/μm and 400 keV/μm. In parallel, a computer code was developed to simulate both the principle of the electrophoretic detection of DNA double-strand breaks and the action of radiations of different ionization density. The results of the experiments and the calculations are presented here and compared with each other. (orig./HSI)

  17. Effect of Lippia alba and Cymbopogon citratus essential oils on biofilms of Streptococcus mutans and cytotoxicity in CHO cells.

    Tofiño-Rivera, A; Ortega-Cuadros, M; Galvis-Pareja, D; Jiménez-Rios, H; Merini, L J; Martínez-Pabón, M C

    2016-12-24

    Caries is a public health problem, given that it prevails in 60 to 90% of the school-age global population. Multiple factors interact in its etiology, among them dental plaque is necessary to have lactic acid producing microorganisms like Streptococcus from he Mutans group. Existing prevention and treatment measures are not totally effective and generate adverse effects, which is why it is necessary to search for complementary strategies for their management. The study sought to evaluate the eradication capacity of Streptococcus mutans biofilms and the toxicity on eukaryotic cells of Lippia alba and Cymbopogon citratus essential oils. Essential oils were extracted from plant material through steam distillation and then its chemical composition was determined. The MBEC-high-throughput (MBEC-HTP) (Innovotech, Edmonton, Alberta, Canada) assay used to determine the eradication concentration of S. mutans ATCC 35668 strain biofilms. Cytotoxicity was evaluated on CHO cells through the MTT cell proliferation assay. The major components in both oils were Geraniol and Citral; in L. alba 18.9% and 15.9%, respectively, and in C. citratus 31.3% and 26.7%. The L. alba essential oils presented eradication activity against S. mutans biofilms of 95.8% in 0.01mg/dL concentration and C. citratus essential oils showed said eradication activity of 95.4% at 0.1, 0.01mg/dL concentrations and of 93.1% in the 0.001mg/dL concentration; none of the concentrations of both essential oils showed toxicity on CHO cells during 24h. The L. alba and C. citratus essential oils showed eradication activity against S. mutans biofilms and null cytotoxicity, evidencing the need to conduct further studies that can identify their active components and in order to guide a safe use in treating and preventing dental caries. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  18. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells.

    Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

    2017-06-01

    Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn 56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

  20. Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

    Burleigh, Susan C

    2011-10-18

    Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

  1. Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies

    Shridhar Bale

    2018-05-01

    Full Text Available Native flexibly linked (NFL HIV-1 envelope glycoprotein (Env trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan “hole” naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.

  2. FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

    Louie, Salina; Haley, Benjamin; Marshall, Brett; Heidersbach, Amy; Yim, Mandy; Brozynski, Martina; Tang, Danming; Lam, Cynthia; Petryniak, Bronislawa; Shaw, David; Shim, Jeongsup; Miller, Aaron; Lowe, John B; Snedecor, Brad; Misaghi, Shahram

    2017-03-01

    During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Reprogramming amino acid catabolism in CHO cells with CRISPR-Cas9 genome editing improves cell growth and reduces by-product secretion

    Ley, Daniel; Pereira, Sara; Pedersen, Lasse Ebdrup

    2017-01-01

    CHO cells primarily utilize amino acids for three processes: biomass synthesis, recombinant protein production and catabolism. In this work, we disrupted 9 amino acid catabolic genes participating in 7 dierent catabolic pathways, to increase synthesis of biomass and recombinant protein, while red...... reducing production of growth-inhibiting metabolic by-products from amino acid catabolism....

  4. Stable lentiviral transformation of CHO cells for the expression of the hemagglutinin H5 of avian influenza virus in suspension culture

    Alaín González Pose

    2014-09-01

    Full Text Available Avian influenza virus H5N1 has caused extensive damage worldwide among poultry and humans. Effective expression systems are needed for the production of viral proteins required for monitoring this devastating disease. The present study deals with the establishment of a stable expression system for the hemagglutinin H5 (HAH5 of avian influenza virus using CHO cells in suspension culture transduced with a recombinant lentiviral vector. The synthetic gene coding the HAH5 protein was inserted in a lentiviral vector with the aim of performing a stable transduction of CHO cells. After the selection of recombinant clones, the one with the highest expression level was adapted to suspension culture and the HAH5 protein was purified by immunoaffinity chromatography from the culture supernatant. There were no significant differences when this protein, purified or direct from the culture supernatant of CHO or SiHa cells, was utilized in an immunologic assay using positive and negative sera as reference. It was also demonstrated that the HAH5 protein in its purified form is able to bind anti-HAH5 antibodies generated with proper and non-proper folded proteins. The results demonstrate that the CHO cell line stably transduced with a lentiviral vector coding the sequence of the HAH5 protein and cultured in suspension can be a suitable expression system to obtain this protein for diagnostic purpose in a consistent and reliable manner.

  5. High-throughput automated parallel evaluation of zinc-based catalysts for the copolymerization of CHO and CO2 to polycarbonates

    Meerendonk, van W.J.; Duchateau, R.; Koning, C.E.; Gruter, G.J.M.

    2004-01-01

    Copolymn. of CO2 and oxiranes using a high-pressure autoclave typically allows one expt. per reactor per day. A high-throughput parallel setup was developed and validated for the copolymn. of CO2 and cyclohxene oxide (CHO) with two b-diiminato zinc complexes. The catalyst activity is affected by

  6. A physiological threshold for protection against menadione toxicity by human NAD(P)H : quinone oxidoreductase (NQO1) in Chinese hamster ovary (CHO) cells

    Haan, de L.H.J.; Boerboom, A.M.J.F.; Rietjens, I.M.C.M.; Capelle, van D.; Ruijter, de A.J.M.; Jaiswal, A.K.; Aarts, J.M.M.J.G.

    2002-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) has often been suggested to be involved in cancer prevention by means of detoxification of electrophilic quinones. In the present study, a series of Chinese hamster ovary (CHO) cell lines expressing various elevated levels of human NQO1 were generated by

  7. Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

    Cliona M Stapleton

    2011-04-01

    Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

  8. Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

    Sege, R.D.; Kozarsky, K.F.; Krieger, M.

    1986-01-01

    The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene

  9. Dual effects of muscarinic M2 acetylcholine receptors on the synthesis of cyclic AMP in CHO cells: dependence on time, receptor density and receptor agonists

    Michal, Pavel; Lysíková, Michaela; Tuček, Stanislav

    2001-01-01

    Roč. 132, č. 6 (2001), s. 1217-1228 ISSN 0007-1188 R&D Projects: GA ČR GA309/99/0214; GA AV ČR IAA7011910 Institutional research plan: CEZ:AV0Z5011922 Keywords : cyclic AMP * muscarinic receptors * CHO cells Subject RIV: ED - Physiology Impact factor: 3.502, year: 2001

  10. Infrared and Raman spectroscopy and quantum chemistry calculation studies of C-H...O hydrogen bondings and thermal behavior of biodegradable polyhydroxyalkanoate

    Sato, H.; Dybal, Jiří; Murakami, R.; Noda, I.; Ozaki, Y.

    744-747, - (2005), s. 35-46 ISSN 0022-2860 R&D Projects: GA AV ČR IAA4050208 Keywords : infrared and Raman spectroscopy * quantum chemical calculation * C-H...O hydrogen bonding Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.440, year: 2005

  11. Protective activity of a novel resveratrol analogue, HS-1793, against DNA damage in 137Cs-irradiated CHO-K1 cells

    Jeong, Min Ho; Jo, Young Rae; Yang, Kwang Mo; Jeong, Dong Hyeok; Lee, Chang Geun; Oh, Su Jung; Jeong, Soo Kyung; Jo, Wol Soon; Lee, Ki Won

    2014-01-01

    Resveratrol has received considerable attention as a polyphenol with anti-oxidant, anti-carcinogenic, and anti-inflammatory effects. Radiation is an important component of therapy for a wide range of malignant conditions. However, it causes damage to normal cells and, hence, can result in adverse side effects. This study was conducted to examine whether HS-1793, a novel resveratrol analogue free from the restriction of metabolic instability and the high dose requirement of resveratrol, induces a protective effect against radiation-induced DNA damage. HS-1793 effectively scavenged free radicals and inhibited radiation-induced plasmid DNA strand breaks in an in vitro assay. HS-1793 significantly decreased reactive oxygen species and cellular DNA damage in 2 Gy-irradiated Chinese hamster ovary (CHO)-K1 cells. In addition, HS-1793 dose-dependently reduced the levels of phosphorylated H2AX in irradiated CHO-K1 cells. These results indicate that HS-1793 has chemical radioprotective activity. Glutathione levels and superoxide dismutase activity in irradiated CHO-K1 cells increased significantly following HS-1793 treatment. The enhanced biological anti-oxidant activity and chemical radioprotective activity of HS-1793 maintained survival of irradiated CHO-K1 cells in a clonogenic assay. Therefore, HS-1793 may be of value as a radioprotector to protect healthy tissue surrounding tumor cells during radiotherapy to obtain better tumor control with a higher dose. (author)

  12. Comparison of APR1400 safety between brake site and shin-Kori site Due to the difference in the climate conditions

    Yoon, Ho Joon; Lee, Jeong Ik; Lee, Jeong Ik

    2012-01-01

    Brake Nuclear Power Plant (BNPP) is now under the construction based on APR1400 designed by Korean Electric Power Corporation (KEPCO). APR1400 is a two loop pressurized water reactor, the nuclear steam supply system (NSSS) US designed for about put of 4,000 MWt, with a corresponding electrical output of approximately 1,390 MWe. The first APR1400 (SKN 3 and 4) constructed in Shin-Kori, Korea has been modified according to the surrounding environment of the United Arab Emirates. In this paper, authors would like to compare safety issues between B NPP and Skin due to the changes of surroundings, since the site characteristics are very different. For instance, the mean annual air temperature in the UAE is 28 .deg. C and the peak air temperature was recorded as 48.8 .deg. C. Sea temperatures are varying from 17. deg. C in January to 35. deg. C in August, while that of Korea is in 9-16. deg. C range. Hot climate of UAE and the malfunction of HVAC system can lead the increasing of the water temperature in safety injection system (SIS). The heated water in SIS may affect the safety margin of the peak cladding temperature (PCT). The change of PCT and response time according to design basis accident scenarios such as large break LOCA are analyzed in detail. To evaluate such effect, Mars code was utilized to evaluate assumed condition by KAIST and the analyses of the results were carried out by Khalifa Univ.

  13. [Analysis of tension-distraction state in the shin bones fractures in conditions of external fixation with application of apparatuses with different spatially oriented supports].

    Hutsuliak, V I

    2014-09-01

    In Autodesk Inventor 11 program, using method of end-capping elements, a three- dimensional computeric modelling of biomechanical systems of two models was conducted: I - "tibia - Ilizarov's apparatus with concentric location of supports"; II - "tibia - Ilizarov's apparatus with excentric location of supports". The loading, which was applied towards distal fragment in 6 standard degrees of freedom, was modelled for studying of the fixation rigidity of tibial fragments in these systems. Determination of the loading value in various directions, in which the fragment have had shifted by 1 mm, have constituted the main task of the investigation. In a model II a rigidity of the fragments fixation, in comparison with such in a model I, is bigger by 631.43% - while applying a compression loading, by 8.35 - 31.75% - the transversal one and by 19.72% - the rotation loading. While choosing the method of transosteal osteosynthesis of the shin bones the advantage, have the apparatuses with excentric location of supports, what secures the enhanced rigidity of the fragments fixation in comparison with such in apparatuses with concentric location of supports. Although, even in excentric location of supports in the apparatus the fixation rigidity is insufficient for early full loading of the traumatized extremity while walking. It is necessary to elaborate such apparatus, the form of which may be adopted toanatomic configuration of segment.

  14. Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

    Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2018-07-01

    Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

  15. Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

    Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

    2016-07-01

    Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre

  16. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

  17. Dynamic metabolic flux analysis using B-splines to study the effects of temperature shift on CHO cell metabolism

    Verónica S. Martínez

    2015-12-01

    Full Text Available Metabolic flux analysis (MFA is widely used to estimate intracellular fluxes. Conventional MFA, however, is limited to continuous cultures and the mid-exponential growth phase of batch cultures. Dynamic MFA (DMFA has emerged to characterize time-resolved metabolic fluxes for the entire culture period. Here, the linear DMFA approach was extended using B-spline fitting (B-DMFA to estimate mass balanced fluxes. Smoother fits were achieved using reduced number of knots and parameters. Additionally, computation time was greatly reduced using a new heuristic algorithm for knot placement. B-DMFA revealed that Chinese hamster ovary cells shifted from 37 °C to 32 °C maintained a constant IgG volume-specific productivity, whereas the productivity for the controls peaked during mid-exponential growth phase and declined afterward. The observed 42% increase in product titer at 32 °C was explained by a prolonged cell growth with high cell viability, a larger cell volume and a more stable volume-specific productivity. Keywords: Dynamic, Metabolism, Flux analysis, CHO cells, Temperature shift, B-spline curve fitting

  18. Functional expression of Squalus acanthias melanocortin-5 receptor in CHO cells: ligand selectivity and interaction with MRAP.

    Reinick, Christina L; Liang, Liang; Angleson, Josepha K; Dores, Robert M

    2012-04-05

    The melanocortin-5 receptor (MC(5)) of the dogfish Squalus acanthias (SacMC(5) receptor) can be functionally expressed in CHO cells in the absence of the co-expression of an exogenous MRAP cDNA. Both human ACTH(1-24) and dogfish ACTH(1-25) were much better stimulators of the SacMC(5) receptor than any of the mammalian or dogfish MSH ligands that were tested. The order of ligand selectivity for the dogfish melanocortins was ACTH(1-25)>αMSH>γ-MSH=δ-MSH>β-MSH. Unlike mammalian MC(5) receptors, the functional expression of the SacMC(5) receptor was not negatively impacted when the receptor was co-expressed with a cartilaginous fish (Callorhinchus milii) MRAP2 cDNA. However, co-expression with either mouse mMRAP1 or zebrafish zfMRAP1 increased the sensitivity of SacMC(5) receptor for hACTH(1-24) by at least one order of magnitude. Hence, SacMC(5) receptor has the potential to interact with MRAP1 orthologs and in this regard behaved more like a melanocortin MC(2) receptor ortholog than a melanocortin MC(5) receptor ortholog. These observations are discussed in light of the evolution of the melanocortin receptor gene family in cartilaginous fish, and the physiological implications of these observations are considered. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells

    Rebersek, Matej; Kanduser, Masa; Miklavcic, Damijan

    2011-01-01

    Gene electrotransfer is a non-viral gene delivery method that requires successful electroporation for DNA delivery into the cells. Changing the direction of the electric field during the pulse application improves the efficacy of gene delivery. In our study, we tested a pipette tip with integrated electrodes that enables changing the direction of the electric field for electroporation of cell suspension for gene electrotransfer. A new pipette tip consists of four cylindrical rod electrodes that allow the application of electric pulses in different electric field directions. The experiments were performed on cell suspension of CHO cells in phosphate buffer. Plasmid DNA encoding for green fluorescent protein (GFP) was used and the efficiency of gene electrotransfer was determined by counting cells expressing GFP 24 h after the experiment. Experimental results showed that the percentage of cells expressing GFP increased when the electric field orientation was changed during the application. The GFP expression was almost two times higher when the pulses were applied in orthogonal directions in comparison with single direction, while cell viability was not significantly affected. We can conclude that results obtained with the described pipette tip are comparable to previously published results on gene electrotransfer using similar electrode geometry and electric pulse parameters. The tested pipette tip, however, allows work with small volumes/samples and requires less cell manipulation

  20. Effect of medium replenishment or composition on [3H] thymidine incorporation in uv-irradiated CHO-K1 cells

    Newman, C.N.; Miller, J.H.

    1985-03-01

    Because culture medium contains uv-absorbing material, it is usually removed just before uv-irradiation of tissue culture monolayers. However, medium removal and replenishment with fresh medium alone (sham-irradiation) causes up to a 10-fold reduction in the rate of [ 3 H]TdR incorporation in CHO-K1 cells which persists for several hours. This reduction, which is much smaller ( 3 H]TdR pulse-label in conditioned (spent) and in fresh medium; TdR in the former is converted by cells to thymine. When responses of uv-irradiated cells are normalized to responses of corresponding sham-irradiated cultures, considerable variation is observed in replicate experiments because fresh medium appears to induce transient metabolic imbalances in irradiated cells which are not readily controlled. This problem can, in part, be circumvented by replenishing treated cultures with the original spent medium; however, the presence of CdR in the growth medium still causes an anomalous 2-3-fold greater uv-induced reduction in [ 3 H]TdR incorporation than is observed in the absence of CdR. 17 refs., 3 figs., 1 tab

  1. Formation of the prebiotic molecule NH2CHO on astronomical amorphous solid water surfaces: accurate tunneling rate calculations.

    Song, Lei; Kästner, Johannes

    2016-10-26

    Investigating how formamide forms in the interstellar medium is a hot topic in astrochemistry, which can contribute to our understanding of the origin of life on Earth. We have constructed a QM/MM model to simulate the hydrogenation of isocyanic acid on amorphous solid water surfaces to form formamide. The binding energy of HNCO on the ASW surface varies significantly between different binding sites, we found values between ∼0 and 100 kJ mol -1 . The barrier for the hydrogenation reaction is almost independent of the binding energy, though. We calculated tunneling rate constants of H + HNCO → NH 2 CO at temperatures down to 103 K combining QM/MM with instanton theory. Tunneling dominates the reaction at such low temperatures. The tunneling reaction is hardly accelerated by the amorphous solid water surface compared to the gas phase for this system, even though the activation energy of the surface reaction is lower than the one of the gas-phase reaction. Both the height and width of the barrier affect the tunneling rate in practice. Strong kinetic isotope effects were observed by comparing to rate constants of D + HNCO → NHDCO. At 103 K we found a KIE of 231 on the surface and 146 in the gas phase. Furthermore, we investigated the gas-phase reaction NH 2 + H 2 CO → NH 2 CHO + H and found it unlikely to occur at cryogenic temperatures. The data of our tunneling rate constants are expected to significantly influence astrochemical models.

  2. Overexpression of metallothionein in CHO cells and its effect on cell killing by ionizing radiation and alkylating agents

    Lohrer, H.; Robson, T.

    1989-01-01

    Metallothionein protein protects cells from the toxic effects of heavy metal ions. To establish its protective function against ionizing radiation and alkylating agents, a model system was created by transfecting two CHO cell lines (wild-type, K1-2 and X-ray sensitive, xrs-2 subclone Bc11) with the human metallothionein II-A (hMTII-A) gene integrated in a bovine papilloma derived autonomously replicating vector. The isolated transfectants are cadmium-resistant (Cd 1 ), due to the overexpression of the hMTII-A gene. Their steady-state level of hMTII-A mRNA can be increased up to 40-fold after Cd treatment and 20-fold after induction with ionizing radiation. The transfected cell lines proved to be as sensitive as the recipient cell lines to ionizing radiation and bleomycin but the transfectants were significantly more resistant to N-methyl-nitro-nitrosoguanidine (MNNG) and mitomycin C (MMC). These results lead to the conclusion that the MT protein does provide a defence mechanism to protect cells from monofunctional alkylating and cross-linking agents but not from free radicals. (author)

  3. Overexpression of metallothionein in CHO cells and its effect on cell killing by ionizing radiation and alkylating agents

    Lohrer, H.; Robson, T. (Newcastle upon Tyne Univ. (UK). Cancer Research Unit)

    1989-12-01

    Metallothionein protein protects cells from the toxic effects of heavy metal ions. To establish its protective function against ionizing radiation and alkylating agents, a model system was created by transfecting two CHO cell lines (wild-type, K1-2 and X-ray sensitive, xrs-2 subclone Bc11) with the human metallothionein II-A (hMTII-A) gene integrated in a bovine papilloma derived autonomously replicating vector. The isolated transfectants are cadmium-resistant (Cd{sup 1}), due to the overexpression of the hMTII-A gene. Their steady-state level of hMTII-A mRNA can be increased up to 40-fold after Cd treatment and 20-fold after induction with ionizing radiation. The transfected cell lines proved to be as sensitive as the recipient cell lines to ionizing radiation and bleomycin but the transfectants were significantly more resistant to N-methyl-nitro-nitrosoguanidine (MNNG) and mitomycin C (MMC). These results lead to the conclusion that the MT protein does provide a defence mechanism to protect cells from monofunctional alkylating and cross-linking agents but not from free radicals. (author).

  4. Use of track-end alpha particles from 241Am to study radiosensitive sites in CHO cells

    Datta, R.; Cole, A.; Robinson, S.

    1976-01-01

    Monolayers of CHO cells placed on membrane filters were irradiated with alpha particles from a 241 Am source. Particle penetration into the cells was controlled by placing the cell sample at various distances from the source. Dosimetric and spectrometric measurements were performed at comparable positions using a parallel plate ionization chamber and a scintillation crystal spectrometer. Cell survival, as measured by conventional cloning techniques, was single hit in form. A pronounced minimum in mean lethal dose of 29 rad was observed for alpha particle beams that penetrated only about 3 μm into the cell. A pronounced maximum in inactivation cross section of 90 μm 2 , equal to about half the projected area of the nucleus, occurred for beams that penetrated only 5 to 7 μm into the cell. Thus, a single alpha particle penetration several micrometers within the cell nucleus was effective in killing the cell, while fully penetrating beams were actually less efficient; the latter beams required multiple particle traversals and about three times the cell dose to achieve the same effect. These results support the proposal that radiosensitive sites are located in a thin peripheral region of the nucleus

  5. Improved modification for the density-functional theory calculation of thermodynamic properties for C-H-O composite compounds.

    Liu, Min Hsien; Chen, Cheng; Hong, Yaw Shun

    2005-02-08

    A three-parametric modification equation and the least-squares approach are adopted to calibrating hybrid density-functional theory energies of C(1)-C(10) straight-chain aldehydes, alcohols, and alkoxides to accurate enthalpies of formation DeltaH(f) and Gibbs free energies of formation DeltaG(f), respectively. All calculated energies of the C-H-O composite compounds were obtained based on B3LYP6-311++G(3df,2pd) single-point energies and the related thermal corrections of B3LYP6-31G(d,p) optimized geometries. This investigation revealed that all compounds had 0.05% average absolute relative error (ARE) for the atomization energies, with mean value of absolute error (MAE) of just 2.1 kJ/mol (0.5 kcal/mol) for the DeltaH(f) and 2.4 kJ/mol (0.6 kcal/mol) for the DeltaG(f) of formation.

  6. SNARE-mediated trafficking of α5β1 integrin is required for spreading in CHO cells

    Skalski, Michael; Coppolino, Marc G.

    2005-01-01

    In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cell spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of α 5 β 1 integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading

  7. Effects of hyperthermia and x irradiation on sister chromatid exchange (SCE) frequency in Chinese hamster ovary (CHO) cells

    Livingston, G.K.; Dethlefsen, L.A.

    1979-01-01

    The BrdUrd labeling method was used to evaluate the effects of hyperthermia, x irradiation, and the combined treatment on the incidence of sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells. Cells cultured in McCoy's 5A media containing 10 μM 5-bromodeoxyuridine were synchronized after one cell cycle by mitotic shake-off. Early-G 1 cells were heated by submerging culture flasks in a 44 +- 0.05 0 C water bath for periods of 20, 40, and 60 min. By the same method, other cultures were x irradiated at doses of 100, 200, 400, and 600 rad. A third protocol involved combined treatment of 20 min at 44 0 C followed immediately by one of the above radiation doses. A fourth protocol reversed the sequence of the combined treatment applying x irradiation (200 or 400 rad) followed immediately by hyperthermia. The data showed that hyperthermia and x irradiation both elevated the frequency of SCEs significantly whether applied separately or together. The combined treatment (heat: 20 min at 44 0 C plus varying x-radiation doses) produced results suggestive of a synergistic interaction. The sequence of the heat and x irradiation did not appear to have a significant effect on the production of SCE

  8. Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells.

    Almeida, Aline G; Pinto, Rodrigo C V; Smales, C Mark; Castilho, Leda R

    2017-08-01

    To develop a recombinant human factor IX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4-8 °C. NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation for a wide range of formulations tested. Particular devised formulations maintained rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix). rFIX remained active after 3 months when stored at 4 °C, though this was not the case with samples stored at 40 °C. Interestingly, particular formulations had an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix formulation in terms of particle size distribution and cake appearance. Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, L-histidine as buffering agent, and NaCl added in the reconstitution liquid at 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.

  9. Origin and evolution of binucleated cells and binucleated cells with micronuclei in cisplatin-treated CHO cultures.

    Rodilla, V

    1993-08-01

    It has recently been described that cisplatin is an agent able to induce binucleated cells (BC) in cultured CHO cells. Both the origin and the significance of those cells within a population are unknown although several hypothesis have been suggested such as blocking of cytokinesis or cell fusion. Using interval photography we have found that at least two mechanisms are involved in the production of BC. These cells can arise in a culture as a result of an incomplete process of cell division, i.e. karyokinesis with incomplete cytokinesis or as a result of the mitotic division of a pre-existent BC. The mitotic division of a BC can give rise to different types of daughter cells. These BC sometimes enter mitosis but fail to divide and as a consequence they remain BC. When the process of division is successful (in the vast majority of cases), the results that have been found are either two mononucleated cells or one mononucleated and one binucleated cell. The possible implications and significance of BC and BC with micronuclei in a given population are discussed.

  10. Cytoskeleton, endoplasmic reticulum and nucleus alterations in CHO-K1 cell line after Crotalus durissus terrificus (South American rattlesnake venom treatment

    B. P. Tamieti

    2007-01-01

    Full Text Available Snake venoms are toxic to a variety of cell types. However, the intracellular damages and the cell death fate induced by venom are unclear. In the present work, the action of the South American rattlesnake Crotalus durissus terrificus venom on CHO-K1 cell line was analyzed. The cells CHO-K1 were incubated with C. d. terrificus venom (10, 50 and 100g/ml for 1 and 24 hours, and structural alterations of actin filaments, endoplasmic reticulum and nucleus were assessed using specific fluorescent probes and agarose gel electrophoresis for DNA fragmentation. Significant structural changes were observed in all analyzed structures. DNA fragmentation was detected suggesting that, at the concentrations used, the venom induced apoptosis.

  11. Use of damaged plasmid to study DNA repair in X-ray sensitive (xrs) strains of Chinese hamster ovary (CHO) cells

    Smith-Ravin, J.; Jeggo, P.A.

    1989-01-01

    The effect of γ-irradiation of pSV2gpt DNA on its transfection frequency has been analysed using radiosensitive CHO xrs mutants showing a defect in double-strand break (dsb) rejoining. At low doses a sharp decrease in relative transfection frequency, i.e. transfection frequency of irradiated plasmid relative to untreated plasmid, as observed in xrs mutants compared with the parent line K1. Electrophoresis of irradiated plasmid DNA showed the decrease in transfection frequency in the xrs mutants correlated with the change of supercoiled molecules into open-circular forms. In the parent line CHO-K1, open-circular and supercoiled molecules have the same transfection frequency. The effect of linearization of pSV2gpt DNA by restriction enzymes on transfection frequency in xrs and wild-type strains was also examined. No difference in the relative transfection frequency between xrs and wild-type strains was detected. (author)

  12. Production and repair of chromosome damage in an X-ray sensitive CHO mutant visualized and analysed in interphase using the technique of premature chromosome condensation

    Iliakis, G.E.; Pantelias, G.E.

    1990-01-01

    Production of chromosome damage per unit of absorbed radiation dose was in xrs-5 cells larger by a factor of 2.6 than in CHO cells (5.2 breaks per cell per Gy). Changes in chromatin structure, associated with the radiation-sensitive pheno-type of xrs-5 cells, that increase the probability of conversion of a DNA double-strand break (dsb) to a chromosome break are invoked to explain this. Repair of chromosome breaks as measured in plateau-phase G 1 cells was deficient in xrs-5 cells and the number of residual chromosome breaks practically identical to the number of lethal lesions calculated from survival data, suggesting that non-repaired chromosome breaks are likely to be manifestations of lethal events in the cell. The yield of ring chromosomes scored after a few hours of repair was higher by a factor of three in xrs-5 compared with CHO cells. (author)

  13. Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in CHO cells

    Kashino, Genro; Prise, Kevin M.; Schettino, Giuseppe; Folkard, Melvyn; Vojnovic, Borivoj; Michael, Barry D.; Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami

    2004-01-01

    This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells

  14. Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone.

    Aghili, Zahra Sadat; Zarkesh-Esfahani, Sayyed Hamid

    2018-02-01

    Growth hormone deficiency results in growth retardation in children and the GH deficiency syndrome in adults and they need to receive recombinant-GH in order to rectify the GH deficiency symptoms. Mammalian cells have become the favorite system for production of recombinant proteins for clinical application compared to prokaryotic systems because of their capability for appropriate protein folding, assembly, post-translational modification and proper signal. However, production level in mammalian cells is generally low compared to prokaryotic hosts. Taguchi has established orthogonal arrays to describe a large number of experimental situations mainly to reduce experimental errors and to enhance the efficiency and reproducibility of laboratory experiments.In the present study, rhGH was produced in CHO cells and production of rhGH was assessed using Dot blotting, western blotting and Elisa assay. For optimization of rhGH production in CHO cells using Taguchi method An M16 orthogonal experimental design was used to investigate four different culture components. The biological activity of rhGH was assessed using LHRE-TK-Luciferase reporter gene system in HEK-293 and compared to the biological activity of prokaryotic rhGH.A maximal productivity of rhGH was reached in the conditions of 1%DMSO, 1%glycerol, 25 µM ZnSO 4 and 0 mM NaBu. Our findings indicate that control of culture conditions such as the addition of chemical components helps to develop an efficient large-scale and industrial process for the production of rhGH in CHO cells. Results of bioassay indicated that rhGH produced by CHO cells is able to induce GH-mediated intracellular cell signaling and showed higher bioactivity when compared to prokaryotic GH at the same concentrations. © Georg Thieme Verlag KG Stuttgart · New York.

  15. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  16. Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures

    Vergara, Mauricio; Berrios, Julio; Mart?nez, Irene; D?az-Barrera, Alvaro; Acevedo, Cristian; Reyes, Juan G.; Gonzalez, Ramon; Altamirano, Claudia

    2015-01-01

    Background Chinese hamster ovary (CHO) cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in ...

  17. Linear antenna microwave plasma CVD diamond deposition at the edge of no-growth region of C-H-O ternary diagram

    Potocký, Štěpán; Babchenko, Oleg; Hruška, Karel; Kromka, Alexander

    2012-01-01

    Roč. 249, č. 12 (2012), s. 2612-2615 ISSN 0370-1972 R&D Projects: GA ČR(CZ) GBP108/12/G108; GA ČR GAP205/12/0908 Institutional research plan: CEZ:AV0Z10100521 Keywords : C-H-O phase diagram * nanocrystalline diamond * plasma enhanced CVD * Raman spectroscopy * SEM Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.489, year: 2012

  18. Promotion of Cholera Awareness Among Households of Cholera Patients: A Randomized Controlled Trial of the Cholera-Hospital-Based-Intervention-for-7 Days (CHoBI7) Intervention.

    Saif-Ur-Rahman, K M; Parvin, Tahmina; Bhuyian, Sazzadul Islam; Zohura, Fatema; Begum, Farzana; Rashid, Mahamud-Ur; Biswas, Shwapon Kumar; Sack, David; Sack, R Bradley; Monira, Shirajum; Alam, Munirul; Shaly, Nusrat Jahan; George, Christine Marie

    2016-12-07

    Previous studies have demonstrated that household contacts of cholera patients are highly susceptible to cholera infections for a 7-day period after the presentation of the index patient in the hospital. However, there is no standard of care to prevent cholera transmission in this high-risk population. Furthermore, there is limited information available on awareness of cholera transmission and prevention among cholera patients and their household contacts. To initiate a standard of care for this high-risk population, we developed the Cholera-Hospital-Based-Intervention-for-7-Days (CHoBI7), which delivers a handwashing with soap and water treatment intervention to household contacts during the time they spend with the admitted cholera patient in the hospital and reinforces these messages through home visits. To test CHoBI7, we conducted a randomized controlled trial among 302 intervention cholera patient household members and 302 control cholera patient household members in Dhaka, Bangladesh. In this study, we evaluated the effectiveness of the CHoBI7 intervention in increasing awareness of cholera transmission and prevention, and the key times for handwashing with soap. We observed a significant increase in cholera knowledge score in the intervention arm compared with the control arm at both the 1-week follow-up {score coefficient = 2.34 (95% confidence interval [CI] = 1.96, 2.71)} and 6 to 12-month follow-up period (score coefficient = 1.59 [95% CI = 1.05, 2.13]). This 1-week hospital- and home-based intervention led to a significant increase in knowledge of cholera transmission and prevention which was sustained 6 to 12 months post-intervention. These findings suggest that the CHoBI7 intervention presents a promising approach to increase cholera awareness among this high-risk population. © The American Society of Tropical Medicine and Hygiene.

  19. Die teologiese skool in die P.U.K vir C.H.O.: met die betekenis van die teologiese skool vir die Christelike wetenskap

    F. Postma

    1944-03-01

    Full Text Available Die Teologiese Skool van die Gereformeerde Kerk gedink op 29 November 1944 sy vyf-en-sewentigjarige bestaan.Die Raad en Senaat van die P.U.K. vir C.H.O. wil ook langs hierdie weg die Teologiese Skool van harte gelukwens en die versekering gee dat daar by die outoriteite van die P.U.K. ’n diepgevoelde dankbaarheid heers dat hierdie inrigting sovele jare deur onse God en Vader gedra en gespaar is.

  20. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    Tetsushi Sakuma

    2015-10-01

    Full Text Available Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc gene, in Chinese hamster ovary (CHO cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  1. Reaction dynamics of the four-centered elimination CH2OH + --> CHO + +H2: Measurement of kinetic energy release distribution and classical trajectory calculation

    Lee, Tae Geol; Park, Seung C.; Kim, Myung Soo

    1996-03-01

    Mass-analyzed ion kinetic energy (MIKE) spectrum of CHO+ generated in the unimolecular dissociation of CH2OH+ was measured. Kinetic energy release distribution (KERD) was evaluated by analyzing the spectrum according to the algorithm developed previously. The average kinetic energy release evaluated from the distribution was extraordinarily large, 1.63 eV, corresponding to 75% of the reverse barrier of the reaction. A global analytical potential energy surface was constructed such that the experimental energetics was represented and that various features in the ab initio potential energy surface were closely reproduced. Classical trajectory calculation was carried out with the global analytical potential energy surface to investigate the causes for the extraordinarily large kinetic energy release. Based on the detailed dynamical calculations, it was found that the strained bending forces at the transition state and strengthening of the CO bond from double to triple bond character were mainly responsible for such a significant kinetic energy release. In addition, the dissociation products H2 and CHO+ ion were found to be rotationally excited in the trajectory calculations. This was attributed to the asymmetry of the transition state and the release of asymmetric bending forces. Also, the bending vibrational modes of CHO+ and the H2 stretching mode, which are coupled with the bending coordinates, were found to be moderately excited.

  2. A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

    Ustav Mart

    2002-04-01

    Full Text Available Abstract Background The rationale of using bovine papillomavirus-1 (BPV-1 derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. Results The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Conclusion Bovine papillomavirus type-1 (BPV-1-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

  3. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

    Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

    2015-10-09

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  4. Rejoining of DNA double-strand breaks in X-irradiated CHO cells studied by constant- and graded-field gel electrophoresis

    Dahm-Daphi, J.; Dikomey, E.

    1996-01-01

    Induction and repair of double-strand breaks (dsb) were measured in exponentially growing CHO-10A cells using the constant- and graded-field gel electrophoresis. Dsb repair was studied after an X-ray dose of 60Gy. The repair curve obtained was biphasic with the respective half-times of τ 1 = 3.8 ± 0.9 and τ 2 = 118 ± 30 min. The number of non-reparable dsb was measured for X-ray doses up to 180 Gy and was found to be only a small fraction (14%) of all non-rejoinable breaks determined previously using the alkaline unwinding technique. The ratio of non-reparable dsb to the number of lethal events calculated from survival curves is 0.14:1. This result indicates that for CHO cells non-reparable dsb represent only a small fraction of lethal damage. This is in line with the cytogenic observation that cell killing mainly results from mis-rejoined events (i.e. exchange aberrations, translocations, interstitial delections). The kinetics of dsb rejoining were found to be independent of the size of the fragments involved (between 1 and 10 Mbp). In addition, the rejoining kinetics of DNA fragments ≤ 1 Mbp did not show the formation of new DNA fragments with time after irradiation indicating the absence of programmed cell death in irradiated CHO cells. (author)

  5. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin; Influencia do hospedeiro (Cho) e da estrategia de cultivo nas estruturas glicidicas e propriedades moleculares da tireotrofina humana

    Oliveira, Joao Ezequiel de

    2007-07-01

    A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of {approx} 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO{sub 2} conditions from 5% CO{sub 2} to air environment (0.03% CO{sub 2}). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (Thyrogen{sup R}) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing {approx} 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO{sub 2} and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 {+-} 10% was in the {alpha} 2,6 and 32 {+-} 10% in the {alpha}2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5

  6. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin

    Oliveira, Joao Ezequiel de

    2007-01-01

    A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of ∼ 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO 2 conditions from 5% CO 2 to air environment (0.03% CO 2 ). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (Thyrogen R ) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing ∼ 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO 2 and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 ± 10% was in the α 2,6 and 32 ± 10% in the α2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5.39 - 7.35 pl range. A considerably different

  7. Evidence of heritable lethal mutations in progeny of X-irradiated CHO cells by micronucleus count in clon-cells

    Hagemann, G.; Kreczik, A.; Treichel, M.

    1996-01-01

    Low doses of ionizing radiation reduce the growth rates of clones following irradiation of the progenitor cells. Such reductions of clone growth have been proven by means of measurements of clone size distributions. The medians of such distributions can be used to quantify the radiation damage. Prolongations of generation times and cell death as result of heritable lethal mutations have been discussed as causes for the reduction of clone growth. The cell number of a clone of hypotetraploid CHO-cells was compared to the frequency of micronucleated binucleated cells in the same clone using the cytokinesis-block-micronucleus method. The dose dependent reduction of clone sizes is measured by the difference of the medians (after log transformation) of the clone size distributions. At cytochalasin-B concentrations of 1 μg/ml and after an incubation time of 16 h a yield of binucleated cells of about 50% was obtained. Median clone size differences as a measure of clonal radiation damage increased linearly with incubation times of 76, 100, 124, and 240 h following irradiation with 3, 5, 7, and 12 Gy. The frequency of binucleated clone cells with micronuclei strongly increased with decreasing clone size by a factor up to 20 following irradiation with 3, 5, and 7 Gy. The frequency of micronucleated binucleated clone cells was found to be independent of incubation time after irradiation. Radiation induced clone size reductions result from cell losses caused by intraclonal expression of micronuclei which have its origin in heritable lethal mutations. Measurements of clone size distributions can be done automatically. They can serve as predictive test for determination of median cell loss rates of surviving cell clones. (orig./MG) [de

  8. Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

    Handlogten, Michael W; Lee-O'Brien, Allison; Roy, Gargi; Levitskaya, Sophia V; Venkat, Raghavan; Singh, Shailendra; Ahuja, Sanjeev

    2018-01-01

    A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally

  9. High-level theoretical characterization of the vinoxy radical (•CH2CHO) + O2 reaction

    Weidman, Jared D.; Allen, Ryan T.; Moore, Kevin B.; Schaefer, Henry F.

    2018-05-01

    Numerous processes in atmospheric and combustion chemistry produce the vinoxy radical (•CH2CHO). To understand the fate of this radical and to provide reliable energies needed for kinetic modeling of such processes, we have examined its reaction with O2 using highly reliable theoretical methods. Utilizing the focal point approach, the energetics of this reaction and subsequent reactions were obtained using coupled-cluster theory with single, double, and perturbative triple excitations [CCSD(T)] extrapolated to the complete basis set limit. These extrapolated energies were appended with several corrections including a treatment of full triples and connected quadruple excitations, i.e., CCSDT(Q). In addition, this study models the initial vinoxy radical + O2 reaction for the first time with multireference methods. We predict a barrier for this reaction of approximately 0.4 kcal mol-1. This result agrees with experimental findings but is in disagreement with previous theoretical studies. The vinoxy radical + O2 reaction produces a 2-oxoethylperoxy radical which can undergo a number of unimolecular reactions. Abstraction of a β-hydrogen (a 1,4-hydrogen shift) and dissociation back to reactants are predicted to be competitive to each other due to their similar barriers of 21.2 and 22.3 kcal mol-1, respectively. The minimum-energy β-hydrogen abstraction pathway produces a hydroperoxy radical (QOOH) that eventually decomposes to formaldehyde, CO, and •OH. Two other unimolecular reactions of the peroxy radical are α-hydrogen abstraction (38.7 kcal mol-1 barrier) and HO2• elimination (43.5 kcal mol-1 barrier). These pathways lead to glyoxal + •OH and ketene + HO2• formation, respectively, but they are expected to be uncompetitive due to their high barriers.

  10. Combined effects of x irradiation and hyperthermia on CHO cells for various temperatures and orders of application

    Sapareto, S.A.; Hopwood, L.E.; Dewey, W.C.

    1978-01-01

    The survival of CHO cells to hyperthermic treatment combined with radiation indicates that heat given either immediately before or immediately after irradiation radiosensitizers S-phase cells more than G1 cells, thus resulting in similar absolute levels of survival for each phase. No difference in effect was observed for different temperatures (42.0 to 45.5 0 C) applied before irradiation in either G1 or S when times of heating were adjusted to obtain the same survival (0.5 to 0.6) from heat alone. When heat was administered after irradiation and the time between treatments was increased, repair during G1 of radiation damage which interacted with subsequent heat damage occurred over a 2-hr period. Survival increased from a synergistic level to an independent level with kinetics similar to those seen for repair between split x-ray doses. For this experiment, the heat treatments were administered at either 42.5 or 45.5 0 C with times of heating adjusted to obtain the same survival (0.15) from heat alone. When cells were treated similarly in S phase using either 42.5 or 45.5 0 C (survival from heat alone was 0.2), recovery from a synergistic level of survival was similar to that observed in G1; however, survival did not reach an independent level by 120 min between treatments. When relatively sublethal heat doses at either 42.5 or 45.5 0 C were applied either before, during, or after irradiation, the maximum reduction in survival of asynchronous cells occurred when heat was present during and immediately following irradation, presumably due to heat increasing the fixation of radiation damage. A sixfold difference in survival was observed with about a 5-min change in the timing of radiation with respect to heating. This sensitivity of survival to changes in protocol may have considerable implications in the combined use of hyperthermia and radiation for cancer therapy

  11. Supplementation of serum free media with HT is not sufficient to restore growth properties of DHFR-/- cells in fed-batch processes - Implications for designing novel CHO-based expression platforms.

    Florin, Lore; Lipske, Carolin; Becker, Eric; Kaufmann, Hitto

    2011-04-10

    DHFR-deficient CHO cells are the most commonly used host cells in the biopharmaceutical industry and over the years, individual substrains have evolved, some have been engineered with improved properties and platform technologies have been designed around them. Unexpectedly, we have observed that different DHFR-deficient CHO cells show only poor growth in fed-batch cultures even in HT supplemented medium, whereas antibody producer cells derived from these hosts achieved least 2-3 fold higher peak cell densities. Using a set of different expression vectors, we were able to show that this impaired growth performance was not due to the selection procedure possibly favouring fast growing clones, but a direct consequence of DHFR deficiency. Re-introduction of the DHFR gene reproducibly restored the growth phenotype to the level of wild-type CHO cells or even beyond which seemed to be dose-dependent. The requirement for a functional DHFR gene to achieve optimal growth under production conditions has direct implications for cell line generation since it suggests that changing to a selection system other than DHFR would require another CHO host which - especially for transgenic CHO strains and tailor-suited process platforms - this could mean significant investments and potential changes in product quality. In these cases, DHFR engineering of the current CHO-DG44 or DuxB11-based host could be an attractive alternative. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian; Wagtberg Sen, Jette; Rasmussen, Søren Kofoed; Kontoravdi, Cleo; Weilguny, Dietmar; Andersen, Mikael Rørdam

    2015-03-01

    Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans

  13. The B cell death function of obinutuzumab-HDEL produced in plant (Nicotiana benthamiana L. is equivalent to obinutuzumab produced in CHO cells.

    Jin Won Lee

    Full Text Available Plants have attracted attention as bio-drug production platforms because of their economical and safety benefits. The preliminary efficacy of ZMapp, a cocktail of antibodies produced in N. benthamiana (Nicotiana benthamiana L., suggested plants may serve as a platform for antibody production. However, because the amino acid sequences of the Fab fragment are diverse and differences in post-transcriptional processes between animals and plants remain to be elucidated, it is necessary to confirm functional equivalence of plant-produced antibodies to the original antibody. In this study, Obinutuzumab, a third generation anti-CD20 antibody, was produced in N. benthamiana leaves (plant-obinutuzumab and compared to the original antibody produced in glyco-engineered Chinese hamster ovary (CHO cells (CHO-obinutuzumab. Two forms (with or without an HDEL tag were generated and antibody yields were compared. The HDEL-tagged form was more highly expressed than the non-HDEL-tagged form which was cleaved in the N-terminus. To determine the equivalence in functions of the Fab region between the two forms, we compared the CD20 binding affinities and direct binding induced cell death of a CD20-positive B cells. Both forms showed similar CD20 binding affinities and direct cell death of B cell. The results suggested that plant-obinutuzumab was equivalent to CHO-obinutuzumab in CD20 binding, cell aggregation, and direct cell death via binding. Therefore, our findings suggest that Obinutuzumab is a promising biosimilar candidate that can be produced efficiently in plants.

  14. Aryl- and alkyl-phosphorus-containing flame retardants induced mitochondrial impairment and cell death in Chinese hamster ovary (CHO-k1) cells

    Huang, Chao; Li, Na; Yuan, Shengwu; Ji, Xiaoya; Ma, Mei; Rao, Kaifeng; Wang, Zijian

    2017-01-01

    Phosphorus-containing flame retardants (PFRs) are increasingly in demand worldwide as replacements for brominated flame retardants (BFRs), but insufficient available toxicological information on PFRs makes assessing their health risks challenging. Mitochondria are important targets of various environmental pollutants, and mitochondrial dysfunction may lead to many common diseases. In the present study, mitochondria impairment-related endpoints were measured by a high content screening (HCS) assay for 11 selected non-halogen PFRs in Chinese hamster ovary (CHO-k1) cells. A cluster analysis was used to categorize these PFRs into three groups according to their structural characteristics and results from the HCS assay. Two groups, containing long-chain alkyl-PFRs and all aryl-PFRs, were found to cause mitochondrial impairment but showed different mechanisms of toxicity. Due to the high correlation between cell death and mitochondrial impairment, two PFRs with different structures, trihexyl phosphate (THP) and cresyl diphenyl phosphate (CDP), were selected and compared with chlorpyrifos (CPF) to elucidate their mechanism of inducing cell death. THP (an alkyl-PFR) was found to utilize a similar pathway as CPF to induce apoptosis. However, cell death induced by CDP (an aryl-PFR) was different from classical necrosis based on experiments to discriminate among the different modes of cell death. These results confirm that mitochondria might be important targets for some PFRs and that differently structured PFRs could function via distinct mechanisms of toxicity. - Highlights: • Mitochondrial impairment induced by PFRs was observed in CHO-k1 cells. • THP (an alkyl-PFR) induced a caspase-mediated apoptosis in CHO-k1 cells. • The cell death induced by CDP (an aryl-PFR) was not traditional apoptosis or necrosis.

  15. miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

    Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

    2017-07-01

    In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. Effects of γ (60Co) and β (90Sr) radiations in Chinese hamster ovarian cells (CHO-K1): induction of micronuclei and cell death

    Murakami, Daniella

    2003-01-01

    Among various types of ionising radiation, the beta emitter radionuclides are involved in many sectors of human activity, such as nuclear medicine, nuclear industries and biomedicine, with a consequent increased risk of accidental, occupational or therapeutic exposure. Despite their recognized importance, there is little information about the effect of beta particles at the cellular level when compared to other types of ionizing radiation. Thus, the objective of the present study was to evaluate the genotoxic and cytotoxic effects of 90 Sr, a pure, highly energetic beta source, on CHO-K1 cells and to compare them with data obtained with 60 Co. CHO-K1 cells irradiated with different doses (1.0, 2.5, 5.0, 7.5 Gy) of 60 Co (0.34 Gy.min -1 ) and 90 Sr (0.23 Gy.min -1 ) were processed for analysis of clonogenic death, induction of micronuclei (MN) and necrotic and apoptotic death. The survival curves obtained for both types of radiation were better fitted by the linear-quadratic model and were similar. However, the cytogenetic results showed that both the proportion of micronucleated cells and the magnitude of radioinduced lesions demonstrated by the analysis of MN distribution were significantly higher in cells irradiated with 60 Co than in cells irradiated with 90 Sr, whereas 90 Sr was more damaging than 60 Co in terms of cell death induction. Necrosis was the major type of death observed in CHO-K1 cells. The data obtained suggest that the low incidence of micronucleated cells after exposure to 90 Sr may be a consequence of selective elimination of severely damaged cells from the population by the necrotic process at a higher rate than observed with 60 Co exposure. The data obtained also demonstrated the need to use several parameters for a better estimate of cellular sensitivity to the action of genotoxic agents, which would be important in terms of radiobiology, oncology and therapeutics. (author)

  17. Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells

    Di Virgilio, A.L. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata (1900) (Argentina); Reigosa, M. [Instituto Multidisciplinario de Biologia Celular (IMBICE), Calle 526 y Camino Gral. Belgrano (entre 10 y 11), La Plata 1900 (Argentina); Arnal, P.M. [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata 1900 (Argentina); Fernandez Lorenzo de Mele, M., E-mail: mmele@inifta.unlp.edu.ar [Instituto de Investigaciones Fisicoquimicas Teoricas y Aplicadas (INIFTA), Diag. 113 y 64, Correo 16, Suc. 4, La Plata 1900 (Argentina)

    2010-05-15

    The aim of this study was to analyze the cytotoxicity and genotoxicity of titanium oxide (TiO{sub 2}) and aluminium oxide (Al{sub 2}O{sub 3}) nanoparticles (NPs) on Chinese hamster ovary (CHO-K1) cells using neutral red (NR), mitochondrial activity (by MTT assay), sister chromatid exchange (SCE), micronucleus (MN) formation, and cell cycle kinetics techniques. Results showed a dose-related cytotoxic effect evidenced after 24 h by changes in lysosomal and mitochondrial dehydrogenase activity. Interestingly, transmission electronic microscopy (TEM) showed the formation of perinuclear vesicles in CHO-K1 cells after treatment with both NPs during 24 h but no NP was detected in the nuclei. Genotoxic effects were shown by MN frequencies which significantly increased at 0.5 and 1 {mu}g/mL TiO{sub 2} and 0.5-10 {mu}g/mL Al{sub 2}O{sub 3}. SCE frequencies were higher for cells treated with 1-5 {mu}g/mL TiO{sub 2}. The absence of metaphases evidenced cytotoxicity for higher concentrations of TiO{sub 2}. No SCE induction was achieved after treatment with 1-25 {mu}g/mL Al{sub 2}O{sub 3}. In conclusion, findings showed cytotoxic and genotoxic effects of TiO{sub 2} and Al{sub 2}O{sub 3} NPs on CHO-K1 cells. Possible causes of controversial reports are discussed further on.

  18. Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 Cells

    Hirano, Seishiro, E-mail: seishiro@nies.go.jp [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Fujitani, Yuji; Furuyama, Akiko [Environmental Nanotoxicology Project, RCER, National Institute for Environmental Studies (Japan); Kanno, Sanae [Department of Legal Medicine, St. Marianna School of Medicine (Japan)

    2012-02-15

    The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20 nm–1 μm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC{sub 50} value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1 μg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure. -- Highlights: ►Carbon nanotubes (CNTs) were tethered to MARCO in vitro. ►CNTs were taken up rapidly into the cell body via MARCO by membrane ruffling. ►The incomplete inclusion of CNTs by membranes caused cytotoxicity.

  19. Evaluation of the radio modifier effect of propolis on chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60-CO

    Santos, Geyza Spigoti

    2011-01-01

    In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60 Co γ radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400μ/ml) in PC3 cells irradiated with 5 Gγ. The survival curves obtained were adequately fitted by a linear-quadratic model, where the α coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a

  20. Stimulation of {sup 125}I-3-iodo-{alpha}-methyl-L-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters

    Shikano, Naoto [Department of Radiological Sciences, Center for Medical Sciences and Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki (Japan)], E-mail: sikano@ipu.ac.jp; Ogura, Masato; Sagara, Jun-ichi; Nakajima, Syuichi [Department of Radiological Sciences, Center for Medical Sciences and Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki (Japan); Kobayashi, Masato [Division of Health Science, Graduate School of Health Sciences, Kanazawa University, Kanazawa, Ishikawa (Japan); Baba, Takeshi; Yamaguchi, Naoto; Iwamura, Yukio; Kubota, Nobuo [Department of Radiological Sciences, Center for Medical Sciences and Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki (Japan); Kawai, Keiichi [Division of Health Science, Graduate School of Health Sciences, Kanazawa University, Kanazawa, Ishikawa (Japan)

    2010-02-15

    Introduction: Transport of the amino acid analog {sup 123}I-3-iodo-{alpha}-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of {sup 125}I-3-iodo-{alpha}-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. Methods: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37 deg. C or under ice-cold conditions. Results: Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of L-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. Conclusions: The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and L-Tyr ethyl or methyl esters to examine

  1. Effect of Soil Moisture Content on Growth, Crude Drug "Cho-to-ko" Yield and Oxindole Alkaloid Content of Uncaria rhynchophylla (B. LIVING SCIENCE)

    川添, 禎浩; 小林, 茂樹; 水上, 元; 大橋, 裕; SADAHIRO, KAWAZOE; SHIGEKI, KOBAYASHI; HAJIME, MIZUKAMI; HIROMU, OHASHI; Laboratory of Environmetal Health and Toxicology, Department of Food Science and Nutrition, Kyoto Prefectural University; Department of Medicinal Plant Research, Faculty of Pharmaceutical Sciences, Nagasaki University; Department of Medicinal Plant Research, Faculty of Pharmaceutical Sciences, Nagasaki University; Department of Medicinal Plant Research, Faculty of Pharmaceutical Sciences, Nagasaki University

    1992-01-01

    Uncaria rhynchophylla was cultivated by using soil with various moisture contents (20,40,60,80 or 100% of the maximum moisture content retained by soil). Both growth and crude drug "Cho-to-ko" (dried stem with hooks of U. rhynchophylla) yield of the plant were in the following order : 60%> 80%> 40%> 100%> 20%, while oxindole alkaloid content of the stem was in the following order : 20%> 60%> 100%> 40%> 80%. It is concluded that moderately wet soil is suitable for cultivation of U. rhynchophyl...

  2. Fiscal 1999 geothermal energy development promotion survey. Report on resource assessment for Shiramizugoe area; 1999 nendo chinetsu kaihatsu sokushin chosa hokokusho. Shiramizugoe chiiki shigen hyoka

    NONE

    2000-03-01

    Under an assumed plan of building a geothermal power station in the Shiramizugoe area of Makizono-cho, Aira-gun, Kagoshima Prefecture, investigations will be conducted under a 4-year program into the amount of geothermal resources, the dimensions and cost performance of a geothermal power station under consideration, and the environmental impact that the locating of such will incur, through for example identifying areas containing geothermal reservoirs suitable for geothermal power generation. In fiscal 1999, activities were conducted in the three fields of (1) survey plan preparation, (2) comprehensive analysis, and (3) environmental assessment. Under item (1), a survey and coordination implementation plan was prepared, existing data were analyzed for the construction of geothermal models out of which a suitable geothermal structure was specified for digging, and a list was drafted of locations for borehole digging for fiscal 2000. Under item (2), it was found that most of the past surveys covered the Ogiri area and that the Shiramizugoe area was but poorly covered. Discussion was made on the geological structure, geothermal structure, geothermal water hydraulic structure, and the geothermal water system. Under item (3), the plan was summarily explained to the local administration, associations of hot spring hotels, and inhabitants in the vicinity, and their consent was obtained. (NEDO)

  3. Report on field test project for wind power development at Nagashima-cho. Detailed wind characteristics survey; Nagashimacho ni okeru furyoku field test jigyo (fukyo seisa) hokokusho

    NONE

    1999-11-01

    A detailed wind characteristics survey was conducted to study the feasibility of a wind power generation system for Nagashima-cho, Izumi-gun, Kagoshima Prefecture. Observation instruments were installed at the top of a hill approximately 80m above the sea level situated to the northwest of the Nagashima-cho town hall and, in the period October 1998 through September 1999, data were collected at a point 20m above ground, such as the average wind speed and direction, wind velocity standard deviation, and the maximum instantaneous wind velocity. The data were analyzed, and findings were obtained, as mentioned below. The annual average wind speed was 5.0m, strong in winter and weak in summer. The annual wind direction occurrence rate was 61.8%, turbulence intensity was 0.17 at wind speeds of 4m/s and more, these not presenting any particular problem. Wind energy density was 148W/m{sup 2}. Both wind speed conditions and energy density were slightly lower than the reference levels indicated for evaluation. Studies were made on the assumption that three classes of wind turbines (150, 300, and 750kW) would be introduced, and then it was found that both operating factors and facility availability rates exceeded the required levels. Since there were no detrimental factors in the surrounding conditions, it was concluded that possibilities were high that wind power generation at the site would be practical. (NEDO)

  4. Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

    Meleady, Paula

    2011-07-24

    Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS\\/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  5. Communication: Photodissociation of CH3CHO at 308 nm: Observation of H-roaming, CH3-roaming, and transition state pathways together along the ground state surface

    Li, Hou-Kuan; Tsai, Po-Yu; Hung, Kai-Chan; Kasai, Toshio; Lin, King-Chuen

    2015-01-01

    Following photodissociation of acetaldehyde (CH3CHO) at 308 nm, the CO(v = 1-4) fragment is acquired using time-resolved Fourier-transform infrared emission spectroscopy. The CO(v = 1) rotational distribution shows a bimodal feature; the low- and high-J components result from H-roaming around CH3CO core and CH3-roaming around CHO radical, respectively, in consistency with a recent assignment by Kable and co-workers (Lee et al., Chem. Sci. 5, 4633 (2014)). The H-roaming pathway disappears at the CO(v ≥ 2) states, because of insufficient available energy following bond-breaking of H + CH3CO. By analyzing the CH4 emission spectrum, we obtained a bimodal vibrational distribution; the low-energy component is ascribed to the transition state (TS) pathway, consistent with prediction by quasiclassical trajectory calculations, while the high-energy component results from H- and CH3-roamings. A branching fraction of H-roaming/CH3-roaming/TS contribution is evaluated to be (8% ± 3%)/(68% ± 10%)/(25% ± 5%), in which the TS pathway was observed for the first time. The three pathways proceed concomitantly along the electronic ground state surface.

  6. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles.

    Davami, Fatemeh; Eghbalpour, Farnaz; Nematollahi, Leila; Barkhordari, Farzaneh; Mahboudi, Fereidoun

    2015-01-01

    The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.

  7. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

    Lena Thoring

    Full Text Available Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

  8. Effect of hepatocyte growth factor on radiation response of HeLa, V79, CHO and primary cultured parenchymal hepatocyte in vitro

    Yamazaki, Hideya; Inoue, Takehiro; Nose, Takayuki; Murayama, Shigeyuki; Teshima, Teruki; Ozeki, Syuji; Koizumi, Masahiko; Inoue, Toshihiko.

    1996-01-01

    Hepatocyte growth factor (HGF) is a multipotent cytokine enhancing regeneration of injured organs as liver, kidney and lung after injury. HGF enhances proliferation of various type of cells, inhibits proliferation of carcinoma cells, enhances motility of epithelial cells. We examined three cell lines (CHO, HeLa, V79) and primary cultured normal rat parenchymal hepatocytes to determine the effect of HGF on radiation response. HGF diminished survival of CHO and V79 cells determined by colony formation assay, whereas no significant change of survival was found in HeLa cells. No synergistic changes of survival were found when these three cell lines were irradiated with the addition of HGF. Thus, HGF did not enhance the radiation effect. We also analyzed the impact of irradiation with HGF on primary cultured normal rat parenchymal hepatocytes. At first, the release of glutamic-oxaloacetic amino-transaminase (GOT) in the supernatant was estimated. Irradiation (40 Gy) with or without HGF did not change GOT release in acute phase by 4 days after irradiation compared with the unirradiated control. Second, the DNA synthesis of rat parenchymal hepatocytes was analyzed using radioactive iodine-labeled deoxyuridine incorporation. HGF counteracted the suppression of DNA synthesis induced by irradiation. Thus, HGF may act as a mitogen even for irradiation-damaged normal cells. (author)

  9. Feeling of reality, bodily sensation, and pleasant perspiration. Let's play and learn town of natural energy (New energy vision in Shin-asahi Town in Shiga Prefecture); Jikkan taikan kaikan. Asonde manabo shizen energy no machi (Shinasahicho energy vision)

    NONE

    2001-03-01

    With an objective to build a resource circulation type society, a basic guideline was established for introducing new energies upon investigating the status of new energy existence, problems and countermeasure thereon to encounter when setting and introducing the targeted introduction values, and specific measures for promoting the introduction. The activity achievements were put into order by the following seven items: 1) the background and purpose, 2) summary of Shin-asahi Town, 3) energy demand and supply conditions in Shin-asahi Town, 4) natural energies available in Shin-asahi Town, 5) directionality of the energy vision, 6) specific targets and introduction scenario, and 7) the energy vision promotion systems. In item 6, the target of introduction in fiscal 2010 was set to 36,300 Gcal/year (reduction of CO2 emission of 15% of the 1999 criterion). The scenario discusses energy efficiency improvement, solar energy, wind power generation, small hydropower generation, and bio-mass. For the efficiency improvement, construction of a natural energy park with wind mills was taken up for the purpose of enlightenment and education of the residents. (NEDO)

  10. Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures.

    Mauricio Vergara

    Full Text Available Chinese hamster ovary (CHO cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in the endoplasmic reticulum.In order to evaluate the effect of the mild hypothermia on processing/endoplasmatic reticulum-associated degradation (ERAD processes, batch cultures of CHO cells producing recombinant human tissue plasminogen activator (rht-PA were carried out at two temperatures (37°C and 33°C and treated with specific inhibitors of glycosylation and ERAD I (Ubiquitin/Proteasome system or ERAD II (Autophagosoma/Lisosomal system pathways. The effect of mild hypothermia was analysed separately from its indirect effect on specific cell growth rate. To do this, chemostat cultures were carried out at the same incubation conditions as the batch cultures, controlling cell growth at high (0.017 h-1 and low (0.012 h-1 dilution rates. For a better understanding of the investigated phenomenon, cell behaviour was also analysed using principal component analysis (PCA.Results suggest that rht-PA is susceptible to degradation by both ERAD pathways studied, revealing that processing and/or ERAD processes are sensitive to temperature cultivation in batch culture. Moreover, by isolating the effect of culture temperature from the effect of cell growth rate verifyed by using chemostat cultures, we have found that processing and/or ERAD processes are more sensitive to reduction in specific growth rate than low temperature, and that temperature reduction may have a positive effect on protein processing. Interestingly, PCA indicated that the integrated performance displayed by CHO

  11. Comparison of [(11)C]Choline ([(11)C]CHO) and [(18)F]Bombesin (BAY 86-4367) as Imaging Probes for Prostate Cancer in a PC-3 Prostate Cancer Xenograft Model.

    Schwarzenböck, Sarah Marie; Schmeja, Philipp; Kurth, Jens; Souvatzoglou, Michael; Nawroth, Roman; Treiber, Uwe; Kundt, Guenther; Berndt, Sandra; Graham, Keith; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Ziegler, Sibylle I; Dinkelborg, Ludger; Wester, Hans-Jürgen; Krause, Bernd Joachim

    2016-06-01

    Carbon-11- and fluorine-18-labeled choline derivatives are commonly used in prostate cancer imaging in the clinical setting for staging and re-staging of prostate cancer. Due to a limited detection rate of established positron emission tomography (PET) tracers, there is a clinical need for innovative tumor-specific PET compounds addressing new imaging targets. The aim of this study was to compare the properties of [(18)F]Bombesin (BAY 86-4367) as an innovative biomarker for prostate cancer imaging targeting the gastrin-releasing peptide receptor and [(11)C]Choline ([(11)C]CHO) in a human prostate tumor mouse xenograft model by small animal PET/X-ray computed tomography (CT). We carried out a dual-tracer small animal PET/CT study comparing [(18)F]Bombesin and [(11)C]CHO. The androgen-independent human prostate tumor cell line PC-3 was implanted subcutaneously in the flanks of nu/nu NMRI mice (n = 10) (PET/CT measurements of two [(11)C]Choline mice could not be analyzed due to technical reasons). [(18)F]Bombesin and [(11)C]CHO PET/CT imaging was performed about 3-4 weeks after the implantation of PC-3 cells on two separate days. After the intravenous tail vein injection of 14 MBq [(18)F]Bombesin and 37 MBq [(11)C]CHO, respectively, a dynamic study over 60 min was acquired in list mode using an Inveon animal PET/CT scanner (Siemens Medical Solutions). The sequence of [(18)F]Bombesin and [(11)C]CHO was randomized. Image analysis was performed using summed images as well as dynamic data. To calculate static and dynamic tumor-to-muscle (T/M), tumor-to-blood (T/B), liver-to-blood (L/B), and kidney-to-blood (K/B) ratios, 4 × 4 × 4 mm(3) volumes of interest (VOIs) of tumor, muscle (thigh), liver, kidney, and blood derived from transversal slices were used. The mean T/M ratio of [(18)F]Bombesin and [(11)C]CHO was 6.54 ± 2.49 and 1.35 ± 0.30, respectively. The mean T/B ratio was 1.83 ± 0.79 for [(18)F]Bombesin and 0.55 ± 0.10 for [(11)C]CHO

  12. Use of a transfected and amplified Drosophila heat shock promoter construction for inducible production of toxic mouse c-myc proteins in CHO cells

    Wurm, F.M.; Gwinn, K.A.; Papoulas, O.; Pallavicini, M.; Kingston, R.E.

    1987-01-01

    After transfection and selection with methotrexate, CHO cell lines were established which contained up to 2000 copies of an expression vector for c-myc protein. The vector contained the Drosophila heat shock protein 70 (hsp70) promoter fused with the coding region of the mouse c-myc gene. Incubation of cells for up to 3 hours at 43 0 C resulted in at least a 100-fold induction of recombinant c-myc mRNA. When cells were shifted back to 37 0 C, within 1 to 4 hours, this RNA was translated into protein to yield about 250 μg per 10 9 cells. Cells died a few hours later, suggesting that high concentrations of intracellular c-myc are cytotoxic. 47 refs., 5 figs

  13. A multi-pronged investigation into the effect of glucose starvation and culture duration on fed-batch CHO cell culture

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian

    2015-01-01

    to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells......In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity...... and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary...

  14. Photodissociation of CH3CHO at 248 nm by time-resolved Fourier-transform infrared emission spectroscopy: Verification of roaming and triple fragmentation

    Hung, Kai-Chan; Tsai, Po-Yu; Li, Hou-Kuan; Lin, King-Chuen

    2014-02-01

    By using time-resolved Fourier-transform infrared emission spectroscopy, the HCO fragment dissociated from acetaldehyde (CH3CHO) at 248 nm is found to partially decompose to H and CO. The fragment yields are enhanced by the Ar addition that facilitates the collision-induced internal conversion. The channels to CH2CO + H2 and CH3CO + H are not detected significantly. The rotational population distribution of CO, after removing the Ar collision effect, shows a bimodal feature comprising both low- and high-rotational (J) components, sharing a fraction of 19% and 81%, respectively, for the vibrational state v = 1. The low-J component is ascribed to both roaming pathway and triple fragmentation. They are determined to have a branching ratio of 0.06, respectively, relative to the whole v = 1 population. The CO roaming is accompanied by a highly vibrational population of CH4 that yields a vibrational bimodality.

  15. Partitioning of the nonfixed excess energy and the reverse critical energy in CH2OH + --> CHO + +H2: A classical trajectory study

    Lee, Tae Geol; Kim, Myung Soo; Park, Seung C.

    1996-04-01

    Dynamics of the four-centered elimination reaction CH2OH+→CHO++H2 has been investigated over the internal energy range 4.6-5.9 eV using the classical trajectory method. A realistic semiempirical potential reported previously [J. Chem. Phys. (in press, 1996)] has been used for the calculation. It has been found that the disposal of the nonfixed excess energy at the transition state and of the reverse critical energy can be considered independently as manifest in the sum rule analysis. The former is determined statistically while the latter dynamically. Based on the above idea, a method to determine the kinetic energy release distribution originating only from the reverse critical energy has been developed.

  16. Gamma-ray induced DNA breaks and repair studied by immuno-labelling of poly(ADP-ribose) polymerase (PARP) in chinese hamster ovary cells (CHO)

    Bidon, N.; Noel, G.; Averbeck, D.; Varlet, P.; Salamero, J.; DeMurcia, G.

    1998-01-01

    The poly(ADP-ribose)polymerase is a nuclear ubiquitous enzyme capable of binding to DNA breaks. Chinese hamster ovary cells were (CHO-K1) cultured on slides and γ-irradiated ( 137 Cs) at a high (12.8 Gy/min) or medium dose rate (5 Gy/min), and immuno-labelling against (ADP-ribose) polymers immediately or three hours after irradiation. Quantification and localisation of γ-ray induced breaks was performed by confocal microscopy. The results show a dose effect relationship, a dose-rate effect and the signal disappearance after 3 hours at 37 deg.C. The presence of PARP activity appears to reflect γ-rays induced DNA fragmentation. (authors)

  17. Radiation-induced DNA breaks detected by immuno labelling of poly(ADP-ribose) in CHO cells. Standardization by pulsed-field gel electrophoresis

    Varlet, P.; Bidon, N.; Noel, G.; Averbeck, D.; Salamero, J.; DeMurcia, G.

    1998-01-01

    The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos)ylation to follow and estimate γ-ray-induced DNA fragmentation at the level of isolated cells after γ-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immuno-labelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after γ-irradiation. (authors)

  18. COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

    Vasila Packeer Mohamed

    2014-05-01

    Full Text Available ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples was involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada and RNeasy mini kit (with DNase treatment; Qiagen, USA respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 µg/µl; p=0.004 than Total RNA purification kit (0.177 ± 0.0243 µg/µl. However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. Total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp.   Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen is recommended if higher yields is the primary concern and Total RNA Purification kit (Norgen is recommended if time and cost are concerned. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat.  Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil.  Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem.  Tujuan kajian dijalankan

  19. Influence of incorporated bromodeoxyuridine on the induction of chromosomal alterations by ionizing radiation and long-wave UV in CHO cells.

    Zwanenburg, T S; van Zeeland, A A; Natarajan, A T

    1985-01-01

    Incorporation of BrdUrd into nuclear DNA sensitizes CHO cells (1) to the induction of chromosomal aberrations by X-rays and 0.5 MeV neutrons and (2) to induction of chromosomal aberrations and SCEs by lw-UV. We have attempted to establish a correlation between induced chromosomal alterations and induced single- or double-strand breaks in DNA. The data show that while DSBs correlate very well with X-ray-induced aberrations, no clear correlation could be established between lw-UV induced SSBs (including alkali-labile sites) and chromosomal alterations. In addition the effect of 3-aminobenzamide (3AB) on the induction of chromosomal aberrations and SCEs induced by lw-UV has been determined. It is shown that 3AB is without any effect when lw-UV-irradiated cells are posttreated with this inhibitor. The significance of these results is discussed.

  20. Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones.

    Lattenmayer, Christine; Loeschel, Martina; Schriebl, Kornelia; Steinfellner, Willibald; Sterovsky, Thomas; Trummer, Evelyn; Vorauer-Uhl, Karola; Müller, Dethardt; Katinger, Hermann; Kunert, Renate

    2007-04-15

    In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development. (c) 2006 Wiley Periodicals, Inc.

  1. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Mas-Oliva, Jaime; Navarro-Vidal, Enrique; Tapia-Vieyra, Juana Virginia

    2014-01-01

    Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+)-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2) associated to a lethal influx of Ca(2+) in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line) transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  2. Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content.

    Kuwae, Shinobu; Miyakawa, Ichiko; Doi, Tomohiro

    2018-01-11

    A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10 6  cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10 7 to 1.8 × 10 7  cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.

  3. Evaluation of the radio modifier effect of propolis on chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60-CO; Avaliacao do efeito radiomodificador da propolis em celulas de ovario de hamster chines (CHO-K1) e em celulas tumorais de prostata (PC3), irradiadas com CO-60

    Santos, Geyza Spigoti

    2011-07-01

    In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with {sup 60}Co {gamma} radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 {mu}g/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400{mu}/ml) in PC3 cells irradiated with 5 G{gamma}. The survival curves obtained were adequately fitted by a linear-quadratic model, where the {alpha} coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 {mu}g/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data

  4. Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells.

    Minshall, R D; Tan, F; Nakamura, F; Rabito, S F; Becker, R P; Marcic, B; Erdös, E G

    1997-11-01

    Part of the beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [3H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B2 receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 mumol/L) increased the total number of low-affinity [3H]BK binding sites on the cells at 37 degrees C, but not at 4 degrees C, from 18.4 +/- 4.3 to 40.3 +/- 11.9 fmol/10(6) cells (P potentiated the release of [3H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP3) induced by BK and [Hyp3-Tyr(Me)8]BK. Moreover, enalaprilat (1 mumol/L) completely and immediately restored the response of the B2 receptor, desensitized by the agonist (1 mumol/L [Hyp3-Tyr(Me)8]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70 +/- 9% to 45 +/- 9% (P desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.

  5. PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p

    Okumoto, K.; Shimozawa, N.; Kawai, A.; Tamura, S.; Tsukamoto, T.; Osumi, T.; Moser, H.; Wanders, R. J.; Suzuki, Y.; Kondo, N.; Fujiki, Y.

    1998-01-01

    Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11-20, 1997), using a transient transfection assay and

  6. An analysis of the dispute process regarding high-level nuclear waste repository siting in Toyo-cho, Japan: Decisive factors in the dispute and roles of the governments and experts

    Komatsuzaki, Shunsaku; Horii, Hideyuki (Dept. of Civil Engineering, Univ. of Tokyo (Japan)); Saigo, Takahiro (Mitsubishi Research Institute, Inc. (Japan))

    2010-09-15

    The siting policy of HLW repository in Japan was 'application-based' until 2007 and Toyo-cho is the only municipality which applied for the Literature Survey. In Toyo-cho, however, a serious antagonism among citizens occurred and the application was withdrawn after the mayor was replaced by election. Our detailed analysis of the process based on the methods of political science and psychology shows five decisive factors: 1) opposing activists both in the town and from outside successfully changed citizens' perceptions of HLW by rhetorical expressions, 2) the mayor lacks careful actions and effective policy adviser, 3) NUMO, an organization which runs HLW projects, didn't effectively coordinate Toyo-cho and stakeholders, 4) the municipal government and council exercised very limited influences on the dispute despite their political authority, and 5) the existence of grant adversely influenced the citizens since it causes criticism that Toyo-cho applies a repository for grant. We finally conclude that the substantial problems, caused by the five decisive factors, were the propagation of enthusiastic opposition and the lack of peaceful deliberation based on local governance. In order to avoid enthusiastic opposition and to realize responsible decision making, or negotiation, we suggest that A) active and prompt response of experts, especially political/administrative ones, to radical opposing activities, B) solution to the adverse influence of the grant by the government's agenda

  7. An analysis of the dispute process regarding high-level nuclear waste repository siting in Toyo-cho, Japan: Decisive factors in the dispute and roles of the governments and experts

    Komatsuzaki, Shunsaku; Horii, Hideyuki; Saigo, Takahiro

    2010-09-01

    The siting policy of HLW repository in Japan was 'application-based' until 2007 and Toyo-cho is the only municipality which applied for the Literature Survey. In Toyo-cho, however, a serious antagonism among citizens occurred and the application was withdrawn after the mayor was replaced by election. Our detailed analysis of the process based on the methods of political science and psychology shows five decisive factors: 1) opposing activists both in the town and from outside successfully changed citizens' perceptions of HLW by rhetorical expressions, 2) the mayor lacks careful actions and effective policy adviser, 3) NUMO, an organization which runs HLW projects, didn't effectively coordinate Toyo-cho and stakeholders, 4) the municipal government and council exercised very limited influences on the dispute despite their political authority, and 5) the existence of grant adversely influenced the citizens since it causes criticism that Toyo-cho applies a repository for grant. We finally conclude that the substantial problems, caused by the five decisive factors, were the propagation of enthusiastic opposition and the lack of peaceful deliberation based on local governance. In order to avoid enthusiastic opposition and to realize responsible decision making, or negotiation, we suggest that A) active and prompt response of experts, especially political/administrative ones, to radical opposing activities, B) solution to the adverse influence of the grant by the government's agenda

  8. A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

    Gulce Iz, Sultan; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, Nazli

    2018-01-01

    Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid

  9. Investigation of geothermal development and promotion for fiscal 1997. Investigation report on geological/alteration zone (No. B-5 Musadake area); 1997 nendo chinetsu kaihatsu sokushin chosa. Chishitsu henshitsutai chosa hokokusho (No.B-5 Musadake chiiki)

    NONE

    1998-03-01

    This investigation was intended to clarify the possibility of existence of geothermal reservoir layer by elucidating the underground structure and thermal source forming the geothermal system around Musadake, in the area extending over Nakashibetu-cho and Shibetu-cho, Shibetu district, Hokkaido, and further by grasping the distribution and properties of the geothermal alteration zone. The geothermal system of the subject area seems to be governed by Musadake - Mt. Shitabanupuri fault, its incidental bent zone, and the graben extending to the southeast side. The thermal source is presumably a magma reservoir that evolved Musa volcano. The depth hot water is presumed to exist in the underground depth around Mt. Imora; and it seems that the thermal conduction and volcanic ejecta are caused by a chain of volcano-related magma reservoirs around Musadake, the thermal source presumably. The depth hot water is estimated to be not less than 250 degrees C and a high Cl concentration. Within the graben, mudstones are distributed such as Kawakita layer and Yokoushigawa geological layer. Moreover, it is possible that an impermeable layer due to alteration is spread. (NEDO)

  10. Investigation of geothermal development and promotion for fiscal 1997. Fluid geochemical investigation (hot-spring gas) report (No. B-5 Musadake area); 1997 nendo chinetsu kaihatus sokushin chosa. Ryutai chikagaku chosa (onsen gas) hokokusho (No.B-5 Musadake chiiki)

    NONE

    1998-03-01

    This investigation elucidated the possible existence of geothermal reservoir layer in the subject area by studying and analyzing the hot-spring gasses of Musadake. The Musadake area is the one extending over Shibetu-cho and Nakashibetu-cho, Shibetu district, Hokkaido. The sampling of the hot-spring gasses was carried out at three natural gusher sites and one hot spring well site. The gasses in the Kawakita hot spring is most affected by volcanic gasses. The origin of the volcanic gasses is a magmatic gas of andesite nature the {sup 3}He/{sup 4}He ratio of which is 8X10{sup -6} or about. As a result of the analysis, the hot-spring water is Na-Cl type, high salt concentrated, and 200 degrees C in temperature; from the result of a gas geochemical thermometer, it is estimated to be not less than 250 degrees C. In the tectonic viewpoint, the depth hot water is derived from the meteorite water that flows in through a bent zone incident to the Musadake-Shitabanupuri mountain fault and from the fossil sea water that exists in the underground depth; the depth hot water is formed by conduction heat from a magma reservoir that formed Musadake and by volcanic ejecta. This depth hot water rises along Kawakita south, Urappu River fault, etc., mixing with the meteorite water and forming the shallow reservoir layer. (NEDO)

  11. Wind power development field test project at Okkobe-cho. Close survey on wind conditions; Okkobecho ni okeru furyoku kaihatsu field test jigyo (fukyo seisa) hokokusho

    NONE

    1999-11-01

    A survey was made on regional wind conditions on the assumption that a wind power generator was installed around Okkobe Rakuno-no-oka, Okkobe-cho, Monbetsu-gun, Hokkaido. The survey period was one year from Oct., 1998 to Sept., 1999. The observations were carried out on the average wind velocity, average wind direction, standard deviation of velocity, and the maximum instantaneous wind velocity. With a fixed point observation at 20 m above ground, and with the minimum observation time unit of 10 minutes, an average value during the 10 minutes was determined as the measurement of each category. However, the maximum instantaneous wind velocity was determined on the measurement with the minimum observation time unit of 2 seconds. The average wind velocity was 4.8 m/s, the maximum wind velocity during the period was 29.9 m/s, the prevailing wind direction was WSW (17.1%), the wind axis was WSW-ENE, and the total occurrence rate of wind direction was 51.1%. The intensity of turbulence was 0.19 at a wind velocity of 2.0 m/s or above and was 0.16 at 4.0 m/s or above. An estimated annual operation rate of a windmill was 59-77% using the rated value of a 150 kW, 300 kW and 750 kW class windmills. (NEDO)

  12. Wind power development field test project at Hirashima, Sakito-cho. Detailed wind characteristics survey; Sakitocho Hirashima ni okeru furyoku kaihatsu field test jigyo (fukyo seisa) hokokusho

    NONE

    1999-11-01

    A detailed wind characteristics survey was conducted at Hirashima, Sakito-cho, Nishisonogi-gun, Nagasaki Prefecture, on the assumption that a wind power generation system would be constructed. The survey was a 1-year project from October 1998 through September 1999, and wind characteristics such as the average wind speed, average wind direction, standard deviation of wind velocity, and the maximum instantaneous wind speed were observed. The observation point was fixed at 20m above ground, the minimum time unit for observation was 10 minutes, and the 10-minute average value was defined as the measured value. For the maximum instantaneous wind speed, the minimum time unit for observation was set to be 2 seconds. The yearly average wind speed was 7.1m/s and the maximum wind speed in the period was 37m/s. Winds came prevalently from N (13.9%), and then from NNE (13.3%), NW (12.2%), and NE (10.7%). The total wind direction occurrence rate involving the 4 directions was 50.1%. Turbulence intensity was 0.14 at wind speed 2.0m/s or more and 0.12 at wind speed 4.0m/s or more. Estimated wind turbine yearly operating factors of 77-87% were obtained using rated values of a 150kW, 300kW, and 750kW-class wind turbines. (NEDO)

  13. Wind power development field test project at Ashibe-cho. Detailed wind characteristics survey; Ashibecho ni okeru furyoku kaihatsu field test jigyo (fukyo seisa) hokokusho

    NONE

    1999-11-01

    A detailed wind characteristics survey was conducted at Ashibe-cho, Iki-gun, Nagasaki Prefecture, on the assumption that a wind power generation system would be constructed. The survey was a 1-year project from October 1998 through September 1999, and wind characteristics such as the average wind speed, average wind direction, standard deviation of wind velocity, and the maximum instantaneous wind speed were observed. The observation point was fixed at 20m above ground, the minimum time unit for observation was 10 minutes, and the 10-minute average value was defined as the measured value. For the maximum instantaneous wind speed, the minimum time unit for observation was set to be 2 seconds. The yearly average wind speed was 5.8m/s and the maximum wind speed in the period was 35m/s. Winds came from the prevailing direction of NE (21.3%), and then from NNE (14.7%) and ENE (7.9%). The wind axis was in the NE-SW direction, and the total wind direction occurrence rate from the 6 directions was 60.2%. Turbulence intensity was 0.19 at wind speed 2.0m/s or more and 0.17 at wind speed 4.0m/s or more. Estimated wind turbine yearly operating factors of 66-84% were obtained using rated values of a 150kW, 300kW, and 750kW-class wind turbines. (NEDO)

  14. Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells.

    Moguilevsky, N; Varsalona, F; Guillaume, J P; Noyer, M; Gillard, M; Daliers, J; Henichart, J P; Bollen, A

    1995-01-01

    A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

  15. Assessment of cytotoxic and cytogenetic effects of a 1,2,5-thiadiazole derivative on CHO-K1 cells. Its application as corrosion inhibitor

    Grillo, C.A.; Mirifico, M.V.; Morales, M.L.; Reigosa, M.A.; Mele, M. Fernandez Lorenzo de

    2009-01-01

    This work focuses on the possible use of phenanthro[9,10-c]-1,2,5-thiadiazole 1,1-dioxide (TDZ) as a harmless corrosion inhibitor. TDZ range-dose providing minimum adverse effects to the environment and human health, with satisfactory corrosion-inhibiting properties was evaluated. Cytotoxicity and genotoxicity of TDZ at 0.57-12.50 μM concentration range were tested by neutral red, chromosomal aberrations, mitotic index, and colony formation assays. Results showed a significant increase of chromatid-type aberrations for the highest concentration of TDZ assayed (12.50 μM). Additionally, a reduction in the proliferative rate for lower concentrations was detected by the MI assay. We concluded that TDZ should be used at concentrations lower than 1.16 μM. Corrosion assays performed showed good inhibition effect (ca. 50%) at low (0.65 μM) TDZ concentration. Consequently, our results indicated that TDZ induced a time- and dose-dependent genotoxic and cytotoxic response on CHO-K1 cells. Short assays should be complemented with long exposure tests to simulate chronic contact with TDZ since lower threshold levels may be found for shorter exposures and a wrong safety range could be determined.

  16. Lack of induction of tissue transglutaminase but activation of the preexisting enzyme in c-Myc-induced apoptosis of CHO cells.

    Balajthy, Z; Kedei, N; Nagy, L; Davies, P J; Fésüs, L

    1997-07-18

    The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through epsilon(gamma-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

  17. Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in repair deficient CHO cells

    Kashino, Genro; Suzuki, Keiji; Prise, K.M.

    2005-01-01

    Evidence is accumulating that irradiated cells produce some signals which interact with non-exposed cells in the same population. Here, we analysed the mechanism of such a bystander effect from targeted cells to non-targeted cells. Firstly, in order to investigate the bystander effect in Chinese hamster ovary (CHO) cell lines we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population, of double strand break repair deficient xrs5 cells, was targeted with 1 Gy of Al-K soft X-rays, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells. The induction of micronuclei was also observed when conditioned medium was transferred from irradiated to non-irradiated xrs5 cells. These results suggest that DNA double strand breaks are caused by factors secreted in the medium from irradiated cells. To clarify the involvements of radical species in the bystander response, cells were treated with 0.5%DMSO 1 hour before irradiation and then bystander effects were estimated in xrs5 cells. The results showed clearly that DMSO treatment during X-irradiation suppress the induction of micronuclei in bystander xrs5 cells, when conditioned medium was transferred from irradiated xrs5 cells. Therefore, it is suggested that radical species induced by ionizing radiation are important for producing bystander signals. (author)

  18. Differences in inhibition by beta-arabinofuranosyladenine (araA) of radiation induced DNA damage repair in exponentially growing and plateau-phase CHO-cells

    Iliakis, G.; Seaner, R.

    1988-01-01

    The effect of beta-arabinofuranosyladenine (araA) on the repair of radiation induced DNA damage, as measured by the DNA unwinding technique, was studied in exponentially growing and plateau-phase CHO-cells after exposure to X-rays. Induction of DNA damage by radiation was found to be similar in exponentially growing and plateau-phase cells. In the absence of araA, repair of radiation induced DNA damage proceeded with similar kinetics in exponentially growing and plateau-phase cells. AraA at concentrations between 0-1500 μM inhibited DNA repair both in exponentially growing and in plateau-phase cells. However, the degree of inhibition was significantly higher (by a factor of 3) in plateau-phase cells. A similar degree of repair inhibition by araA was observed in plateau-phase cells treated in their conditioned medium, as well as in plateau-phase cells that were transferred in fresh growth medium just before treatment initiation. These results indicate the importance of biochemical parameters associated with alterations in the growth state of the cells for the inhibitory effect of araA and may help in the elucidation of the molecular mechanism(s) underlying repair inhibition by inhibitors of DNA replication. (orig.)

  19. Radiosensitization of CHO cells by the combination of glutathione depletion and low concentrations of oxygen: The effect of different levels of GSH depletion

    Clark, E.P.; Epp, E.R.; Zachgo, E.A.; Biaglow, J.E.

    1984-01-01

    Recently, the authors have examined the effect of GSH depletion by BSO on CHO cells equilibrated with oxygen at various concentrations (0.05-4.0%) and irradiated with 50 kVp x-rays. This is of interest because of the uncertain radiosensitizing effect GSH depletion may have on cells equilibrated with low oxygen concentrations. GSH depletion (0.1 mM BSO/24 hrs reduced [GSH] ≅ 10% of control) enhanced the radiosensitizing action of moderate (0.4-4.0%) concentrations of oxygen, i.e., GSH depletion reduced the [O/sub 2/] necessary to achieve an equivalent ER by ≅ 2-3 fold. However, GSH depletion was much more effective as a rediosensitizer when cells were equilibrated with low (<0.4%) concentrations of oxygen, i.e., GSH depletion reduced the [O/sub 2/] necessary to achieve an equivalent ER by 8-10 fold. Furthermore, while the addition of exogenous 5 mM GSH restored the ER to that observed when GSH was not depleted, the intracellular [GSH] was not increased. The results of these studies carried out at different levels of GSH depletion are presented

  20. Changes in Al and Fe associated with amorphous soil minerals over one year after a wildfire at Pico Cho Marcial (Tenerife, Canary Islands, Spain

    J. Notario

    2013-05-01

    Full Text Available Amorphous-linked Al, Fe and Si were determined both in burned and unburnt soil samples after a wildfire close to Pico Cho Marcial (Tenerife, Canary Islands, Spain that affected 7.1 ha of high mountain Teide broom scrub along four successive samplings held in September 2003 (three months after the wildfire, February 2004, June 2004 and October 2004. Soils in the area are Lithic Xerorthents, with a scarcely developed AC-type profile. The chemical elements under study were extracted using selective dissolutions (0.2M acid ammonium oxalate and 0.1N sodium pyrophosphate, and determined by Atomic Absorption Spectrophotometry. The average contents of total amorphous Al (oxalate-extractable and organo-metallic complexes-bound Al (pyrophosphate extractable were significantly higher in burned samples along the study. Also, the oxalate-extractable Al varied significantly along the different samplings, and so consequently did the Alp:Alox ratio. A progressive decrease in the Al:Si ratio in allophanes was also found throughout the study period. No differences were found for oxalate-extractable Fe, either between sample groups or samplings, which was also applicable to the (Alox+0.5Feox percentage.

  1. The systematic study of the electroporation and electrofusion of B16-F1 and CHO cells in isotonic and hypotonic buffer.

    Usaj, Marko; Kanduser, Masa

    2012-09-01

    The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells' response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41 ± 9 % yield, while in isotonic buffer 32 ± 11 % yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1 % in isotonic buffer to 10 ± 4 % in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.

  2. Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

    Godfrey, Charlotte L; Mead, Emma J; Daramola, Olalekan; Dunn, Sarah; Hatton, Diane; Field, Ray; Pettman, Gary; Smales, C Mark

    2017-08-01

    mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

    Niessen, Markus; Jaschinski, Frank; Item, Flurin; McNamara, Morgan P.; Spinas, Giatgen A.; Trueb, Thomas

    2007-01-01

    Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the β-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission

  4. Intracellular pH and 42.00 C heat response of CHO cells cultured at pH 6.6

    Cook, J.A.; Fox, M.H.

    1987-01-01

    The authors previously reported that cells under chronic low pH (6.6) conditions have altered thermotolerance. They further characterized both the doubling time (t/sub d/) and the internal pH (pH/sub 1/) of CHO cells continuously cultured at pH 6.6 for times greater than one year. The following differences were noted: 1) A t/sub d/ of 16 hr compared to a t/sub d/ of 12 hr for cells at normal pH (7.3) and a t/sub d/ of 25 hr for the acute low pH cells (pH = 6.6; incubation time = 4 hr). 2) A pH/sub i/ 0.1-0.15 pH units > normal cells and 0.3 pH units > acute low pH cells. 3) Survival at 42.0 0 C which differed from both normal and acute low pH cells. The chronic culture was still quite sensitive to 42.0 0 C treatments during the first 5 hr, but developed tolerance at a higher level than cells under acute low pH conditions. The pH/sub i/ of the chronic culture responded to 42.0 0 C heating in a manner similar to that for acute low pH cells. Whether this culture represents a normal response to long term low pH exposure, or was the response of a mutant population is at the present unknown

  5. Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

    Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

    2015-01-01

    The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in

  6. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

    2012-01-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO 3 ). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  7. Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling.

    Grom, Matic; Kozorog, Mirijam; Caserman, Simon; Pohar, Andrej; Likozar, Blaž

    2018-04-15

    Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) - an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins. Packing breakthrough curves were recorded at an array of prolonged residence times. A mathematical simulation model was developed, applied and validated in combination with non-linear regression algorithms on bed effluent concentrations to determine the previously-unknown binding properties of stationary phase materials. Apart from the columns' differential partitioning, the whole external system was also integrated. It was confirmed that internal pore diffusion is the global rate-limiting resistance of the compound retention process. Immobilizing substrate characteristics, obtained in this engineering study, are indispensable for the scale-up of the periodic counter-current control with mechanistic load, elution and wash reduction. Furthermore, unit's volumetric flow screening measurements revealed dynamic effect correlation to eluate quality parameters, like the presence of aggregates, the host cell-related impurities at supernatant's extended feeding, and titre. Numerical sensitivity outputs demonstrated the impacts of fluidics (e.g. axial dispersion coefficient), thermodynamics (Langmuir adsorption) and mass transfer fluxes. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Frederick Yi-Tung Cho (1939-2011) : His PhD days in Biophysics, the Photosynthesis Lab, and his patents in engineering physics.

    Govindjee; Munday, John C; Papageorgiou, George C

    2017-06-01

    We present here a Tribute to Frederick Yi-Tung Cho (1939-2011), an innovative and ingenious biophysicist and an entrepreneur. He was one of the 4 earliest PhD students [see: Cederstrand (1965)-Carl Nelson Cederstrand; coadvisor: Eugene Rabinowitch; Papageorgiou (1968)-George C. Papageorgiou (coauthor of this paper); and Munday (1968)-John C. Munday Jr. (also a coauthor of this paper)] of one of us (Govindjee) in Biophysics at the University of Illinois at Urbana-Champaign (UIUC) during the late 1960s (1963-1968). Fred was best known, in the photosynthesis circle for his pioneering work on low temperature (down to liquid helium temperature, 4 K) absorption and fluorescence spectroscopy of photosynthetic systems; he showed temperature independence of excitation energy transfer from (i) chlorophyll (Chl) b to Chl a and (ii) from Chl a 670 to Chl a 678; and temperature dependence of energy transfer from the phycobilins to Chl a and from Chl a 678 to its suggested trap. After doing research in biophysics of photosynthesis, Fred shifted to do research in solid-state physics/engineering in the Government Electronics Division (Group) of the Motorola Company, Scottsdale, Arizona, from where he published research papers in that area and had several patents granted. We focus mainly on his days at the UIUC in context of the laboratory in which he worked. We also list some of his papers and most of his patents in engineering physics. His friends and colleagues have correctly described him as an innovator and an ingenious scientist of the highest order. On the personal side, he was a very easy-going and amiable individual.

  9. A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies.

    Roy, Gargi; Martin, Tom; Barnes, Arnita; Wang, Jihong; Jimenez, Rod Brian; Rice, Megan; Li, Lina; Feng, Hui; Zhang, Shu; Chaerkady, Raghothama; Wu, Herren; Marelli, Marcello; Hatton, Diane; Zhu, Jie; Bowen, Michael A

    2018-04-01

    The conserved glycosylation site Asn 297 of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn 297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX™ Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.

  10. Effect of adenovirus infection on transgene expression under the adenoviral MLP/TPL and the CMVie promoter/enhancer in CHO cells

    Mohamed A. El-Mogy

    2017-06-01

    Full Text Available The adenovirus major late promoter (MLP and its translational regulator – the tripartite leader (TPL sequence – can actively drive efficient gene expression during adenoviral infection. However, both elements have not been widely tested in transgene expression outside of the adenovirus genome context. In this study, we tested whether the combination of MLP and TPL would enhance transgene expression beyond that of the most widely used promoter in transgene expression in mammalian cells, the cytomegalovirus immediate early (CMVie promoter/enhancer. The activity of these two regulatory elements was compared in Chinese hamster ovary (CHO cells. Although transient expression was significantly higher under the control of the CMVie promoter/enhance compared to the MLP/TPL, this difference was greater at the level of transcription (30 folds than translation (11 folds. Even with adenovirus infection to provide additional elements (in trans, CMVie promoter/enhancer exhibited significantly higher activity relative to MLP/TPL. Interestingly, the CMVie promoter/enhancer was 1.9 folds more active in adenovirus-infected cells than in non-infected cells. Our study shows that the MLP-TPL drives lower transgene expression than the CMVie promoter/enhancer particularly at the transcription level. The data also highlight the utility of the TPL sequence at the translation level and/or possible overwhelming of the cellular translational machinery by the high transcription activity of the CMVie promoter/enhancer. In addition, here we present data that show stimulation of the CMVie promoter/enhancer by adenovirus infection, which may prove interesting in future work to test the combination of CMVie/TPL sequence, and additional adenovirus elements, for transgene expression.

  11. G1- and S-phase syntheses of histones H1 and H1o in mitotically selected CHO cells: utilization of high-performance liquid chromatography

    D'Anna, J.A.; Thayer, M.M.; Tobey, R.A.; Gurley, L.R.

    1985-01-01

    The authors have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [ 3 H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O 4 ; the total HC1O 4 -soluble, Cl 3 CCO 2 H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure

  12. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

    Bajinskis, Ainars, E-mail: ainars.bajinskis@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden); Olsson, Gunilla; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden)

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO{sub 3}). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  13. Fiscal 2000 report on geothermal energy development promotion survey. Phase 1. Report on environmental impact survey in No. C-5 Appi district (Hot spa fluctuation); 2000 nendo chinetsu kaihatsu sokushin chosa hokokusho. No. C-5. Appi chiiki kankyo chosa (onsen hendo) dai 1 ji

    NONE

    2001-07-01

    For the assessment of the environmental impact to be produced by geothermal exploration well boring and short-term outburst tests, water in hot spas, fountains, and rivers in and around the Appi survey district was inspected for fluctuation. A survey was conducted of multiple components to help analysis of the geothermal reservoir structure. Distributed in the alteration zone in this district are the hot spas of Kusanoyu, Shin-Kusanoyu, Appi, and so forth, and, in the vicinity, numerous hot spas including Iwahatanoyu belonging to the Appi hot spa. In the multiple component survey, 12 hot spas, 1 fountain, 8 rivers, and 2 gas wells were subjected to measurement. In the fluctuation survey, 5 hot spas, 1 fountain, 5 rivers, and the amount of precipitation were subjected to measurement. In the survey of river bottom quality and river bed rocks, river bottom quality was checked at 18 spots and river bed rocks at 12 spots. It was found that fluctuation was great in the amount of welling water at Kusanoyu, Shin-kusanoyu, and Appi, with the coefficient of fluctuation recording 10 or more. A negative correlation was detected between the welling amount and water temperature. It is deemed that the hot water spurts out of natural fountains or shallow wells and that its temperature tends to grow lower with an increase in meteoric water by which it is diluted. (NEDO)

  14. Ultraprecision machining. Cho seimitsu kako

    Suga, T [The Univ. of Tokyo, Tokyo (Japan). Research Center for Advanced Science and Technology

    1992-10-05

    It is said that the image of ultraprecision improved from 0.1[mu]m to 0.01[mu]m within recent years. Ultraprecision machining is a production technology which forms what is called nanotechnology with ultraprecision measuring and ultraprecision control. Accuracy means average machined sizes close to a required value, namely the deflection errors are small; precision means the scattered errors of machined sizes agree very closely. The errors of machining are related to both of the above errors and ultraprecision means the combined errors are very small. In the present ultraprecision machining, the relative precision to the size of a machined object is said to be in the order of 10[sup -6]. The flatness of silicon wafers is usually less than 0.5[mu]m. It is the fact that the appearance of atomic scale machining is awaited as the limit of ultraprecision machining. The machining of removing and adding atomic units using scanning probe microscopes are expected to reach the limit actually. 2 refs.

  15. Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

    Schlatter, Stefan; Senn, Claudia; Fussenegger, Martin

    2003-07-20

    Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210-225, 2003.

  16. Bystander effect-induced mutagenicity in HPRT locus of CHO cells following BNCT neutron irradiation: Characteristics of point mutations by sequence analysis

    Kinashi, Yuko [Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka (Japan)], E-mail: kinashi@rri.kyoto-u.ac.jp; Suzuki, Minoru; Masunaga, Shinichiro; Ono, Koji [Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka (Japan)

    2009-07-15

    To investigate bystander mutagenic effects induced by alpha particles during boron neutron capture therapy (BNCT), we mixed cells that were electroporated with borocaptate sodium (BSH), which led to the accumulation of {sup 10}B inside the cells, with cells that did not contain the boron compound. BSH-containing cells were irradiated with {alpha} particles produced by the {sup 10}B(n,{alpha}){sup 7}Li reaction, whereas cells without boron were only affected by the {sup 1}H(n,{gamma}){sup 2}H and {sup 14}N(n,{rho}){sup 14}C reactions. The frequency of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus was examined in Chinese hamster ovary (CHO) cells irradiated with neutrons (Kyoto University Research Reactor: 5 MW). Neutron irradiation of 1:1 mixtures of cells with and without BSH resulted in a survival fraction of 0.1, and the cells that did not contain BSH made up 99.4% of the surviving cell population. Using multiplex polymerase chain reactions (PCRs), molecular structural analysis indicated that most of the mutations induced by the bystander effect were point mutations and that the frequencies of total and partial deletions induced by the bystander effect were lower than those resulting from the {alpha} particles produced by the {sup 10}B(n,{alpha}){sup 7}Li reaction or the neutron beam from the {sup 1}H(n,{gamma}){sup 2}H and {sup 14}N(n,{rho}){sup 14}C reactions. The types of point mutations induced by the BNCT bystander effect were analyzed by cloning and sequencing methods. These mutations were comprised of 65.5% base substitutions, 27.5% deletions, and 7.0% insertions. Sequence analysis of base substitutions showed that transversions and transitions occurred in 64.7% and 35.3% of cases, respectively. G:C{yields}T:A transversion induced by 8-oxo-guanine in DNA occurred in 5.9% of base substitution mutants in the BNCT bystander group. The characteristic mutations seen in this group, induced by BNCT {alpha} particles

  17. Expression of human FcgammaRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding.

    Armour, Kathryn L; Smith, Cheryl S; Clark, Michael R

    2010-03-31

    The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcgammaRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcgammaRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcgammaRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcgammaRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcgammaRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcgammaRIII and the cell lines were shown to have slightly higher levels of receptor than FcgammaRIII-positive peripheral blood mononuclear cells. Because the affinity of FcgammaRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcgammaRIIIa-158V-antibody interaction is 3

  18. The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate.

    Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

    2012-03-01

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO(3)). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Picosecond real time study of the bimolecular reaction O(3P)+C2H4 and the unimolecular photodissociation of CH3CHO and H2CO

    Abou-Zied, Osama K.; McDonald, J. Douglas

    1998-07-01

    The bimolecular reaction of O(3P) with ethylene and the unimolecular photodissociation of acetaldehyde and formaldehyde have been studied using a picosecond pump/probe technique. The bimolecular reaction was initiated in a van der Waals dimer precursor, C2H4ṡNO2, and the evolution of the vinoxy radical product monitored by laser-induced fluorescence. The NO2 constituent of the complex was photodissociated at 266 nm. The triplet oxygen atom then attacks a carbon atom of C2H4 to form a triplet diradical (CH2CH2O) which subsequently dissociates to vinoxy (CH2CHO) and H. The rise time of vinoxy radical production was measured to be 217 (+75-25) ps. RRKM theory was applied and a late high exit barrier was invoked in order to fit the measured rise time. The structure and binding energy of the van der Waals complex have been modeled using Lennard-Jones type potentials and the results were compared with other systems. The unimolecular side of the potential energy surfaces of this reaction has been investigated by photodissociating acetaldehyde at the same pump energy of 266 nm. The resulting photoproducts, acetyl radical (CH3CO) and formyl radical (HCO), have been monitored by resonance enhanced multiphoton ionization (REMPI) combined with a time-of-flight mass spectrometer. The similarity in the measured evolution times of both radicals indicates the same photodissociation pathway of the parent molecule. The photodissociation rate of acetaldehyde is estimated from RRKM theory to be very fast (3×1012s-1). The T1←S1 intersystem crossing (ISC) rate is found to be the rate determining step to photodissociation and increases with energy. The REMPI mechanism for the production of CH3CO+ is proposed to be the same as that of HCO+(2+1). The HCO product from the photodissociation of formaldehyde at 266 nm reveals a faster T1←S1 ISC rate than in acetaldehyde.

  20. Sustained Uptake of a Hospital-Based Handwashing with Soap and Water Treatment Intervention (Cholera-Hospital-Based Intervention for 7 Days [CHoBI7]): A Randomized Controlled Trial.

    George, Christine Marie; Jung, Danielle S; Saif-Ur-Rahman, K M; Monira, Shirajum; Sack, David A; Mahamud-ur Rashid; Mahmud, Md Toslim; Mustafiz, Munshi; Rahman, Zillur; Bhuyian, Sazzadul Islam; Winch, Peter J; Leontsini, Elli; Perin, Jamie; Begum, Farzana; Zohura, Fatema; Biswas, Shwapon; Parvin, Tahmina; Sack, R Bradley; Alam, Munirul

    2016-02-01

    Diarrhea is the second leading cause of death in children under 5 years of age globally. The time patients and caregivers spend at a health facility for severe diarrhea presents the opportunity to deliver water, sanitation, and hygiene (WASH) interventions. We recently developed Cholera-Hospital-Based Intervention for 7 days (CHoBI7), a 1-week hospital-based handwashing with soap and water treatment intervention, for household members of cholera patients. To investigate if this intervention could lead to sustained WASH practices, we conducted a follow-up evaluation of 196 intervention household members and 205 control household members enrolled in a randomized controlled trial of the CHoBI7 intervention 6 to 12 months post-intervention. Compared with the control arm, the intervention arm had four times higher odds of household members' handwashing with soap at a key time during 5-hour structured observation (odds ratio [OR]: 4.71, 95% confidence interval [CI]: 2.61, 8.49) (18% versus 50%) and a 41% reduction in households in the World Health Organization very high-risk category for stored drinking water (OR: 0.38, 95% CI: 0.15, 0.96) (58% versus 34%) 6 to 12 months post-intervention. Furthemore, 71% of observed handwashing with soap events in the intervention arm involved the preparation and use of soapy water, which was promoted during the intervention, compared to 9% of control households. These findings demonstrate that the hospital-based CHoBI7 intervention can lead to significant increases in handwashing with soap practices and improved stored drinking water quality 6 to 12 months post-intervention. © The American Society of Tropical Medicine and Hygiene.

  1. Evaluation of cytotoxic effect of methanolic extracts isolated from endemic plants of Chaharmahal va Bakhtiari province on PC-3, MCF-7, Hep G2, CHO and B16-F10 cell lines

    Z. Tayarani-Najaran

    2017-11-01

    Full Text Available Background and objectives: To date, thousands of secondary metabolites have been isolated from plants and microorganisms and there is an unprecedented attention towards potential biomedical applications of natural compounds. In this study, cytotoxic properties of methanol extracts of Stachys obtusicrena, Aristolochia olivieri, Linum album, Dionysia sawyeri, Ajuga chamaecistus, Achillea kellalensis, Nepeta glomerulosa, Phlomis aucheria, Tanacetum dumosum, Dianthus orientalis, Scutellaria multicaulis, Cicer oxyodon and Picris oligocephalum which are widely grown in Iran, were investigated on PC-3 (prostat cancer, MCF-7 (breast cancer, Hep-G2 (liver cancer, CHO (ovarian cancer and B16-F10 (melanoma cell lines. Methods: The cancer cells were cultured in RPMI-1640 and incubated with different concentrations of the plant extracts. Cell viability was quantitated by Alamar blue® assay. The apoptotic cells were determined by PI coloring and Flow Cytometry (Sub-G1 peak. Results: The methanol extracts of D. sawyeri, S. obtusicrena, and C. oxyodon significantly decreased the viability of CHO cells. The Methanol extract of D. sawyer and L. album had cytotoxic effects on B16-F10 cells, whereas no toxicity was observed in MCF-7, Hep-G2 and PC-3 cell lines after incubation of the cancer cells with the plant extracts. The PI staining results showed that D. sawyeri, S. obtusicrena, and C. oxyodon in CHO cancer cells could induce apoptosis in a concentration-dependent manner. Conclusion: Screening plants to find the most cytotoxic extract showed D. sawyeri, S. obtusicrena, C. oxyodon and L. album had the potential for further analysis toward finding active phytochemicals with cytotoxic activity.

  2. Carbon isotope fractionation during diamond growth in depleted peridotite: Counterintuitive insights from modelling water-maximum CHO fluids as multi-component systems

    Stachel, T.; Chacko, T.; Luth, R. W.

    2017-09-01

    Because of the inability of depleted cratonic peridotites to effectively buffer oxygen fugacities when infiltrated by CHO or carbonatitic fluids, it has been proposed recently (Luth and Stachel, 2014) that diamond formation in peridotites typically does not occur by rock-buffered redox reactions as previously thought but by an oxygen-conserving reaction in which minor coexisting CH4 and CO2 components in a water-rich fluid react to form diamond (CO2 + CH4 = 2C + 2H2O). In such fluid-buffered systems, carbon isotope fractionation during diamond precipitation occurs in the presence of two dominant fluid carbon species. Carbon isotope modelling of diamond precipitation from mixed CH4- and CO2-bearing fluids reveals unexpected fundamental differences relative to diamond crystallization from a single carbon fluid species: (1) irrespective of which carbon fluid species (CH4 or CO2) is dominant in the initial fluid, diamond formation is invariably associated with progressive minor (diamond in 13C as crystallization proceeds. This is in contrast to diamond precipitation by rock-buffered redox processes from a fluid containing only a single carbon species, which can result in either progressive 13C enrichment (CO2 or carbonate fluids) or 13C depletion (CH4 fluids) in the diamond. (2) Fluid speciation is the key factor controlling diamond δ13 C values; as XCO2 (XCO2 = CO2/[CO2 + CH4]) in the initial fluid increases from 0.1 to 0.9 (corresponding to an increase in fO2 of 0.8 log units), the carbon isotope composition of the first-precipitated diamond decreases by 3.7‰. The tight mode in δ13C of - 5 ± 1 ‰ for diamonds worldwide places strict constraints on the dominant range of XCO2 in water-rich fluids responsible for diamond formation. Specifically, precipitation of diamonds with δ13C values in the range -4 to -6‰ from mantle-derived fluids with an average δ13C value of -5‰ (derived from evidence not related to diamonds) requires that diamond-forming fluids were

  3. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    Kramer, J.M.

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs

  4. Effect of ethanolic extract of propolis on cell viability of chinese hamster ovary cells (CHO-K1) irradiated with {sup 60}CO gamma-rays using differential staining technique

    Castro, Marcos P.M. de; Castro, Renato F. de; Okazaki, Kayo; Vieira, Daniel P., E-mail: dpvieira@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    The objective of present study was to assess the effect of Brazilian propolis (AF-08) on CHO-K1 cells irradiated with {sup 60}Co, through the differential staining technique, using acridine orange and ethidium bromide. The cells were pre-incubated with different concentrations of propolis (50, 100 and 200 μg/mL) for 24h and irradiated with 5 Gy, analyzed at 24 and 48h after exposure. This technique is based on the cell capacity to incorporate fluorescent DNA dyes, where the viable (green), apoptotic (orange/yellow) and necrotic (red) cells can be identified through fluorescence microscopy. Digital high-resolution images were acquired from at least 5 visualization fields, and cells were analyzed using ImageJ and Flowing software. This approach permitted to analyze a large number of cells/sample with the time reduction, much easier and faster, proportioning more statistical power of the technique. The treatment with propolis only was not cytotoxic at 24 and 48h, except for the higher concentration of 200 μg/mL associated or not with radiation, increasing apoptotic and mainly necrotic cells (p<0.001). The data showed a promising use of propolis as well as technique used, pointing out that 200 μg/mL of propolis was cytotoxic, but at lower one (50 μg/mL) presented a radioprotective effect in irradiated CHO-K1 cells. (author)

  5. Effect of ethanolic extract of propolis on cell viability of chinese hamster ovary cells (CHO-K1) irradiated with 60CO gamma-rays using differential staining technique

    Castro, Marcos P.M. de; Castro, Renato F. de; Okazaki, Kayo; Vieira, Daniel P.

    2013-01-01

    The objective of present study was to assess the effect of Brazilian propolis (AF-08) on CHO-K1 cells irradiated with 60 Co, through the differential staining technique, using acridine orange and ethidium bromide. The cells were pre-incubated with different concentrations of propolis (50, 100 and 200 μg/mL) for 24h and irradiated with 5 Gy, analyzed at 24 and 48h after exposure. This technique is based on the cell capacity to incorporate fluorescent DNA dyes, where the viable (green), apoptotic (orange/yellow) and necrotic (red) cells can be identified through fluorescence microscopy. Digital high-resolution images were acquired from at least 5 visualization fields, and cells were analyzed using ImageJ and Flowing software. This approach permitted to analyze a large number of cells/sample with the time reduction, much easier and faster, proportioning more statistical power of the technique. The treatment with propolis only was not cytotoxic at 24 and 48h, except for the higher concentration of 200 μg/mL associated or not with radiation, increasing apoptotic and mainly necrotic cells (p<0.001). The data showed a promising use of propolis as well as technique used, pointing out that 200 μg/mL of propolis was cytotoxic, but at lower one (50 μg/mL) presented a radioprotective effect in irradiated CHO-K1 cells. (author)

  6. Histones H10a and H10b are the same as CHO histones H1(III) and H1(IV):new features of H10 phosphorylation during the cell cycle

    D'Anna, J.A.; Gurley, L.R.; Becker, R.R.

    1981-01-01

    Two histone H1 fractions [H1(I) and H1(II) and two histone H1 0 fractions (H1 0 a and H1 0 b) have been isolated from butyrate-treated Chinese hamster (line CHO) cells by guanidine hydrochloride gradient chromatography on Bio-Rex 70 ion-exchange resin. The fractions have been identified by electrophoresis and amino acid analyses. Electrophoretic analysis of cyanogen bromide treated H1 0 in long acid-urea-polyacrylamide gels suggests that H1 0 a and H1 0 b differ, at least, within the 20-30 residue fragment(s) removed by the cyanogen bromide clevage. Shallow-gradient Bio-Rex 70 chromatography indicates that histones H1 0 a and H1 0 b are the same as the respective CHO histones, H1(III) and H1(IV). This identification and the phosphate incorporation data of Gurley et al. (1975) reveal new features about H1 0 phosphorylation: (1) following release from G 1 arrest, H1 0 a and H1 0 b become phosphorylated in late G 1 prior to DNA synthesis; (2) H1 0 a and H1 0 b are phosphorylated at similar rates throughout the cell cycle. These and other data demonstrate that histone H1 0 is phosphorylated in a cell cycle dependent fashion which mimics that of histone H1

  7. Fiscal 1999 survey report. Survey of environmental effect pertaining to survey for geothermal development and promotion (Hakusuigoe area); 1999 nendo chinetsu kaihatsu sokushin chosa no uchi kankyo eikyo chosa (Hakusuigoe chiiki) hokokusho

    NONE

    2000-03-01

    This survey of environmental effect was intended to grasp actual state concerning some environmental factors that should agree with survey/development and also to assess its effect on the surrounding environment, particularly as a part of the 'stage C' in the 'survey for geothermal development and promotion', which was implemented by the Government for the purpose of guiding geothermal development by enterprises. This report compiles the plan details of the environmental effect survey to be conducted in the Hakusuigoe district, and presents proposals. The survey area is in the north of Makizono-cho, Aira-gun, in Kagoshima prefecture and situated about 3 km west of Onaminoike in Karakunidake, one of the peaks in the Kirishima mountain range. The Ogiri geothermal power plant is in the west of the site, a tourist spot Ebino Heights about 4 km northeast, and the Kirishima hot-spring about 4 km southeast. The location is in the national park, with a part designated as No. 2 and 3 class special areas. The environmental effect survey was divided broadly into such categories as fauna/flora, hot-spring water alteration and inland water, landscape, noise/vibration/subsoil, and air/water, and was further subdivided in accordance with the actual operations. (NEDO)

  8. Geothermal development promotion survey. No.B-4 'Kuenohira-yama area'; 1997 nendo chinetsu kaihatsu sokushin chosa data shori hokokusho. No.B-4 Kuenohira-yama chiiki

    NONE

    1999-01-01

    The above-named survey had been scheduled to be conducted in fiscal years 1996-1998 for an approximately 10km{sup 2}-large area of Kokonoe-cho, Kusu-gun, Oita Prefecture. In fiscal 1996, surface surveys were conducted, such as geological and alteration zone surveys, gravity prospecting, and electromagnetic survey. In fiscal 1997, an exploration well (N9-KH-1: depth 2,222.0m) was drilled. Since the maximum temperature in the well was found to be so low as 78.5 degrees C, the survey was given up in the second year. The survey conducted at N9-KH-1 showed that deep-seated and shallow-seated ruptures developed there in the absence of aquicludes, and so it was inferred that it was a recharge area, as predicted by the temperature logging profile, with surface water flowing into the depth. The above showed that there was no deep-seated blind geothermal system which the survey was after. Since it was not likely that the shallow-seated geothermal system located in the southern part of the Mizuwake pass would be found expanding into the area of the current survey, it was decided that the survey not be carried forward to the following fiscal year. (NEDO)

  9. Geothermal development promotion survey. No.B-4 'Kuenohira-yama area'; 1997 nendo chinetsu kaihatsu sokushin chosa data shori hokokusho. No.B-4 Kuenohira-yama chiiki

    NONE

    1999-01-01

    The above-named survey had been scheduled to be conducted in fiscal years 1996-1998 for an approximately 10km{sup 2}-large area of Kokonoe-cho, Kusu-gun, Oita Prefecture. In fiscal 1996, surface surveys were conducted, such as geological and alteration zone surveys, gravity prospecting, and electromagnetic survey. In fiscal 1997, an exploration well (N9-KH-1: depth 2,222.0m) was drilled. Since the maximum temperature in the well was found to be so low as 78.5 degrees C, the survey was given up in the second year. The survey conducted at N9-KH-1 showed that deep-seated and shallow-seated ruptures developed there in the absence of aquicludes, and so it was inferred that it was a recharge area, as predicted by the temperature logging profile, with surface water flowing into the depth. The above showed that there was no deep-seated blind geothermal system which the survey was after. Since it was not likely that the shallow-seated geothermal system located in the southern part of the Mizuwake pass would be found expanding into the area of the current survey, it was decided that the survey not be carried forward to the following fiscal year. (NEDO)

  10. Research report on promotion of geothermal energy exploitation for fiscal 1997. Fluid geochemical research (No.B-6 Tsujinodake district); 1997 nendo chinetsu kaihatsu sokushin chosa. Ryutai chikagaku chosa hokokusho (No.B-6 Tsujinodake chiiki)

    NONE

    1998-07-01

    The research covers the Tsujinodake district spreading over Yamakawa-cho and Ibusuki-shi, Kagoshima Prefecture. Hot spring water in the district is of the neutral Cl type, heated mainly by conduction and containing seawater. The thermal water layer of the highest geothermal potential exists in the vicinity of the Nishi district of Ibusuki-shi. It spreads widely in the westward part and may be linked with the Unagiike district. The temperature of the deep-seated thermal water layer is estimated at 220-230 degrees C, yet it may be higher. In addition to the layer in the vicinity of the Nishi and Unagiike districts, there is a shallow-seated hot spring aquifer in the Nagasakibana district, where the deep-seated thermal water is probably prevented from lifting in the presence of an aquiclude. In the Narikawa district, possibilities are low that the thermal water just beneath will come up. As for hot spring water in the Oyama and Okachogamizu districts, there are no signs of high-temperature thermal water infiltration. In these two districts, it appears that hot spring water is heated by conduction over a large area. For the disclosure of linkage of hot water systems and deep-seated geothermal structures which cannot be detected by geochemical investigations conducted into hot spring water, etc., it is necessary to resort to geological and geophysical researches, and to carry out test drilling. (NEDO)

  11. Diverse bone scan abnormalities in shin splints

    Spencer, R.P.; Levinson, E.D.; Baldwin, R.D.; Sziklas, J.J.; Witek, J.T.; Rosenberg, R.

    1979-01-01

    Four young patients who presented with pain over the anterior compartment of the legs, gave a recent athletic history suggesting stress fractures. Although radiographs were initially normal in all four cases, the bone scintigrams were positive. The individual findings, however, were quite different. In one there was a single focal area of increased radioactivity in each of the tibias; the second patient had uneven uptake of radiotracer and several foci of accumulation in the fibulas; the third showed diffuse linear tibial uptake suggesting periosteal lesions; and the fourth case revealed uptake in the lateral malleolus and in bones of the foot

  12. Radiosensitive xrs-5 and parental CHO cells show identical DNA neutral filter elution dose-response: implications for a relationship between cell radiosensitivity and induction of DNA double-strand breaks

    Iliakis, George; Okayasu, Ryuichi; Seaner, Robert

    1988-01-01

    The purpose of this work was to investigate a possible correlation between DNA elution dose-response and cell radiosensitivity. For this purpose neutral (pH 9.6) DNA filter elution dose-response curves were measured with radiosensitive xrs-5 and the parental Chinese hamster ovary (CHO) cells in the logarithmic and plateau phase of growth. No difference was observed between the two cell types in the DNA elution dose-response curves either in logarithmic or plateau phase, despite the dramatic differences in cell radiosensitivity. This observation indicates that the shape of the DNA elution dose-response curve and the shape of the cell survival curve are not causally related. It is proposed that the shoulder observed in the DNA elution dose-response curve reflects either partial release of DNA from chromatin, or cell cycle-specific alterations in the physicochemical properties of the DNA. (author)

  13. Ethanol oxidation reactions catalyzed by water molecules: CH3CH2OH+n H2O→ CH3CHO+ H2+n H2O (n=0,1,2)

    Takahashi, H.; Hisaoka, S.; Nitta, T.

    2002-09-01

    Ab initio density functional theory calculations have been performed to investigate the catalytic role of water molecules in the oxidation reaction of ethanol: CH3CH2OH+n H2O→ CH3CHO+ H2+n H2O (n=0,1,2) . The results show that the potential energy barrier for the reaction is 88.0 kcal/mol in case of n=0, while it is reduced by ˜34 kcal/mol when two water molecules are involved ( n=2) in the reaction. As a result, the rate constant increases to 3.31×10 -4 s-1, which shows a significant catalytic role of water molecules in the ethanol oxidation reactions.

  14. 125IdUrd-induced chromosome fragments, assayed by premature chromosome condensation, and DNA double-strand breaks have similar repair kinetics in G1-phase CHO-cells

    Iliakis, George; Pantelias, G.E.; Okayasu, Ryuichi; Seaner, Robert

    1987-01-01

    The effect of 125 I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G 1 -phase CHO-cells. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragments was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation. (author)

  15. Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells.

    Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

    Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.

  16. Wnt-11 signaling leads to down-regulation of the Wnt/β-catenin, JNK/AP-1 and NF-κB pathways and promotes viability in the CHO-K1 cells

    Railo, Antti; Nagy, Irina I.; Kilpelaeinen, Pekka; Vainio, Seppo

    2008-01-01

    The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical β-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical β-catenin mediated Wnt signaling but also JNK/AP-1 and NF-κB signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-κB pathway. Consistent with the central role of Akt, JNK and NF-κB in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways

  17. Evaluation of genotoxic effect of prozac (fluoxetine without and with addition of vitamins A and C by means of the comet assay in culture of CHO-K1 cells

    Noélle Giacomini Lemos

    2005-12-01

    Full Text Available The fluoxetine, commercially named Prozac, is efficient against depression and anxiety, with lower risk of collateral effects. However, the possible genotoxic effects are still unknown. The use of vitamins as protectors against damages on cells and DNA has been evaluated, mainly for vitamins A and C. Furthermore, the associative effect of vitamins with several medicines demands studies. The evaluations of genotoxic effect of Prozac and vitamins A and C protective effect were carried out in culture of Chinese hamster ovary cells, CHO-K1, by means of the comet test. The Prozac was used, in liquid formulation, diluted in 5µg, 1µg and 0.2 µg/mL of culture medium. The vitamins were used, in liquid formulation, at the concentrations of 3µg and 880,5 µg/mL of culture medium to vitamins A and C, respectively. The treatments were carried out during 1 hour. The obtained data demonstrated that only the highest concentration of Prozac (5 µg is genotoxic and both vitamins A and C reduced such genotoxicity. The data suggest a follow-up on patients who use Prozac and the possibility of vitamins A and C association in order to minimize the collateral genotoxic effects.

  18. Comparison of DNA double-strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose gradient centrifugation and non-unwinding filter elution in irradiated plateau-phase CHO cells

    Iliakis, G.; Metzger, L.; Pantelias, G.

    1991-01-01

    The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral sucrose gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. The results suggest all major techniques currently used for assaying rejoining of DNA dsb give similar results, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established. (author)

  19. Wind power development field test project at Aoyama Heights, Aoyama-cho. Detailed wind characteristics survey; Aoyamacho Aoyama Kogen ni okeru furyoku kaihatsu field test jigyo (fukyo seisa) hokokusho

    NONE

    1999-11-01

    A detailed wind characteristics survey was conducted at Aoyama Heights, Aoyama-cho, Naga-gun, Mie Prefecture, on the assumption that a wind power generation system would be constructed. The survey was a 1-year project from October 1998 through September 1999, and wind characteristics such as the average wind speed, average wind direction, standard deviation of wind velocity, and the maximum instantaneous wind speed were observed. The observation point was fixed at 20m above ground, the minimum time unit for observation was 10 minutes, and the 10-minute average value was defined as the measured value. For the maximum instantaneous wind speed, the minimum time unit for observation was set to be 2 seconds. The yearly average wind speed was 5.3m/s and the maximum wind speed in the period was 32m/s. Winds came prevalently from WNW (31.6%), and then from NW (16.8%), ESE (20.9%), and E (9.9%). The wind axis was in the direction of WNW-ESE, and the total wind direction occurrence rate was 89.0%. Turbulence intensity was 0.25 at wind speed 2.0m/s or more and 0.23 at wind speed 4.0m/s or more. Estimated wind turbine yearly operating factors of 70-84% were obtained using rated values of a 150kW, 300kW, and 750kW-class wind turbines. (NEDO)

  20. Journal of Genetics | Indian Academy of Sciences

    Author Affiliations. Shin-Aeh Lee1 Seung-Gon Lee1 Kyoung-Oh Cho2 Changbaig Hyun1. Section of Internal Medicine, School of Veterinary Medicine, Kangwon National University, Chuncheon 200 701, Korea; Institute of Veterinary Medicine, College of Veterinary Medicine, Chonnam National University, Kwangju 500 757, ...

  1. The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion.

    Brunner, Matthias; Braun, Philipp; Doppler, Philipp; Posch, Christoph; Behrens, Dirk; Herwig, Christoph; Fricke, Jens

    2017-07-01

    Due to high mixing times and base addition from top of the vessel, pH inhomogeneities are most likely to occur during large-scale mammalian processes. The goal of this study was to set-up a scale-down model of a 10-12 m 3 stirred tank bioreactor and to investigate the effect of pH perturbations on CHO cell physiology and process performance. Short-term changes in extracellular pH are hypothesized to affect intracellular pH and thus cell physiology. Therefore, batch fermentations, including pH shifts to 9.0 and 7.8, in regular one-compartment systems are conducted. The short-term adaption of the cells intracellular pH are showed an immediate increase due to elevated extracellular pH. With this basis of fundamental knowledge, a two-compartment system is established which is capable of simulating defined pH inhomogeneities. In contrast to state-of-the-art literature, the scale-down model is included parameters (e.g. volume of the inhomogeneous zone) as they might occur during large-scale processes. pH inhomogeneity studies in the two-compartment system are performed with simulation of temporary pH zones of pH 9.0. The specific growth rate especially during the exponential growth phase is strongly affected resulting in a decreased maximum viable cell density and final product titer. The gathered results indicate that even short-term exposure of cells to elevated pH values during large-scale processes can affect cell physiology and overall process performance. In particular, it could be shown for the first time that pH perturbations, which might occur during the early process phase, have to be considered in scale-down models of mammalian processes. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Investigation of the interactions of critical scale-up parameters (pH, pO2 and pCO2) on CHO batch performance and critical quality attributes.

    Brunner, Matthias; Fricke, Jens; Kroll, Paul; Herwig, Christoph

    2017-02-01

    Understanding process parameter interactions and their effects on mammalian cell cultivations is an essential requirement for robust process scale-up. Furthermore, knowledge of the relationship between the process parameters and the product critical quality attributes (CQAs) is necessary to satisfy quality by design guidelines. So far, mainly the effect of single parameters on CQAs was investigated. Here, we present a comprehensive study to investigate the interactions of scale-up relevant parameters as pH, pO 2 and pCO 2 on CHO cell physiology, process performance and CQAs, which was based on design of experiments and extended product quality analytics. The study used a novel control strategy in which process parameters were decoupled from each other, and thus allowed their individual control at defined set points. Besides having identified the impact of single parameters on process performance and product quality, further significant interaction effects of process parameters on specific cell growth, specific productivity and amino acid metabolism could be derived using this method. Concerning single parameter effects, several monoclonal antibody (mAb) charge variants were affected by process pCO 2 and pH. N-glycosylation analysis showed positive correlations between mAb sialylation and high pH values as well as a relationship between high mannose variants and process pH. This study additionally revealed several interaction effects as process pH and pCO 2 interactions on mAb charge variants and N-glycosylation pattern. Hence, through our process control strategy and multivariate investigation, novel significant process parameter interactions and single effects were identified which have to be taken into account especially for process scale-up.

  3. Vitamin C (Vit C) added after irradiation reduces the number and alters the spectrum of CD59- mutants in human/CHO AL cells exposed to high LET carbon ions

    Vannais, D.B.; Hirai, Y.; Waldren, C.A.; Ueno, A.

    2003-01-01

    Full text: Miyazaki, Watanabe, Kumagai and colleagues discovered the existence in mammalian cells of long-lived radicals (LLR) with half-lives of minutes to hours. They further showed that concentrations of LLR were increased in a dose dependent manner by X-rays; that LLR were transforming and mutagenic but not clastogenic or lethal; that they were scavenged by Vit C but not by DMSO, and that they occured mainly (>99.8%) in proteins from which they escape by atomic tunneling. They also showed that Vit C added after radiation (but not DMSO) eliminated HPRT mutants in human cells exposed to X-rays. Following on their work, we found that Vit C (5 mM) added 30 min after radiation significantly reduced, but did not eliminate, induction of CD59- mutants in human-CHO hybrid AL cells exposed to high LET carbon beam radiation (NIRS-HIMAC, 290 MeV/nucleon, LET 100 KeV/μ: m). Lethality of the carbon beam was not affected by Vit C. DMSO decreased mutation and killing, only when present during radiation. Lycopene, reported to reduce spontaneous mutation, did not affect radiation killing or mutagenesis. Our findings with Vit C for high LET generally support the results reported for X-rays. Analysis of the spectrum of mutations in CD59- mutant cells isolated after carbon beam irradiation (2.5 Gy), indicates a substantial reduction by post-radiation Vit C in mutants with small mutations and those displaying genomic instability, seen as increased levels of translocations. Our results substantiate a role for LLR in radiation mutagenesis and implicate them in radiation-induced genomic instability

  4. Supernatural in society. (Cho) to shakai

    Watanabe, S. (Agency of Industrial Science and Technology, Tokyo (Japan))

    1992-10-05

    The technologies prefixed with 'super' have offered the conveniences in the various aspects of the society including the industrial field up to present. Firstly a conception 'super high speed transportation system' has realized the 'supersonic aircraft' and 'ultra super express train', etc. such as a linear motor car, and moreover is developing the 'superconducting propulsive ship' and so forth. In the information oriented society, the information processing speed of computer has made a rapid progress by the development of very high speed large scale integrated circuit etc. In the field of manufacture, there are the magnetic head of video tape recorder and the lens of camera as products close to us manufactured by the 'super precision working.' As for the products adhered closely to ous lives, there are medical equipments, wedling machine, fish detector, etc. utilizing the 'ultrasonic wave', as well as 'supermultistoried building,' etc. Furthermore, turning a loon on the world of culture and art, the activities of the 'surrealism' are giving a substantial effect. It is important that the technology prefixed with 'super' will be let developed, and not only the conveniences, but also the harmony between the human being and nature will be realized in a better form in the future as well.

  5. Fiscal 1999 survey for promotion of geothermal energy development. Survey report of environmental impact survey - Hot spring fluctuation (No. B-5, Musadake district); 1999 nendo chinetsu kaihatsu sokushin chosa. Kankyo eikyo chosa hokokusho (onsen hendo) (No.B-5 Musadake chiiki)

    NONE

    2000-12-01

    Research boreholes N11-MD-3 and -4 were drilled in Shibetsu-cho, Shibetsu-gun, Hokkaido, in the period July 1999 through December 1999, and thermal water was examined at the sites in July and November 1999. For the purpose of detecting the impact of the said work on the environment, local hot springs were examined for their status before, during, and after the drilling period. Measurements were conducted in the period August 5, 1998, through December 31, 2000. It was found that no fluctuation in water temperature or yield attributable to the drilling was detected. Hot springs were examined for nine items including water temperature and pH level at four spots which were Kaiyo-dai, River Tenyu, and Otoko-yu and Onna-yu of Kawakita Hot Spring, when 23-31 measurements were performed from each of the four. (NEDO)

  6. Radioiodinated 4-iodo-L-meta-tyrosine, a system L selective artificial amino acid: molecular design and transport characterization in Chinese hamster ovary cells (CHO-K1 cells)

    Shikano, Naoto, E-mail: sikano@ipu.ac.j [Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Kotani, Takashi; Nakajima, Syuichi; Ogura, Masato; Nakazawa, Shinya [Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Sagara, Jun-ichi [Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Kobayashi, Masato [Division of Health Science, Graduate School of Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 9200-942 (Japan); Baba, Takeshi; Yamaguchi, Naoto [Center for Medical Science, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Kubota, Nobuo [Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Kawai, Keiichi [Division of Health Science, Graduate School of Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 9200-942 (Japan)

    2010-11-15

    Introduction: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of {sup 14}C(U)-L-tyrosine ({sup 14}C-Tyr), {sup 125}I-4-iodo-L-meta-tyrosine (4-{sup 125}I-mTyr), {sup 125}I-6-iodo-L-meta-tyrosine (6-{sup 125}I-mTyr), {sup 125}I-3-iodo-{alpha}-methyl-L-tyrosine ({sup 125}I-IMT) and {sup 125}I-3-iodo-L-tyrosine (3-{sup 125}I-Tyr) using Chinese hamster ovary cells (CHO-K1). Methods: Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na{sup +} at 37{sup o}C or 4{sup o}C. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na{sup +}-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na{sup +}-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN{sub 3} and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and L- and D-isomers of natural amino acids. Results: {sup 14}C-Tyr exhibited affinity for systems L, A and ASC. 4-{sup 125}I-mTyr and 3-{sup 125}I-Tyr exhibited high specificity for system L, whereas 6-{sup 125}I-mTyr and {sup 125}I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-{sup 125}I-mTyr was markedly reduced by incubation at 4 {sup o}C, and was not significantly inhibited by NaN{sub 3}, DNP, PAH or TEA. The inhibition profiles of the L- and D-isomers of natural amino acids indicated that system L mediates the transport of 4-{sup 125}I-mTyr. Conclusions: 4-{sup 125}I-mTyr exhibited the greatest system L specificity (93.46{+-}0.13%) of all of the tested amino acids.

  7. Report on the results of research and development under a consignment from NEDO of glycoconjugate production utilizing technologies. Development of technologies to fix and effectively utilize carbon dioxide by applying glycoconjugates; 1997 nendo sangyo kagaku gijutsu kenkyu kaihatsu jigyo Shin energy Sangyo Gijutsu Sogo Kaihatsu Kiko itaku. Fukugo toshitsu seisan riyo gijutsu no kenkyu kaihatsu (fukugo toshitsu oyo nisanka tanso koteika yuko riyo gijutsu kaihatsu) seika hokokusho

    NONE

    1998-03-01

    This paper reports research results in fiscal 1997 for the `research and development of glycoconjugate production utilizing technologies`. In synthesizing, utilizing and remodeling technologies for glycoconjugates by means of chemical synthesis, studies were performed on developing methods to synthesize Gal {beta}1-3Gal NA(c {alpha})1-0-Serine in preparative scale, synthesizing high mannose type sugars of natural type without protection groups, and linking GlcNA or GalNAc onto partial peptide of fibroblast growth factor (FGF). In synthesizing, utilizing and remodeling technologies for glycoconjugates by using biological methods, studies were carried out, with regard to glycoconjugate synthesizing, utilizing and remodeling technologies utilizing animal cells, on identifying sugar structures of IFN-{gamma} produced from CHO cell line, and isolating CHO cell lines introduced with genes of sugar transferred enzyme GnTIV and/or GnTV. Furthermore, studies were conducted on glycoconjugate synthesizing, utilizing and remodeling technologies utilizing microorganisms, and glycoconjugate structure analyzing technologies. In addition, overall investigation was made on glycoconjugate production utilizing technologies. 113 refs., 76 figs., 18 tabs.

  8. ON THE FORMATION AND ISOMER SPECIFIC DETECTION OF PROPENAL (C{sub 2}H{sub 3}CHO) AND CYCLOPROPANONE (c-C{sub 3}H{sub 4}O) IN INTERSTELLAR MODEL ICES—A COMBINED FTIR AND REFLECTRON TIME-OF-FLIGHT MASS SPECTROSCOPIC STUDY

    Abplanalp, Matthew J.; Borsuk, Aleca; Jones, Brant M.; Kaiser, Ralf I., E-mail: ralfk@hawaii.edu [W. M. Keck Research Laboratory in Astrochemistry, University of Hawaii at Manoa, Honolulu, Hawaii, HI, 96822 (United States)

    2015-11-20

    The formation routes of two structural isomers—propenal (C{sub 2}H{sub 3}CHO) and cyclopropanone (c-C{sub 3}H{sub 4}O)—were investigated experimentally by exposing ices of astrophysical interest to energetic electrons at 5.5 K thus mimicking the interaction of ionizing radiation with interstellar ices in cold molecular clouds. The radiation-induced processing of these ices was monitored online and in situ via Fourier Transform Infrared spectroscopy and via temperature programmed desorption exploiting highly sensitive reflectron time-of-flight mass spectrometry coupled with single photon ionization in the post irradiation phase. To selectively probe which isomer(s) is/are formed, the photoionization experiments were conducted with 10.49 and 9.60 eV photons. Our studies provided compelling evidence on the formation of both isomers—propenal (C{sub 2}H{sub 3}CHO) and cyclopropanone (c-C{sub 3}H{sub 4}O)—in ethylene (C{sub 2}H{sub 4})—carbon monoxide (CO) ices forming propenal and cyclopropanone at a ratio of (4.5 ± 0.9):1. Based on the extracted reaction pathways, the cyclopropanone molecule can be classified as a tracer of a low temperature non-equilibrium chemistry within interstellar ices involving most likely excited triplet states, whereas propenal can be formed at ultralow temperatures, but also during the annealing phase via non-equilibrium as well as thermal chemistry (radical recombination). Since propenal has been detected in the interstellar medium and our laboratory experiments demonstrate that both isomers originated from identical precursor molecules our study predicts that the hitherto elusive second isomer—cyclopropanone—should also be observable toward those astronomical sources such as Sgr B2(N) in which propenal has been detected.

  9. Separation of two major chalcones from Angelica keiskei by high-speed counter-current chromatography.

    Kil, Yun-Seo; Nam, Joo-Won; Lee, Jun; Seo, Eun Kyoung

    2015-08-01

    Angelica keiskei (Shin-sun cho) is an edible higher plant with the beneficial preventive effects on cancer, hypertension, and coronary heart disease. Two bioactive chalcones of Shin-sun cho, xanthoangelol (1) and 4-hydroxyderricin (2), were separated simultaneously by using high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-EtOAc-MeOH-H2O (9:5:9:4). Only nonconsuming processes, solvent fractionations and Sephadex LH-20 column chromatography, were conducted as presteps. Xanthoangelol (1, 35.9 mg, 99.9 % purity at 254 and 365 nm) and 4-hydroxyderricin (2, 4.4 mg, 98.7 % purity at 254 nm and 98.8 % purity at 365 nm) were successfully purified from 70 mg of the processed extract from A. keiskei. The structures of two compounds were confirmed by (1)H- and (13)C-NMR analysis.

  10. New alloys for structural application. Kikai kozoyo shin kinzoku zairyo

    Suzuki, T [Kogakuin Univ., Tokyo (Japan)

    1993-01-15

    As recent advance on the scientific technologies is amazing, the materials having new function or excellent performance have been developed. Speaking on some recent examples, they can be divided roughly to one group belonging to invention of materials themselves such as oxide superconductors and compounds semi-conductors, and the other belonging to the products as invention of epoc-making material manufacturing methods such as noncrystalline alloys and particle dispersion reinforced alloys (ODS). In this report, first, some new metallic materials introduced relatively recently and converting many technical innovations in wide range of machinery field, such as titanium alloys, refractory alloys having nickel group, aluminium-lithium alloys, HSLA steels composed of reduced weight of alloy elements and having higher strengths than those of carbon-steels and various properties, intermetallic materials such as Ni3Al and others are described, and next, mechanical alloying and its particle dispersion reinforced alloys, liquid phase rapid cooling process and its noncrystalline alloy-rapid cooling cohesion powder sintering alloys, and others are summarized under the paths from their birth to present status. 31 refs., 12 figs., 5 tabs.

  11. The medial tibial stress syndrome. A cause of shin splints.

    Mubarak, S J; Gould, R N; Lee, Y F; Schmidt, D A; Hargens, A R

    1982-01-01

    The medial tibial stress syndrome is a symptom complex seen in athletes who complain of exercise-induced pain along the distal posterior-medial aspect of the tibia. Intramuscular pressures within the posterior compartments of the leg were measured in 12 patients with this disorder. These pressures were not elevated and therefore this syndrome is a not a compartment syndrome. Available information suggests that the medial tibial stress syndrome most likely represents a periostitis at this location of the leg.

  12. (nitro substituted-phenyl)-hydrazonomethyl

    7.14 (d, 1H, ArH), δ 7.36 (m, 2H, ArH), δ 7.47–7.51 (m,. 2H, ArH), δ 7.77 ..... Phys. Chem. A 115 1985; (d) Kang Y, Gwon K, Shin J H, ... (a) Zhang J F, Lim C S, Bhuniya S, Cho B R and Kim. J S 2011 Org. ... Chem. Rev. 240 1; (c) Sessler J L and.

  13. Experimental and molecular dynamics simulation study of the sublimation and vaporization energetics of iron metalocenes. crystal structures of Fe(eta5-C5H4CH3)2 and Fe[(eta5-(C5H5)(eta5-C5H4CHO)].

    Lousada, Claudio M; Pinto, Susana S; Lopes, José N Canongia; da Piedade, M Fatima Minas; Diogo, Hermínio P; da Piedade, Manuel E Minas

    2008-04-03

    The standard molar enthalpies of sublimation of ferrocene, 1,1'-dimethylferrocene, decamethylferrocene, ferrocenecarboxaldehyde and alpha-methylferrocenemethanol, and the enthalpy of vaporization of N,N-dimethyl(aminomethyl)ferrocene, at 298.15 K, were determined by Calvet-drop microcalorimetry and/or the Knudsen effusion method. The obtained values were used to assess and refine our previously developed force field for metallocenes. The modified force field was able to reproduce the deltasubHdegreesm and deltavapHdegreesm values of the test-set with an accuracy better than 5 kJ.mol-1, except for decamethylferrocene, in which case the deviation between the calculated and experimental deltasubHdegreesm values was 16.1 kJ.mol-1. The origin of the larger error found in the prediction of the sublimation energetics of decamethylferrocene, and which was also observed in the estimation of structural properties (e.g., density and unit cell dimensions), is discussed. Finally, the crystal structures of Fe(eta5-C5H4CH3)2 and Fe[(eta5-(C5H5)(eta5-C5H4CHO)] at 293 and 150 K, respectively, are reported.

  14. Structure and bonding of transition metal-boryl compounds. Theoretical study of [(PH3)2(CO)ClOs-BR2] and [(PH3)2(CO)2ClOs-BR2] (BR2 = BH2, BF2, B(OH)2, B(OCH=CHO), Bcat).

    Giju, K T; Bickelhaupt, F M; Frenking, G

    2000-10-16

    Quantum chemical DFT calculations using the B3LYP functionals have been carried out for the electronically unsaturated 16 VE five-coordinate osmium boryl-complexes [(PH3)2(CO)ClOs-BR2] and the 18 VE six-coordinate complexes [(PH3)2(CO)2ClOs-BR2] with BR2 = BH2, BF2, B(OH)2, B(OHC=CHO), and Bcat (cat = catecholate O2C6H4). The bonding situation of the Os-BR2 bond was analyzed with the help of the NBO partitioning scheme. The Os-B bond dissociation energies of the 16 VE complexes are very high, and they do not change very much for the different boryl ligands. The 18 VE complexes have only slightly lower bond energies than the 16 VE species. The Os-B bond in both classes of compounds is provided by a covalent sigma-bond which is polarized toward osmium and by strong charge attraction. Os-->B pi-donation is not important for the Os-B binding interactions, except for the Os-BH2 complexes. The stability of the boryl complexes [Os]-BR2 comes mainly from BB pi-donation. The intraligand charge distribution of the BR2 group changes little when the Os-B bond is formed, except for BH2. The CO ligand in [(PH3)2(CO)2ClOs-BR2] which is trans to BR2 has a relatively weak bond to the osmium atom.

  15. 2-Aminoanthracene, 5-fluorouracil, colchicine, benzo[a]pyrene, cadmium chloride and cytosine arabinoside tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster ovary (CHO) cells at Covance Laboratories, Harrogate UK in support of OECD draft Test Guideline 487.

    Whitwell, James; Fowler, Paul; Allars, Sarah; Jenner, Karen; Lloyd, Melvyn; Wood, Debbie; Smith, Katie; Young, Jamie; Jeffrey, Laura; Kirkland, David

    2010-10-29

    The reference genotoxic agents 2-aminoanthracene (a metabolism dependent weak clastogen), 5-fluorouracil (a nucleoside analogue, characterised by a steep dose response profile), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation), cadmium chloride (an inorganic carcinogen), and cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair) were tested in the in vitro micronucleus assay using the Chinese hamster ovary (CHO) cell line at Covance Laboratories, Harrogate, UK. All chemicals were treated in the absence and presence of cytokinesis block (via addition of cytochalasin B) with this work forming part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 on the In vitro Mammalian Cell Micronucleus Test (MNvit). The toxicity measures used, detecting a possible combination of both cytostasis and cell death (though not cell death directly), were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index in the presence of cytokinesis block. All of the chemicals tested either gave marked positive increases in the percentage of micronucleated cells with and without cytokinesis block, or did not induce micronuclei at concentrations giving approximately 50-60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcome from this series of tests supports the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Hyekyung Cho: Chinas langer Marsch in den Kapitalismus

    Czeskleba-Dupont, Rolf

    2006-01-01

    Chos afhandling fra Berlin refereres i hovedtræk. Tre modsætninger tages op i den: 1) mellem 'transitionen' ind i kapitalismen og herredømmet af et kommunistisk parti, hvor det bl.a.viser sig at particentralen fremmer liberaliseringen i kampen mod statsvirksomhederne; 2) mellem integrationen i ve...

  17. Dicty_cDB: CHO707 [Dicty_cDB

    Full Text Available itdkkivmiiiqkqlivaiprlvnvltnhimlsqvvli*sl v*lgshwwgtggkgmqnxkxxtll Frame C: vllipqsm*miitnvqlihvpkkvv*lilqsilmitm...smpvpnqq vllipxsm*mittnvqlmhvpkxvv*lipqsilmitmpvqlmhvqkkvv*phtsxqy*** qclypltpvxhplafptpqltaxixipcxvxlmf---

  18. Dicty_cDB: CHO775 [Dicty_cDB

    Full Text Available WMLKGRPGRCPTCK--- ---EGNMIENQYITLKTKLTPT**r*csl*nlgrlccq*sr*ti*nqylcsrysft*ere *k*tfhslgkr*e*itlmaw**vn*frid...sft*ere *k*tfhslgkr*e*itlmaw**vn*fridclagikncsticsknwlqiw*rylfc*ly** iiqllfhik*lsnctyvim*sftw*he*iet*ylygrgt

  19. Dicty_cDB: CHO739 [Dicty_cDB

    Full Text Available t*ere* k*tfhslgkr*e*itlmaw**vn*fridclagikncsticsknwlqiw*rylfc*ly**i iqllfhik*lsnctyvim*sftw*he*iet*ylygrgtts...YITLKTKLTPT**r*csl*nlgrlccq*sr*ti*nqylcsrysft*ere* k*tfhslgkr*e*itlmaw**vn*fridclagikncsticsknwlqiw*rylfc*ly

  20. Newly identified CHO ERCC3/XPB mutations and phenotype characterization

    Rybanská, Ivana; Gurský, Ján; Fašková, Miriam; Salazar, Edmund P.; Kimlíčková-Polakovičová, Erika; Kleibl, Karol; Thompson, Larry H.; Piršel, Miroslav

    2010-01-01

    Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron–exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5′ incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages ∼8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER. PMID:19942596