The objective is to investigate which technologies are the most sustainable management and/or amelioration of soils for the agricultural practices and the most stabile sequestration as the soil organic carbon (SOC) from an atmospheric CO₂ in the arid land, low net primary productivity. The capacity of a soil to sequester organic carbon, including humus carbon and microbial biomass carbon, is dependent mainly on climate, edaphic conditions, type of vegetation and soil management imposed by agricultural practice. The background of this research has already reported that the increase of agricultural products became possible with those humified natural organic matters in the following arid lands, irrigated land, alkali/salt-affected lands, and acid-affected land, assumed where are difficult to restore as arable land. It is possible that plant matter is increased by ameliorating with physicochemically stable humic materials in a high risk arid lands, and humus carbon is self-produced by microbial activity in the soil carbon cycling. The aims of this work are to produce humus carbon (A type humic acid) from model substances of plant residues like sugars, carbohydrate, lignin and cellulose using fresh soil or artificial aggregated soil at low moisture conditions, to estimate change of SOC components with incubated time, and to clarify catalytic reaction of mineral components. Humic substances are capable of bringing about the long-term stabilization of soil aggregates. The build up of SOC levels will not only advantageous to the industry but it will also lead to larger inputs of organic carbon into the soil thus promoting higher steady state levels of SOC.

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The series of activated carbon have been prepared by soaking the coffee pulp with phosphoric acid solution and then pyrolysed at 450-500°C. Different concentrations of phosphoric acid were used to evaluate the adsorption capacity : 10 %, 20 %, 30 %, 40 %, and 50 %. The porous of the obtained activated carbon was evaluated by adsorption of methylene blue at room temperature. The concentration of phosphoric acid influences the pore volume of these activated carbons. Soaking the pulp with phosphoric acid solution 30 % give the optimum condition that give the optimum adsorption capacity.

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Activated carbons were prepared from various food wastes, such as kitchen garbage, lactic acid fermentation residuals and saccharification residuals, by steam-activation of carbonized samples. The food wastes were carbonized at 773 K in inert atmosphere and the carbonized samples were activated by steam. Activated carbons, with enough BET surface areas compared with a commercial activated carbon, were successfully prepared. The effect of the heating rates at the carbonization stage on the porous properties of the resultant activated carbons was investigated by using nitrogen adsorption-desorption method. Activated carbons prepared at a slow heating rate showed high mesoporosity compared with the carbons prepared at a fast heating rate. Moreover, the carbonized wastes were also treated by HCl prior to the steam activation in order to improve the mesoporosity of activated carbons. It was found that the pores of the carbons developed by using the pre-treatment method in the region of the pore radii around 2 nm. The surface hydrophobicity of the activated carbons was also investigated by water vapor adsorption. The liquid-phase adsorption properties of phenol, bisphenol-A, and dye (Black-5) on the obtained activated carbons were examined through the long-period adsorption tests. Some of the activated carbons prepared from saccharification residuals showed higher adsorption capacities for phenol and Black-5 than the commercial carbon.

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It is required to sequestrate CO₂ for mitigating global warming at present. Recently, large-scale desert afforestation has been suggested as a measure of CO₂ fixation. The hydraulic water coefficients, depth of soil, salt concentration of soil and water source of trees were measured at a specific site. As the results of these analyses and measurements, it was found that Eucalyptus camaldulensis has the highest carbon fixation ability of 300kg-Carbon/tree. Thus, it is necessary to predict salt and water movement for E. camaldulensis afforestation. Long-term accumulation predictions have been also reported. However, evaluation of relationship between the prediction in soils and growth year of E. camaldulensis has not been reported yet. From this viewpoint, this paper predicted the water and salt movement in soil using simulation software HYDRUS (U. S. salinity laboratory). Furthermore, long term change in salt concentrations in soil of Leonora area were also predicted using the data measured at the specific site such as hydraulic conductivity, salt concentration, depth of soil, precipitation and evaporation in many precipitation patterns. It was predicted that time of salt accumulations in soil surface without E. camaldulensis was from 50 to 90 years.

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With a view to utilizing Rhizopus oryzae cells immobilized within biomass support particles (BSPs) as a whole-cell biocatalyst for biodiesel fuel production, an investigation was made of the effect of cross-linking treatment with glutaraldehyde on the stability of lipase activity. Although the lipase activity of the BSP-immobilized cells decreased considerably in the presence of the methyl esters produced by methanolysis, the activity of cells treated with 0.1% glutaraldehyde solution showed no significant decrease during twenty batch cycles, with the methyl ester content of the reaction mixture reaching 60-83% in each cycle. We also constructed a novel cell-surface display system using as a new type of cell-wall anchor the 3297-bp of the 3' region of the FLO1 gene, and in this system the N-terminus of the Rhizopus oryzae lipase (ROL) was fused to the FLO1 protein. In the use of Saccharomyces cerevisiae cells displaying FLO1 gene as a whole-cell biocatalyst methyl ester content synthesized reached 78.3% after 72 h. These findings indicate that, given the simplicity of the lipase production process, the use of whole cell biocatalysts using either R. oryzae cells immobilized within BSPs or S. cerevisiae cells displaying ROL offers a promising means of biodiesel fuel production for industrial application.

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Biodiesel fuel (BDF) is a cleaner-burning diesel replacement fuel made from renewable sources such as natural oils and fats. An alkali-catalyzed alcoholysis process, to form fatty acid methyl esters (FAME) from vegetable oils, is widely used to produce BDF, however this process requires high costs for purification of by-product and recovery of catalysts. In this study, a new reactor was developed to produce FAME by blowing superheated methanol gas continuously into oils without using any catalysts. High-oleic sunflower oil and methanol were fed into a reactor vessel, and reacted under various reaction conditions. Effects of reaction temperature, methanol feed flow rate, operating pressure, stirring rate, and initial oil volume were investigated. The reaction product inside the reactor (liquid phase) and the one flowing out from the reactor with methanol gas (gas phase) were collected and their composition was analyzed by using thin layer chromatography / flame ionization detection (TLC/FID) and high performance liquid chromatography (HPLC). It has been confirmed that FAME was contained in both the liquid and gas phases. The total production rate of FAME increased with the reaction temperature, while the outflow rate of FAME into the gas phase was maximum at 290°C. Both the total production and the outflow rate increased with the methanol feed flow rate. The total production rate of FAME was maximum at 1.0 MPa, while the outflow rate of FAME was maximum at 0.1 MPa. Increase in initial oil volume and decrease in stirring rate increased both the total production and the outflow rate. As a result, a gas-liquid interface between methanol bubble and surrounding liquid phase appeared to have large effect on the reaction.

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Two areas of concern to man are pollution control and the discovery of renewable resources. The conversion of unutilized biomass to useful chemicals combines both these items. In this sense, we have advocated the Biomass Refinery Social System. The high temperature compressed water decomposition process we developed so far is an important elemental process of the system, which can be operated without any additional catalyst. In order to the implementation of this process, it is necessary to demonstrate the effective continuous decomposition of biomass under high concentration. In this study, we investigated the chemical decomposition of cellulose in newly manufactured continuous high temperature compressed water reactor (25 mm in diameter; 1,500 mm in length) equipped with screw-feeder to allow the feed of high concentration slurry.The decomposition of microcrystalline cellulose was performed to clarify the effects of cellulose concentration, operation temperature, and reaction time, while the operation pressure was set as 15 MPa. It was found from the results that the cellulose was decomposed to valuable chemicals such as furfural, 5-hydroxymethylfurfural (5-HMF), and sugars (mainly glucose) effectively even in high concentration (10wt%). The maximum yields of furfural, 5-HMF, and sugars in this concentration were 0.02, 0.09 and 0.11 gC/gC-cellulose, respectively. The yields were affected by feed concentration, operation temperature and reaction time within the range of 3 to 10 wt%, 240 to 260 degree C, and 7.5 to 20 minutes, respectively. A numerical description of the decomposition mechanism of cellulose in the reactor was also discussed.

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Hazards evaluation of chemical reaction processes plays an important role in the prevention of serious incidents during scale-up. In this study, a hazards evaluation of the synthetic process for 6-benzyloxyindole is described.
First the synthesis of 4-benzyloxy-2-nitropyrrolidinostyrene intermediate, which has a potential exothermic decomposition hazard, was evaluated. Using the results of the reaction calorimetry data, the details of a reaction mechanism were clarified. The issue of a safety temperature margin was solved by the extension of the reaction time and elimination of the concentration.
Secondly, the synthesis of 6-benzyloxyindole was evaluated. The original procedure was a reduction process using hydrazine and Raney Ni. The result of a reaction calorimetry study indicated that the reaction was very exothermic, with significant heat accumulation after the addition of hydrazine. This procedure had a potential hazard of exceeding the boiling point of the solvent resulting in a runaway reaction. Therefore, an alternative procedure was employed using hydrogen gas under catalytic conditions. The results of the reaction calorimetry study, indicated that the heat flow of the alternative procedure could be controlled by the addition rate of hydrogen gas and agitation speed. For the kinetic evaluation, on-line spectroscopic FT-IR monitoring of both chemical processes was also demonstrated and compared with the two procedures. Both the formation of intermediates and overall reaction behavior were detected through the FT-IR. The two reaction procedures mentioned above showed different kinetic profile of these intermediates formation. The exothermic behavior of both procedures could be attributed to the different rates of formation of these intermediates during the course of reactions.

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To prevent system failure or accidents, it is essential to detect their symptoms in their early stage. In this sense, the role of a safety monitoring system is very important. Further, in the safety monitoring system, two types of failure must be considered: failed-dangerous and failed-safe. The former does not issue alarms when it should, leading to a serious accident, while the latter issues alarms when it should not, causing unnecessary maintenance cost. This paper proposes a general framework for the design of a risk-based safety monitoring system of a dynamical system based on the current monitored data, which minimizes the expected loss caused by failure of the safety monitoring system. The overall system is represented by a dynamic Bayesian network, which can consider both transitions of component states such as one from its normal state to failed state, and cause-effect relations among components. Based on the monitored data, the dynamic Bayesian network updates state probabilities and shows their transition. Using this information, the risk caused by the diagnosis decision can be evaluated to determine the optimal logic structure. An illustrative example of a safety monitoring system of a simple dynamical system shows the details and merits of the proposed method.

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Today's technological systems, such as chemical plants, nuclear power and petroleum are characterized by a high level of complexity which presents the great requirements for the reliability and safety of such systems. In fact, most of companies who are planning or have significant investment in assets can not accept malfunction causing a hazardous situation of system due to safety problems. This paper presents a framework for analyzing and improving the total performance of machinery and equipment in a system from the view point of efficiency and safety. The main idea is to employ Artificial Intelligence technique (AI) and multi-variate analysis to provide insight on impact of safety policy on the system availability. The integrated approach discussed in this paper is based on Total Performance Measures (TPMs) and Probabilistic Safety Analysis (PSA). At first Data Envelopment Analysis (DEA) is utilized to recognize the critical hardware, which has most impact on the total performance. Then, Probabilistic Safety Analysis (PSA) is performed to classify its components based on their importance measures to improve total reliability. Finally, Radial Basis Function (RBF) network is used for safety impact assessment. To achieve the objectives of this study, a comprehensive study was conducted to locate the economic and technical indicators which have great influence on machinery and equipment performance. These indicators are related to machine maintainability, efficiency. Standard factors such as down time, time to repair, mean time between failures, operating time were considered as shaping factors. The results of this approach not only show the weak and strong points of system in regard to machinery and equipment but also facilitate senior management with the expert advice to make better strategies in safety and production management.

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The accident at the Union Carbide Plant in Bhopal on the night of December 2-3, 1984 was the worst accident resulting in the immediate death of approximately 4,000 and the subsequent death, over the years, of another over 16,000 with life long injuries suffered by over 200,000 followed by genetic mutations to affect several generations of offsprings. The pollution of water bodies is affecting several hundred thousand more. No other recorded accident anywhere in the world has produced even 10% of the above casualties and life-long effects.
In this paper, we will briefly describe the accident and the significant effects it has had on regulations, process development and design, training, teaching, RD, etc., all impacting process safety.

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Nippon Mitsubishi Oil Corporation was established in April 1999, by merger of the Nippon Oil Company Limited and Mitsubishi Oil Co. Ltd. and renamed to Nippon Oil Corporation on March 2002.
Nippon Mitsubishi Oil Co. Ltd. worked to increase the operating efficiency of its refining network and created optimum structure and integrated the organizational, personnel and the management system. Two companies had each safety policy and management system, but their systems were slightly different. Therefore two safety management systems were integrated to establish enhanced management system.
Author would like to introduce enhanced safety management systems and the difference of the safety management of each company.
And to show "Safety Management System for the chemical process" should be managed in based of all these facts of operation.

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Singapore's industrial development began in the '60s with the building of several Industrial Estates. Many considered it foolhardy to pin Singapore's future on transforming swamps into an industrial estate. But it succeeded. Now, three decades later, Singapore is writing a new chapter in the industrial development with the creation of offshore Chemical Hub made up of several smaller islands. The Chemical Hub is fast developing into a self-sufficient petrochemical complex with superb infrastructure and all the key support facilities. It was targeted that by 2010, the complex will be able to accommodate 5 crackers and 150 companies with a total estimated fixed asset investment of S$40 billion. The companies on it are and will be linked to one another via a network of pipelines, which will transverse a dedicated piece of land known as a "pipeline service corridor". These pipelines serve as a vital artery, supplying feedstock and products from one plant to another. Companies will benefit from over-the-fence selling of products through this linked petroleum and petrochemical cluster concept. This linked cluster concept is vital for the success of Singapore as a chemicals hub. However, in order for these pipelines to operate safely, it is necessary for consistent and appropriate standards to be set. Thus, a working group, which made up of experts from the petrochemical companies, Singapore's government agencies and statutory boards, developed a code of practice based on best industry practices and the appropriate existing internationally accepted codes and standards.

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In this paper, a novel methodology regarding to batch plant safety analysis is proposed. Batch structures are elaborated and categorized into several safety objects that are linked to phase level operation. Batch model is implemented using ISA S88 that naturally represents object-oriented approach. Safety object performance determines safety condition during batch process, if it works in low performance, initiating event will rising into hazardous outcome due to propagation of fault. In the other hand, measured process variable and its liability are important factors that affect safety object performance. To implement safety object performance, raw data is analyzed using fuzzy system approach that starting from knowledge acquisition, connection and mapping the sensor data. From the output of the safety object performance, reasoning mechanism for specific phase level operation can be implemented. The output of safety analysis using fault propagation can be considered as performance index that represents the success or failure of the systems. This research gives significance contribution to improve the batch plant safety with integrates safety management system and information technology. For case study, the polyvinyl chloride (PVC) batch plant that process hazardous material vinyl chloride monomer (VCM) is taken for consideration.

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The approach we present in this paper is to assist the engineer in tackling the HAZOP wicked problem with software wizards that implement a number of functions such as providing to do lists for plant sections, lines and major vessels to be analyzed; recording pros and cons of proposed preventive strategies; displaying sections of the plant with similar pressure values; finding information on previously carried out tests that relate to the consequences of the abnormal situation under study; capturing design rationale. This paper discusses approaches to be implemented by the wizards.

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HAZOP (Hazard and Operability Studies) is one of the safety analysis techniques most widely used in the process industries. Typically, in a HAZOP studies a team of specialists examines the P&IDs of the plant apply a series of guidewords to every pipeline, identify possible hazardous consequences and propose preventive strategies. A number of tools are available in the market to support the documentation of the HAZOP sessions. However, the information stored in these tools is in the form of textual natural language descriptions that limit computer-based extraction of knowledge for the reuse of the HAZOP analyses in other designs or during plant operation. To overcome the limitations of text-based descriptions, a HAZOP ontology has been proposed that provides a basic set of standard concepts and terms The development of the ontology uses an upper ontology based on ISO-15926, that define general-purpose terms and act as a foundation for more specific domains. The ontology is developed so that engineers can build new concepts out from the basic set of concepts. This paper evaluates the proposed ontology by means of use cases that measure the performance in finding relevant information used and produced during the safety analyses. In particular, the extraction of knowledge is performed using JTP (An object oriented Modular Reasoning System) that is used for querying the ontology.

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HAZOP studies are widely used for identifying hazard and operability problems during plant design. However, HAZOP studies are time consuming, labour intensive and expensive. Therefore, major research projects have been carried out to develop systems to automate the HAZOP technique. The basic technology used in the developed systems is built around the idea of fault propagation using signed-directed graph, together with a range of information to capture the possible failures of the equipment, and the susceptibility of the equipment to deviations propagated from elsewhere. A lot of progress has been made in the implementation of the basic fault propagation system and development of signed-directed graphs for basic plant models. However, the limitations of the prototypes resulting from different research groups are twofold. First, they are standalone systems that cannot work with commercially available process design software. Second, they lack facilities for reviewing the results generated automatically by the hazard identification systems. This paper presents a set of novel tools that overcome the identified limitations. These tools include a "data wizard" for extracting data from a process design system and converting the data into a format suitable for the hazard identification software, a semi-automatic tool for mapping the extracted plant items to the available signed-directed graph models and an interactive tool for selectively viewing the HAZOP results in different ways and to provide explanation on how an identified cause reaches an identified consequence.

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Chemical industry's accidents always have a risk to cause serious hazards, such as explosion, fire disaster, leak and spill, which may involve many residents. In terms of sharing such risk information, the U.S. and Europe have developed various systems to promote dialogue between citizens and industries. In Japan, however, in order to build public acceptance on chemical industry's safety, Japanese style systems are required getting along with our unique cultural background. In this report, we examine two systems to form public acceptance: an interactive information system over the Internet between citizens and a company, and a perceptual communication system, e.g. direct talks. Finally, we suggest how to adjust their problems toward a more efficient system.

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Advances in science and technology have improved our well-being and increased our convenience in daily life. However, with this progress have come many problems which shake the foundation of our trust in science and technology. To solve these problems RISTEX(Research Institute of Science and Technology for Society) have established.
With other 10 Mission-Oriented Research Group, "Chemical Process Safety Research Group" have started research works for finding solutions to social issues.
The group is working on research aimed at a safety consensus-building system as the means to achieve this goal. Proto-type model of the system has completed and now is in the stage of application to get more comments for the improvement of the system.

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Process safety issues are not only for the inside of company but also for its compliance and corporate social responsibility. The process industry has spent an enormous amount of its resources to operator training and maintenance work and has taken its responsibility of executing process safety management (PSM). As most of PSM in Japan have been provided on the basis of experiences, individual sub-tasks in responsible PSM have been separately executed. It is very difficult and costly to carry out the PSM with information integrity. In order to plan and execute a reliable and feasible PSM, the process industry has to carefully analyze the social requirements of health, safety, environment and sustainability in terms of plant lifecycle engineering, risk management, personnel task, and cost and decide an executable PSM. This paper proposes a responsible framework of PSM based on safety-conscious plant lifecycle engineering, and a technological information infrastructure (TII) to support its execution.

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In cell fusion and genetic recombination, although the activity of single cell is extremely important, there is no analyzing method of single cell activity. Development of quick analyzing method of single cell activity is desired in various fields.
Dielectrophoresis (DEP) refers to the force exerted on the induced dipole moment of an uncharged dielectric and/or conductive particle by a non-uniform electric field. By applying DEP, we obtained experimentally relationship between the cell activity and the dielectric property Re [K (ω)], and examined how to evaluate the single cell activity by measuring of Re [K (ω)] of single cell. A cone and plate electrode geometry was adapted in order to achieve the feedback-controlled DEP levitation. The single cell is exposed to a non-uniform field induced by the cone and plate electrode, and more polarizable cell moves to the direction of the cone electrode by the DEP force. The cell settles in the position where the DEP force and the gravity are balanced by controlling applied voltage. This settled position where measured on the center axis of the cone electrode depended on the dielectric constant of the cell. From these results, the relationship between the specific growth rates in cell growth phase and the dielectric property Re [K (ω)] was obtained. Furthermore, the effect of various stresses to cell such as concentration of carbon dioxide, temperature etc. on the cell activity was examined.

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For effective screening by biopanning, we propose a new affinity screening method utilizing protein-coupled liposomes (proteoliposomes) as adsorbents. With multilamellar vesicles (MLVs) composed of DPPC : DCP = 10 : 1 (molar ratio), adsorption of nonspecific phage VCSM13 to the liposomes without any blocking was comparable to that on polystyrene tube wall coated with blocking protein. Phages displaying octapeptides specific to an anti-peptide antibody against a peptide antigen (FVNQHLCK) were screened from an octapeptide-displayed phage library by biopanning utilizing liposomes coupled with the antibody (AB-MLVs) or a conventional immunotube coated with the antibody (AB-tube). After 4 rounds of biopanning, all selected phages displayed homological peptides to the antigen peptide by use of AB-MLVs, while only 15 % of the selected phages displayed homological peptides in the conventional biopanning. Thus, protein-coupled liposomes are useful as adsorbents for screening from combinatorial phage libraries.

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We here report a newly developed spheroid microarray technique in which rat hepatocytes form almost the same diameter of spherical multicellular aggregates (spheroids) and are immobilized at high density on a spheroid microarray (SM) chip. The SM chip consisted of 2,800 cavities of 300 µm diameter in triangle arrangement within a 16 mm square on a polystyrene plate. Hepatocytes formed one spheroid in each cavity, in which the spheroid diameter can be controlled by inoculated cell densities. The spheroids stably maintained ammonia removal and albumin secretion, which are markers of detoxification and protein synthesis of liver, for at least 14 days of culture on the SM chip, compared with hepatocytes that had a spreading cellular shape and lost their functions in conventional monolayer culture on a collagen-coated surface. The activation of enzyme P-450 depending on the concentrations of 3-methylcholanthrene, a carcinogen, was detected in the spheroids on the chip by fluorescence of resorufin converted from ethoxyresorufin. Thus cell responses could be evaluated on the chip by using the fluorescent technique. These results suggest that a spheroid microarray on the chip is a promising technique for high-throughput cell-based analysis.

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E.coli alkaline phosphatase (PhoA) is a thermally stable homodimeric metalloenzyme with resistance to many proteases. In previous studies, several loops in PhoA were known to permit peptide insertion without destroying its structure as well as the enzymatic activity. Especially, an epitope insertion near the active site (407-408) allowed modulation of enzymatic activity upon binding with corresponding antibody. Based on this observation, we decided to make a novel 'allosteric' enzyme that can accommodate desired receptor domains, whose activity can be controlled by the binding of its ligand as a molecular biosensor. To this end, construction of circularly permutated PhoAs having new N- and C-terminus at permissive loops (91-93/407-408) near the active center was attempted, with the original termini connected by (G₄S)₃ peptide linker. When the DNA encoding these circular permutants were inserted to pET20b plasmid and expressed in E. coli BL21(DE3,pLysS), an active enzyme was obtained only for the former having new C-terminus at aa 90 of the wild-type PhoA (cp90). Then an epitope of osteocalcin, an important marker of bone metabolism, was inserted to the new N-terminus of cp90, with its original 91 and 93th residues randomized to obtain optimal variants. Interestingly, some resultant fusion proteins showed similar specific activity to that of wild-type PhoA, and showed significant reduction in V_max/Km in the presence of anti-osteocalcin antibody. The result indicates that the epitope-tagged cp90 can act as a molecular sensor to detect specific antibodies, which will enable competitive detection of specific antigen epitopes

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We developed a novel selective cell-separation method based on using a poly(N-isopropylacrylamide)-graft-polypropylene (PNIPAAm-g-PP) membrane containing adsorbed monoclonal antibody specific to the target cell. This membrane was prepared by plasma-induced polymerization and soaking in an antibody solution at 37°C. Poly(propylene) has many advantages as a filter material, such as large specific area, chemical and physical stability, good resistance to biodegradation, and low cost. Poly(N-isopropylacrylamide) has a thermoresponsive phase transition: at 32°C water-insoluble (hydrophobic) and water-soluble (hydrophilic) states interconvert. Adsorption of antibody onto PNIPAAm-g-PP membrane at 37°C and its desorption at 4°C was verified by fluorescence-microscopy of the PNIPAAm-g-PP membrane after soaking it in fluorescein-conjugated goat anti-mouse IgG in phosphate-buffered saline. PNIPAAm-g-PP membranes containing adsorbed anti-mouse CD80 monoclonal antibody preferentially captured mouse-CD80 transfected cells at 37°C compared with membranes lacking antibody or containing anti-mouse CD86 monoclonal antibody. Detachment of captured cells from PNIPAAm-g-PP membranes was facilitated by washing at 4°C because of the thermoresponsive phase transition of PNIPAAm. With this method, mouse CD80- or mouse CD86-transfected cells were enriched from a 1:1 cell suspension to 72% or 66%, simply and with high yield.

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We developed a photoresponsive culture surface (PRCS), of which the cell adhesiveness is controlled with light irradiation. A photoresponsive copolymer p(MMA-NSP) was synthesized from methylmethacrylate and methacrylamide monomer containing a nitrospirobenzopyran chromophore by random radical copolymerization. PRCS was prepared by spin-coating a dilute 1,2-dichloroethane solution containing MMA-NSP and poly(ethylene glycol) onto flat glass plates. 5 minutes after disseminating Balb/3T3 cells on the PCRS, UV light (wavelength 365 nm; intensity 35 mW cm²) was irradiated upon a part of PCRS for 5 min. 12 hours later, many cells were observed to adhere to the irradiated region of the PRCS surface, but very few did so in the unirradiated region. After washed with the phosphate buffer saline, it was observed that the density of remaining cells in the irradiated region was twice as much as that in the unirradiated region. These experimental results indicated that the adhesiveness of PRCS to cells can be enhanced considerably by UV irradiation even after seeding cells. It was suggested strongly that the PRCS we developed in this study should provide a novel and useful method for cell manipulation, cell separation and cell-culture patterning.

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The yeast Rhodotorula glutinis was examined for its ability to remove heavy metals from aqueous solution. Among the heavy metals tested, selectivity of lead ion was very high. Langmuir sorption model provided a good fit throughout the concentration range. The maximum Pb²⁺ sorption capacity (q_max) and Langmuir constant (b) were 73.5 mg/g of biomass and 0.02 l/mg, respectively. This result showed that the biosorption of Pb²⁺ by R. glutinis could be characterized as a monolayer, single site type phenomenon with no interaction between ions adsorbed in neighboring sites. The pseudo second-order equation was well fitted to the experimental data. The correlation coefficients for the linear plots of t/q against t for the second-order equation are 0.999 for all the initial concentrations of biosorbent for contact times of 180 min. The theoretical q_eq value was very close to the experimental q_eq value. The titration data shows that the cell surface of R. glutinis biomass has a variety of cation binding functional group such as carboxyl, phosphoryl, and amine functional groups.

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We report a novel method to prepare nano scale vesicles with high encapsulation efficiency of target nucleotides. First, DNA was extracted from an aqueous solution into a cationic reverse micellar solution consisting of dimethyldistearylammonium bromide (2C18AB). Secondly, small amount of the reverse micellar solution containing DNA was dropped into an aqueous surfactant solution to form the outer-layer of vesicles. We mainly employed polyoxyethylene (20) sorbitan monolaurate (Tween 20) or cetyltrimethylammonium bromide (CTAB) as an outer-layer surfactant. Salt concentration of the aqueous solution affects the solubility of the organic solution droplets into the aqueous surfactant solution. At high NaCl concentration, over 60 % of extracted DNA was re-dispersed in the aqueous solution, while DNA was hardly dispersed in the solution without NaCl. Characterization of the resulting surfactant-DNA complexes was carried out by dynamic light scattering (DLS), and which showed that the average diameters were between 200-300 nm. This technique facilitates high encapsulation efficiency of DNA molecules into liposome, and the resultant lipid-DNA complex is nano-scale gene vector.

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In the present work, we investigated the effects of GC content, mismatching position, and length on the thermostability of double-strand oligo-DNAs in order to develop an affinity chromatography using oligonucleotide-immobilized capillary for SNP detection. We found that a single mismatching base pair appeared on 25 mer fragment of human tp53 tumor suppressor protein gene could reduce the melting temperature by 6.6°C. It seemed to be enough for eluting the mutated fragment separately from the intact one during a course of temperature ascending process.

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Hepatocyte transplantation using porous scaffolds is a potential therapy for both acute and chronic hepatic insufficiency and also for treatment of inborn errors of metabolism affecting liver. However, some authors reported that the hepatocytes located in the center of the scaffold died after transplantation due to insufficient oxygenation to the cells. No reports have been sufficiently addressed to resolve this problem. In this study, we fabricated a scaffold from poly (lacide acid) (PLA) coated with acidic gelatin. Basic fibroblast growth factor (bFGF) adsorbed onto the gelatin, would be released continuously and could induce angiogenesis in the scaffold in vivo. A combination of thickness and cell density in the scaffold was optimized so as to suppress the hepatocytes death in the center of the scaffold in the first stage of implantation. Further, a new centrifugal cell seeding method was developed to immobilize hepatocytes uniformly within the scaffold. The hepatocytes immobilized by the centrifugal cell seeding method in the bFGF-containing scaffold at the cell density optimized were implanted into mesentery of rat received a 70% hepatectomy. From observation of histological section of the scaffold after one week of implantation, it was confirmed that the living hepatocytes were uniformly distributed throughout the section, and exhibited PAS-positive indicating that they had a potential to store glycogen. This result indicates that the technique developed in this study was available for hepatocyte transplantation using a scaffold.

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A biological membrane is the frontier of the cells against the environmental reactive oxygen species (ROS) and is expected to play an important role in the antioxidant system corporately with antioxidant enzymes. The response behaviors of the antioxidant enzymes (superoxide dimutase (SOD) and catalase (CAT)) were investigated in the presence of artificial membrane, liposome, under the oxidative stress condition in order to obtain the fundamental information for the ROS scavenger system. The intracellular antioxidant enzymes, SOD and CAT, are known to make protective system against ROS. Although the SOD is known to catalyze the dismutation of ·O₂- to H₂O₂. The activity of SOD was lost thoroughly in the presence of H₂O₂ at high concentration (>5mM). The SDS-PAGE analysis showed that the SOD was fragmented into several small peptides. SOD has two -helix loops, which introduce the substrate ·O₂- to the activity center of SOD (Cu(II)). From the circular dichroism analysis of SOD in the presence of H₂O₂, the contents of α-helix in the SOD were found to decrease in corresponding with the inactivation of the SOD, suggesting that the conformation of the α-helix loops might be varied. However, the SOD was not inactivated in the presence of POPC liposomes while the contents of α-helix were decreased similarly in the case of that without liposome. Based on these results, the possibility to develop the ROS scavenger system using the SOD and CAT together with liposome was finally discussed.

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Chromatography is used not only for high performance analysis but also for process scale purification of biomolecules. It is known that when a small difference in similar molecules is well recognized by chromatography stationary phase (ligand) a good separation performance can be obtained. Often intended and unintended conformational changes of biomolecules occur before or during chromatography. The former is pretreatment of DNA samples by heating for DNA chromatography. The latter includes denaturation of biomolecules due to adsorption. Other conformational changes during chromatography are refolding of (denatured) proteins which is called "solid-phase refolding" and PEGylation of proteins on chromatography (selective PEGylation and separation). In this study biorecognition and transport phenomena for during chromatography was investigated by using a model developed for ion-exchange chromatography. The samples employed are DNAs, denatured proteins and PEGylated proteins. The information on the number of bindings sites was carefully investigated along with the data obtained by separate experiments (Taylor method, melting-curve analysis, etc.).

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Candida antarctica lipase B (CALB) and Thermomyces lanuginosa lipase (TLL) encapsulated in reverse micelles of sodium bis(2-ethylhexyl) phosphate (NaDEHP) were used to investigate the role of different enzymes, effects of water-to-surfactant molar ratio (Wo) and fatty acids (caprylic acid and oleic acid) in catalytic activity of esterification of fatty with hexanol. The reverse micellar system consisted of NaDEHP, hexanol, fatty acid in isooctane and 1.0M NaCl aqueous system. In the NaDEHP reverse micellar system, the optimum Wo of CALB was 6.67 and 7.08 for oleic acid and caprylic acid, respectively, while the optimum Wo of TLL was 8.44 for oleic acid and 9.12 for caprylic acid, respectively. The water-to-surfactant molar ratio, type and concentration of substrate have strong effects to the specific activity of both lipases and the conversion of the reactions. Both CALB and TLL showed the selectivity regarding to long chain fatty acid, however, encapsulated CALB gave higher specific activity and conversion than the encapsulated TLL.

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The effect of process parameters involved in predispersed solvent extraction (PDSE) on the stability of colloidal liquid aphrons (CLAs) was investigated. Tertiary amine, trioctylamine (TOA) was used as an extractant of succinic acid and 1-octanol was used as diluent for lowering toxicity and viscosity of TOA. CLAs were formulated with an TOA/1-octanol/Tergitol-15-S-3 solvent phase and Succinic acid/Sodium dodecylbenzene sulfonate (SDBS) aqueous phase. Visual observation and light scattering measurements by UV/spectrophotometer were used as a method of obtaining the breakup speed of CLAs. The influence of pH, solvent composition, ion species were investigated based on these measurements. At low pH values, excess hydrogen ions in aqueous phase leaded to protonation of the head-group of the water-soluble surfactant located on the outer soapy shell of CLAs and lowered the stability. As the concentration of TOA increased, the stability of CLAs decreased because of the increasing basicity of aphron core. The stability of CLAs displays a strong dependence on ionic strength of aqueous phase because the energy gap between continuous phase and bilayer of surfactant at outer soapy shell decreases with the contained ion concentration.

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Active β1,3-N-acetylglucosaminyltransferase 2 (β3GnT2) was produced in the baculovirus expression system (BES) and in stably transformed insect Tn-5B1-4 cells. β3GnT2 was expressed as a secreted fusion protein with GFP_uv with prepromelittin signal sequence from honeybee. The β3GnT activities of the culture supernatant in BES (Tn-5B1-4 cells) without or with the addition of the protease inhibitor, leupeptin, were 0.68 and 2.01 mU/ml, respectively. The stably transformed Tn-5B1-4 cells (Tn-pXme11) exhibited a β3GnT activity of 6.83 mU/ml, which was 3.4-fold higher than that observed for BES with the leupeptin addition. The purity of fusion protein purified from the culture supernatant of the Tn-pXme11 was higher than 95% in contrast with that purified from the culture supernatant of the baculovirus-infected cells which contained low-molecular-weight fragments of the fusion protein. The stably transformed cell line is more suitable than BES for the efficient production of the secretory protein, β3GnT2.

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We propose a method derived from the traditional Japanese art form of "Origami" (paper folding) to produce entire organs to be utilized for transplantation, such as in kidney and heart transplants. By considering how the 3D structure is formed by bending and folding, we designed a two-dimensional surface diagram made by unfolding and unbending the 3D structure. According to this surface diagram and folding process, a branch structure, capillary networks and a vascular construct were successfully prepared by combination of the technology of cross-linked gel with transglutaminase, Biocompatible Anchor for Membrane (BAM) and microfabrication technique. This novel method will be widely applied for making more complex 3D structures which can not be created by the previous method like simple layering of cell sheets.

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A biodegradable PLA rod device (1mm in diameter x 3-4mm in length) has been developed for implantable drug delivery systems. Indometacin, an NSAID, incorporated in the PLA rod was released in a bi-phasic manner: the initial bursting due to surface drug dissolution and the second bursting caused by the polymer bulk erosion. The ultrasound (2W/cm², 1MHz) was applied for 30 minutes following the onset of the drug release experiment. The ultrasonic energy significantly enhanced the drug release rate during the application period. After the removal of the ultrasonic energy, however, the enhanced drug release was not appeared and returned to the level under passive transport. This finding may suggest that the drug release rate from the PLA rod implant can be dynamically controlled by the ultrasound application.

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Gene amplification has been developed as an indispensable technique for the bio-pharmaceutical production by recombinant mammalian cells. The most commonly used technique is likely dihydroforate reductase (dhfr) gene amplification system in Chinese hamster ovary (CHO) cell line. In this gene amplification system, high productive CHO cells can be selected by "time-consuming" stepwise increases of methotrexiate (MTX) concentration in culture medium. However, it is of great significance to shorten the time for constructing the stable and productive recombinant CHO cell line in the industrial process. In this study, we focused on a rapid construction method for gene-amplified cell line. We employed the human granulocyte-macrophage colony stimulating factor (hGM-CSF) as a model of glycoprotein and investigated the relationship among the productivity and stability of amplified gene, the location of the amplified gene and the MTX concentration. The distribution of amplified gene on the chromosome was analyzed using fluorescence in situ hybridization (FISH). Based on the FISH image analysis, we speculated the specific chromosome location suitable for gene amplification.

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An artificial gill, which transfers oxygen from water to air, driven by photo energy, was developed with dissolved Mo^IVO(tmp) in o-xylene as an oxygen carrier solution. The oxygen affinity of oxygen carrier solution varies with photo-irradiation. The ratio of the mass transfer coefficient of the oxygen carrier solution to that of the water was 0.746 in oxygen uptake and 0.654 in oxygen release, respectively. The scaling up for a human at rest was estimated with the ratio of mass transfer coefficient. In designing a plant type artificial gill, which is for supplying oxygen to a closed space underwater such as submerged vessel, the required membrane surface area, the seawater flow rate and the reservoir tank volume were 123 m², 0.00533 m³/s, and 5.06 m³, respectively. These values increased as oxygen partial pressure of seawater decreased. However, this artificial gill enables oxygen uptake even at low oxygen partial pressure of water such as 10.0 kPa, and may be useful for human underwater activity.

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The effect of Zn²⁺ to β₂-adrenergic receptor (β₂AR) is experimented in the solution containing Bronchodilator. From the immunohistochemical detection of β₂AR, the result shows the influence of Zn²⁺ on drug-receptor interaction in the Zn²⁺ containing solution on the human bronchial-epithelial cells. G protein-coupled receptors (GPCRs) are the membrane proteins which transmit extracellular signals into intercellular domain by binding of ligands. Artificial ligands are used as drugs and about 50% of current drugs targets GPCRs. Therefore, it is important to investigate the agents which are bound by GPCRs and the influence by the change of conditions on the binding. The effective ligands of GPCRs for the drugs are indicated less side effect. β₂AR is a kind of GPCRs, which has close relationship with the treatment of asthma. The bronchodilators are the ligands of β₂AR, which activate β₂AR and open the bronchi. However, one of disadvantages of the bronchodilators is that the bronchodilators act on the other receptor, β₁-adrenergic receptor, and can cause the serious heart troubles. Various factors have an influence on the interaction of β₂AR with drugs. Therefore, the experiments under the condition that drugs act on more selectively and effectively is valuable for the safe treatment.

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It has been realized that nitric oxide (NO) plays several regulatory roles in vivo. Despite very beneficial effects, NO has also been implicated as a deleterious agent in various pathophysiological conditions. In this study, we estimated immune response by using CD4/CD8 for NHCEC with added nitrate or nitrite ions. The growth medium of NHCEC was prepared by combining one part of Dullbecco-modified Eagle's medium and one part of Ham's nutrient mixture. The medium was further supplemented with cholera toxin, insulin, transferrin, epidermal growth factor, hydrocortisone, penicillin, streptomycin, gentamicin, HEPES and fetal bovine serum 10%FBS. From this three cultures were made. To the first, nitric acid was added. To the second, nitrous acid was added. The third was a control. The NHCEC's suspension was placed into centrifuged tubes and FITC-attached anti-CD4 antibodies and R-PE-attached anti-CD8 antibodies were added. The tubes were incubated for 30 minutes on ice in a dark room. The samples were washed once in phosphate buffered saline (PBS). The cell sediment was resuspended in paraformaldehyde in PBS. And then an immune response to NHCEC was estimated by comparing the fluorescence area ratio CD4/CD8. We could find the effect of nitrate ion and nitrite ion by CD4/CD8. This study is a first step in the investigation immune response using the fluorescent antibody method.

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The respiratory flow-pattern at the human oral airways was examined for various inhalation flow rates, since it is important for the inhalation therapy of oral diseases to estimate particle deposition. The airflow pattern for the inhalation flow rate of 9, 25, 50 l/min was analyzed numerically with 3-D reconstruction of the human oral airways. The human oral airways model was obtained from the images of Magnetic Resonance Imaging (MRI) analyzed by Rhinoceros, which has 3-D CAD modeling function. Numerical analysis of the airflow pattern in this human oral airways model has been performed by the thermodynamic fluid analysis code STAR-CD combined with the mesh generation function of Pro-Modeler. 52 MRI images were taken vertically at intervals of 2mm on the oral cavity, and 52 MRI images were also taken horizontally at intervals of 2mm on the pharynx and larynx, then outlines of airways were extracted. The cross-sectional data were inputted into the Rhinoceros and then arranged in Cartesian coordinate system. In those manners, the 3-D model of the human oral airway was obtained. The numerical results showed that vortex occurred near the larynx for each inhalation flow rate. Furthermore, the airflow pattern was relatively uniform in the oral cavity and the upper side of trachea. Consequently, it was suggested that the airflow pattern in the human oral airways was not changed qualitatively for the inhalation flow rate of 9, 25, 50 l/min.

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Although many bio-active proteins and vectors for gene therapy are produced by mammalian cell culture, its efficiency should be improved. Culture media are very expensive because serum or its derivatives are essential for them, and so there is concern about the risk such as BSE. Therefore inexpensive, safe and potent mitogenic supplement for mammalian cell culture is eagerly desired. In this study, sericin, a protein derived from silkworm, was considered as such a factor. In the presence of sericin, the proliferation of all mammalian cell lines tested were accelerated. In comparison with bovine serum albumin, a widely used supplement in serum-free medium, sericin had better effect on the proliferation of human hepatoma HepG2. Although most of proteins are easily inactivated by autoclave sterilization, mitogenic activity of sericin was not damaged after autoclave. As well as sericin derived from silkworm, recombinant sericin peptide synthesized by E.coli stimulated the proliferation of mammalian cells. In cell therapy or bio-artificial organs, stable storage and supply of the cells are crucial. Therefore the effect of sericin on cryopreservation was tested. Sericin was added to the freezing medium and HepG2 cells, which several groups have used in bio-artificial liver, were frozen at -80°C for three days. Cells were thawed out, cultured for one day and the viable cell number was determined. Supplementation of sericin improved the viable cell number by 27%. These results indicate that sericin is a novel and suitable mitogenic supplement for mammalian cell culture as well as supplement for cyropreservation.

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The expansionable human hepatoma cell lines including HepG2 have potential for use in bio-artificial liver (BAL) systems for liver disease due to the shortage of donation. However, at present, BALs are incomplete and the functions need to be improved or at least maintained for a longer period. In this study, we aimed to establish novel hepatoma cell lines for a longer-term or permanent artificial livers. For this purpose, anti-apoptosis genes such as bcl-2, p35 and crmA were introduced into HepG2 cells. Over-expression of any of these genes significantly inhibited apoptosis. For example, after exposure to 51°C for 10 minutes, the viability of bcl-2, p35 and crmA transfectants were 8%, 27% and 16%, respectively, while that of wild type was 3%. Over-expression of anti-apoptosis gene prolonged the period of the stationary phase in the growth curve and did not affect the growth rate during the exponential phase. To test the liver function, cytochrome P-450 1A1 activity of transfectants were measureed. After induction with 3-methylcholanthrene, the cytochrome activity of bcl-2, p35 and crmA transfectants were 8.7, 9.4 and 7.7 fmol/cell/hour, respectively, while that of wild type was 7.0 fmol/cell/hour. Introduction of anti-apoptosis genes would be effective for the generation of novel hepatoma cell lines for artificial livers.

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Lipid bilayer permeability coefficient of benzoil-tyrosin-p-nitroanilide (Bz-Tyr-pNA) was evaluated using large unilamellar vesicles prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine) and cholesterol containing the entrapped α-chymotrypsin. Alpha-chymotrypsin-catalyzed reactions occurred inside the vesicles after external addition of Bz-Tyr-pNA as a bilayer permeable substrate. From such kinetic measurements, the permeability coefficient could directly be determined by dynamic modeling. Using this method, the permeability coefficient can be determined accurately within 9% deviation. The effects of temperature and cholesterol content on the permeability were examined quantitatively. The permeability increased with temperature in the absence or presence of cholesterol. In the presence of cholesterol, the permeability was highest at 10 mol%. These changes of the permeability coefficient were discussed in relation to the lipid bilayer fluidity measured by steady-state fluorescent anisotropy.

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INTRODUCTION: Dialyzer apparatus have widely been used as an artificial kidney in medical treatment. However, some side effects as amyloidosis and so on have been occurred during the long term treatment. Therefore, we forcused on development of a hybrid artificial kidney with not only filtration but also reabsorption apparatus, but extensive spreading is easily occurred and difficult to maintain uniform monolayer state on collagen coated substrate. The purpose of this study is improvement of the cell adhesion, uniform stable monolayer formation and active transport function by immobilization of RGD on the culture substratum.MATERIALS AND METHODS: Polycarbonate semipermeable membrane separating culture well was coated by collagen, fibronectin, laminin and synthetic polypeptide including RGD (PnF). Cell adhesive and digoxin transport activities were estimated using renal proximal tubule cell line overexpressed p-glycoprotein gene.RESULTS AND DISCUSSION: Immobilized cell densities at initial and confluent conditions on PnF-coated membrane were higher than those on the other conditions in culture well. Transepithelial electrical resistance and digoxin transport activity on PnF coated membrane were the highest of all conditions. This might be caused by uniform cell morphology and high cell density.

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Microcapsules with a range in 500-1000 µm in diameter have been used for mammalian cell-enclosing which have a potency to treat a wide range of diseases. Reduction in capsule diameter presents many advantages, including better cell oxygenation, higher mechanical strength, higher biocompatibility, smaller total implant volume, and accessibility to new implantation sites. The aim of the present study was to develop smaller mammalian cell-enclosing capsules with narrow distribution in size than those have been reported previously., i.e., less than 200 µm in diameter. The diameter of agarose capsules prepared in an immiscible coflowing liquid with laminar flow decreased with increasing the velocity of the ambient fluid flow and decreasing that of a agarose aqueous solution extruded from a nozzle with 300 µm in diameter. The mammalian cell-enclosing capsules of 76 ± 9 µm in diameter, i.e., subsieve-sized capsules, with narrow distribution in size were obtained by extruding the agarose solution at a velocity of 1.2 cm/sec into the ambient liquid paraffin flowing at a velocity of 20.8 cm/sec. The viability measurement based on the percentage of intact mitochondrial system in cell demonstrated that the reduction in diameter from 351 µm to 79.2 µm scarcely hindered the viability of the enclosed cells.

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The roles of metal ions both on the formation of fibril aggregation of amyloid-β-peptide (Aβ) and on the generation of superoxide by a metal-Aβ complex have been studied to clarify the relationship of both phenomena with the cause of Alzheimer's Disease (AD). Among the several metal ions, the addition of Zn(II) or of Cu(II) was found to increase the formation of Aβ fibril. Although Zn(II) promoted the Aβ fibril formation both at neutral and acidic pH levels, Cu(II) could induce the aggregation of Aβ only at acidic pH. In addition, hydrogen peroxide was generated at neutral pH in the absence of Aβ and Cu(II) beyond a stoichiometric amount of Cu(II) ion, suggesting the formation of a catalytic site that can react between Cu(II) and Cu(I). These results indicate that Zn(II) and Cu(II) at different pHs may each contribute to AD in different modes; the promotion of fibril aggregation (Zn(II) at every pH and Cu(II) at acidic pH) and the formation of superoxide (Cu(II) at neutral pH). The most characteristic aspect is the effect of pH on the promotion of aggregation and the formation of superoxide for the Cu(II)-Aβ system. The coordination structure of the Cu(II)-Aβ complex at neutral and acidic pHs was analyzed by using NMR spectroscopy, circular dichroism (CD), and ESR. The formation of a complex of histidine (His) residues and Cu(II) was found, and the coordination number of the His was suggested to be two at neutral pH and three at acidic pH. These results imply that the intramolecular coordination structure of the Aβ -Cu(II) complex at neutral pH could be converted to the intermolecular one by acidification. The pH variation thus led to a change in the complex structure of Cu(II)-Aβ. This finding suggests the important roles of pH to regulate two phenomena, i.e. the aggregation of Aβ (inhibition of superoxide formation) and the formation of superoxide from Cu(II)-Aβ complex.

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To make cultured cells express highly differentiated functions for a long time, a novel culture technique that cultured cells form a multicellular aggregate with tissue-like structure (organoid) has much attentions. It is important to control the thickness of the organoid within 100 µm to prevent growth of dead cells layer caused by oxygen exhaustion. Previously, we developed a culture technique to form a hepatocyte organoids with spherical (spheroid) and cylindrical shape (cylindroid) performed highly liver specific functions for development of hybrid artificial liver. In this study, we have developed a novel method to form an organoid having sheet shape (organoid-sheet). Hepatocytes were inoculated inside gap between parallel positioned porous membranes with spacer by centrifugation, and an organoid-sheet was formed with homogenous thickness and high cell density distribution. Using 150 µm thickness spacer, about 30-40 µm thickness dead cells layer appeared inside the organoid-sheet. On the other hand, using 100-µm thickness spacer, such a layer did not appear. Hepatocyte organoid-sheet using 100 µm thickness spacer could express liver specific functions such as ammonia metabolism and albumin secretion about 1.5 times higher than those of cylindroid and maintained them for at least 1 month. Moreover, we found myocardial cells also formed organoid-sheet, and it expressed spontaneous synchronized contraction to the whole sheet area at least 4 days. These results suggest that using organoid-sheet is expected to applying hybrid artificial organs and patches for graft.

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We will report a targeting and an anti-cancer effect of a lipid vesicle, to which Euchuma Serra Agglutinin (ESA) immobilized, to human colon cancer cell (Colo201) in mouse xenopathic model. The lipid vesicles were mainly composed of Span80 (sorbitan monooleate). First, the specific binding between the lipid vesicle and the cancer cell was studied for the application of the vesicle to DDS. It was preliminary confirmed that the vesicle flew smoothly in superior artery of a rabbit. As a targeting ligand (combined with the vesicle) at a Colo201, Euchuma Serra Agglutinin (ESA) was chosen. The ESA is a new lectin derived from a marine red alga and specifically combines with mannose-rich carbohydrate-chain, which often exists on the surface of cancer cells abundantly. ESA was found to specifically combine with many cancer cells, especially to colon adenocarcinoma (Colo201) and induce an apoptosis to the cancer cell. Secondly, the in vivo experiments toward to colon cancer therapy were carried out administrating the ESA-immobilized vesicles into nude mice in which human colon cancer-cell Colo201 had been implanted. Preliminary autoradiogram experiments showed the free ESA selectively accumulated in the tumor of the mice. Then, we prepared either ESA-immobilized vesicle or the vesicle entrapping anti-tumor drug (Taxotere) for using colon-cancer xenopathic model in nude mouse. Autoradiograms also showed the ESA-immobilized vesicles were specifically accumulated into the tumor rather than the other organ. The most effective anti-tumor effect was observed in using the ESA-immobilized vesicle entrapping Taxotere.

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