Earticle

Surface modification of liposomes with polyethylene glycol (PEG) is a common method to improve their circulation time in vivo. However, the pegylation of liposomes generally suffers from the significant decrease of cellular uptake by hydrophilic PEG components, requiring the use of active targeting moieties in the liposomes. To enhance the distribution of the pegylated liposomes to the target tissues, and to improve the uptake of liposomes by the target cells, we screened targeting moieties to tumor cells. AG73 is a ligand of syndecans, which is highly expressed in various cancer cell lines. AG73-PEG liposomes were formulated with cationic lipid N,N″-dioleylglutamide and pegylated with PEG-DSPE. In syndecan-overexpressing cells such as HeLa and KB cells, the cellular delivery of a fluorescent siRNA was significantly increased after treatment with AG73-PEG liposomes. Enhanced RNAi efficiency was observed by quantitative real-tim e PCR. The anticancer effects by siMcl-1 were enhanced after delivery using AG73-PEG liposomes as compared to PEG liposomes. In vivo molecular imaging revealed the enhanced distribution of AG73-PEG liposomes to tumor tissues. Taken together, our results suggest that AG73-PEG liposomes might be further used to deliver chemical or nucleic acid-based anticancer agents to the tumor tissues.

Dengue virus belongs to the Flavivirus genus of the Flaviviridae family and is the cause for hemorrhagic fever. There are four serotypes of Dengue virus that are genetically similar but not identical; therefore infection of one serotype will not induce immune protection of the patient against other serotypes. It has been surmised that a successful dengue vaccine has to be able to effectively neutralize all four serotypes. Recently the dengue envelop protein domain III has been proved to be a neutralizing antigen. This is a short protein of approximately 100 amino acid and thus is an attractive target for heterologous expression. Various approaches to exploit this new discovery have been attempted. In this study we explored the prospects of expressing a consensus protein obtained from aligning the envelop domain III of all four serotypes in Saccharomyces cerevisiae and its use as a tetravalent vaccine against dengue virus. We have been successful in expressing this protein as a secretory protein and devicing several approaches to purify this protein from the culture filtrate.

Targeting particular mRNAs has become an excellent approach for the silencing of gene expression. Here, we showed a cell-penetrating antibody (transbody) that specifically hydrolyzes targeted mRNAs in the cellular cytosol and leads to targeted gene silencing. We generated a synthetic library on the yeast surface based on 3D8 VL, which is a cell-penetrating and nucleic acid-hydrolyzing single domain antibody. With the cell-penetrating activity, 3D8 VL into the cytosol of living cells can selectively decrease the amount of target sequence-carrying mRNAs. We selected 3D8 VL variants had higher affinity and greater selective hydrolyzing activity for target ss-DNAs than for off targets in the library by using 18-bp single-stranded (ss)-DNAs as target substrates. In particular, one 3D8 VL variant targeting the Her2 sequence showed more efficient downregulation of Her2 expression than a small-interfering RNA targeting the same Her2 sequence, and the variant caused apoptotic cell death of Her2-overexpressing breast cancer cells. Our results demonstrate that the 3D8 VL variants with cell penetrating, nucleic acid-hydrolyzing activity and sequence-selectivity could degrade target mRNAs in the cytosol, which suggests that they would be potential toolds in anti-cancer, anti-viral therapies.

Quantification of RNA provides information crucial for various biological studies. Reverse transcription coupled with real-time PCR is known to be the most accurate method for quantifying nucleic acids, and thus represents the state-of-the-art for RNA quantification. However, the use of real-time PCR for RNA quantification is limited to a single target per analytical run because of reductions in quantification power and lim itations of fluorescence dyes associated with multiplex applications. Capillary electrophoresis-based single-strand conformation polymorphism analysis (CE-SSCP), which separates single stranded DNAs by conformational difference and quantifies them by a simple procedure, is an alternative multiplex RNA quantification method. For the multiplex amplification of RNAs, modified multiplex ligationdependent probe amplification (MLPA) was combined with CE-SSCP analysis so that initial amount of RNAs could be quantified precisely. We have demonstrated that MLPA-CE-SSCP could be used to monitor expression of 57 metabolic genes of Escherichia coli and 20 Arabidopsis response regulators.

Acinetobacter baumannii has emerged as a new clinical threat to human health, particularly to the immunocompromised patients in hospital environment. In this study, we reconstructed the genome-scale metabolic network of this pathogen, and used it for subsequent drug targeting. The in silico model was simulated using constraints-based flux analysis with appropriate constraints according to the specific known conditions. Essential gene/reaction analysis and essential metabolite analysis were performed using this simulation method, each of which identifies essential genes/reactions and metabolites critical to the cell growth. The EMFilter, a framework that filters initially predicted essential metabolites to find the most effective ones as drug targets, was also developed (1). Final drug target candidates obtained by this system framework are presented with discussion. [This work was supported by the Korean Systems Biology Research Project (20090065571) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation (NRF) of Korea. Further supports by the World Class University Program (R32-2008-000-10142-0) of the MEST, LG Chem Chair Professorship, IBM SUR program, and Microsoft are appreciated.]

A new protein chip for fluorescence immunoassay was developed by thermal deposition of modified parylene film on a slide glass for the one-step immobilization of biomolecules. Conventional parylene is a polymer of p-xylene, and it is modified to have aldehyde groups (-CHO) in its molecular structure. The aldehyde groups made covalent bonding with amine group on proteins and peptides by one-step incubation. The efficiency of immobilization and the long-term stability of the modified parylene film were determined to be far improved in comparison to the conventional protein chips by using horseradish peroxidase (HRP) as a model protein. The applicability to the fluorescence immunoassay based on the protein chip was demonstrated by the detection of antibodies against cyclic citrullinated peptide (CCP) in the patient serum. The CCP was known to be an antigne against the autoantibodies of Rheumatoid Arthritis (RA). Therefore, the detection of antibodies agains CCP could be used for the early diagnosis of RA. In this work, CCP was immobilized on the protein chip and the amount of autoantibodies against CCP was quantified by using Cy-5 labeled anti-hIgG antibodies. By comparison with the test results (n=30) from conventional test, the feasibility of RA diagnosis by using the protein chip was confirmed.

The bacterial ghost system is a novel vaccine delivery system. It provides versatile carrier functions for foreign antigens with excellent natural intrinsic adjuvant properties. The optimized semi-defined medium for E.coli K-12/pHCE-InaN-GAPDH-Ghost27 were determined as 10 g/L glucose, 5 g/L yeast extract, 50 m g/L thiamine w ith R iesenberg's semi-defined medium. The optimal batch fermentation parameters were determined as 10% inoculum size, 300 rpm agitation and 1.5 vvm aeration for the highest cell mass production. The optimal fed-batch process for high cell density cultivation of E.coli K-12/pHCE-InaNGAPDH-Ghost27 was developed. The nutrient feeding strategy with Riesenberg's defined medium during fed-batch process was evaluated. The fermentation was conducted in four phases as follow: initial phase, fed-batch phase, temperature increase phase and high temperature holding phase. The maximum bacterial ghost vaccine was obtained about 40 g/L. The expression of antigen GAPDH on the ghost cell with high temperature holding phase was confirmed from outer-membrane fractionation and western blot. This results indicate that there was demages of expressed antigen on the ghost cell surfaces after increasing temperature for the high efficiency of ghost cell formation.

In this work, a highly sensitive immunoassay for human hepatitis B (hHBV) was developed by using SPR biosensor with autodisplayed Z-domains. Hepatitis B virus (HBV) is noncytopathic virus of the class Hepadnaviridae that causes acute and chronic liver diseases and HBV surface antigen (HBsAg) is the sentinel marker for the confirmation of acute HBV infection as it can be detected as early as 6 weeks after exposure. However, the detection of HBsAg is requires very low limit of detection (<1 ng/ml) and a wide detection range for positivity determination. In the previous work, we expressed Z domains of protein A with IgG-binding activity as a fusion protein of AIDA-1 by using autodisplay method. For the preparation of OM layer with Z-domains, the outer membrane with autodisplayed Z-domain was isolated and coated on the gold surface of SPR biosensor. Such an immunoaffinity layer based on the OM layer with Z-domains was reported to far improve the sensitivity and the limit of detection of immunoassays through the orientation control of the immobilized antibodies. For the medical diagnosis of HBsAg, a very sensitive immunoaffinity (IA) layer was formed by using the autodisplayed Z-domains. After the immobilization of anti-HBsAg antibodies to the OM layer, a standard curve was prepared by using standard samples with known HBsAg concentration at the required concentration range for the medical diagnosis of the HBsAg. From the analysis of patient samples (n=20), the feasibility of HBsAg test based on the OM layer with Z-domains was determined by comparison with the positivity test results from the conventional immunoassay.

Mannheimia succiniciproducens LPK7 (knock-out: ldhA, pflB, ptaackA), a powerful succinate-producing strain was used in this study. Organic acids, succinic acid and pyruvic acid produced in fermentation have toxic effects on the cells that inhibit the cellular growth and metabolism. Therefore, experiments were designed and performed to determine harmful effects of the organic acids on fermentation processes. The resistance test of organic acids was composed of two steps; a viable cell count test, and a flask culture test of culturing cells at various organic acid concentrations. To figure out tolerance of the cells against organic acid, cellular growth was measured in several organic acid concentrations; 0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% for succinic acid and 0.25%, 0.75%, and 1.25% (w/v) for pyruvic acid. In the 0.1% succinic acid concentration, cells were not affected significantly by the toxic effect of succinic acid. However, cell growth began to be inhibited when the succinic acid concentration reached 0.2% or higher levels. In addition, cells did not grow at all in the 0.5% medium. For the toxicity test of pyruvic acid, cell concentration was nearly one third in the 0.25% pyruvic acid medium compared to the control.

Modified parylene film which contains aldehyde groups are applied as a linker layer of SPR biosensor. Parylene is a polymer of p-xylylene with good optical properties, chemical resistance against most organic solvents and relatively high mechanical strength which are required for the linker layer of SPR biosensor. In this work, parylene that was modified to have aldehyde groups was deposited on gold surface of the SPR biosensor as thin film. Under the film thickness of 50 nm, the SPR biosensor was correctly performed with no significant change of sensitivity. Trough covalent coupling between aldehyde groups and amine groups, proteins and peptides could be strongly immobilized on th surface of the parylene linker layer. To demonstrate improvement of immobilization efficiency, covalent immobilization of horseradish peroxidase (HRP) on the parylene linker layer was performed and SPR signal change from the immobilized HRP was compared with that from physically adsorbed HRP on gold surface of the SPR biosensor. The SPR biosensor with the modified parylene lanker layer was applied for diagnosis of rheumatoid arthritis. On the linker layer of the SPR biosensor, cyclic citrullinated peptide (CCP) was covalently immobilized and anti-CCP antibodies was detected from RA patient sera. The results from SPR were well corresponded to those from ELISA assay previously performed. Furthermore, regeneration of SPR biosensor was achieved by removal and re-deposition of the parylene linker layer by treating oxygen plasma

A capacitive biosensor was developed for the medical diagnosis of menginitis by detection of S100B protein in the patient cerebrospinal fluid (CSF). S100 protein is a calcium-binding protein and consists of two monomers, and it is overproduced during gliosis in patients with Alzheimer disease, Down syndrome and related dementia. Therefore, overproduction of S100B protein was reported to implicate the developmental brain dysfunction. In this work, a capacitive biosensor based on chronoamperometry was applied for the detection of S100B protein. For the improvement of the capacitive biosensor, the outer membrane (OM) layer with autodisplayed Z-domains was used to immobilize anti-S100B protein. In the previous work, we reported that the immunoaffinity layer based on the OM layer could far improve the sensitivity and limit of detection of immunoassays through the orientation control of the immobilized antibodies. For the comparison of sensitivity, different immunoaffinity layers were prepared: self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid combined with covalent coupling of anti-S100B antibodies, physical adsorption of antibodies to the electrode surface. The immunoassay by using the OM layer with Z-domains showed the limit of detection was improved as much as 100-fold higher than the conventional SAM. For the capacitive sensor based on the OM layer with autodisplayed Z-domains, a standard curve at the S100B protein concentration of 1 pg/ml – 10 ng/ml was prepared by using standard samples by spiking S100B protein to CSF. The feasibility of the capacitive biosensor for the medical diagnosis was confirmed by comparison with the test results (n=20) from the conventional immunoassay.

Pentose phosphate pathway generates reducing equivalents, NADPH, and 5-carbon sugars. The ribose 5-phosphate produced by this pathway can be recycled into glucose 6-phosphate (G6P) by transketolase, transaldolase, and some of the enzymes of the gluconeogenic pathway. Consequently, complete oxidization of 1 mole of G6P to CO2 can generate 12 moles of NADPH. However, in most cases, glycolysis is much more activated than gluconeogenesis to generate energy sufficient for cell survival. Moreover, glycolysis and gluconeogenesis are reciprocally regulated so that they do not take place simultaneously in the same cell to a significant extent. Therefore, it is important to deregulate gluconeogenic pathway to fully oxidize G6P for generation of NADPH. Fructose-1,6-bisphosphatase (FBPase) is a key enzyme that participate in the regulation of gluconeogenesis. It is allosterically inhibited by both AMP and G6P. Here, we engineered FBPase to be deregulated by AMP and G6P based on the 3D-structure. Variants of FBPase purified by Ni-NTA column were characterized in terms of kinetic parameters.

A micelle-fractional precipitation hybrid process was developed for the effective pre-purification of the anticancer agent paclitaxel extracted from plant cell cultures. By removing waxy substances originating from plant cells using the adsorbent sylopute, the efficiency of the subsequent pre-purification process could be remarkably enhanced. Paclitaxel yield was improved and the fractional precipitation time was shortened compared to the control by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite IR120 or Amberlite 200), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96), or glass beads. Most of the paclitaxel (>98%) could be obtained after about 12 h of fractional precipitation using Amberlite 200. Purity increased with increasing fractional precipitation time up to 9 h to about 85%, after which it showed little change. On the other hand, no paclitaxel precipitate was formed using either of the nonionic exchange resins because paclitaxel, which is hydrophobic, was strongly adsorbed on the hydrophobic resin surface. Since high purity paclitaxel can be obtained in high yield and the precipitation time can be reduced by combining micelle formation with fractional precipitation, this hybrid method is expected to significantly enhance the final purification process.

The solvent treatment of paclitaxel is a convenient method for controlling the morphologies of paclitaxel. Amorphous paclitaxel was simply made by dissolving paclitaxel in methylene chloride/methanol (98/2, v/v) and inrelatively non-polar solvents (t-butyl methyl ether, pentane, acetonitrile/hexane (1/2, v/v), methylene chloride, chloroform, toluene). On the other hand, crystalline paclitaxel was made by dissolving paclitaxel in a special polar solvent containing a small amount of water. However, when we used only methanol, we got mixed morphologies of paclitaxel made of both the crystalline and amorphous forms. Their physicochemical properties were investigated by X-ray powder diffraction (XRPD), scanning electron microscopy(SEM), andhigh performance liquid chromatography(HPLC). The initial water content of amorphous paclitaxel and crystalline paclitaxel was determined for 0.65 wt% and 5.85wt%, respectively. The hygroscopic property of crystalline paclitaxel was very changeable in all given humidity (15, 60, 95 RH%) during storage. Dissolution profiles for paclitaxel showed that amorphous paclitaxel measured the highest solubility in water and its solubility held most stable during the measurements. The residual solvent could be reduced to the maximum allowed value (600 ppm for methylene chloride, 3,000 ppm for methanol) of guidance for the International Conference on Harmonization (ICH) by spray drying.

This study describes the evaluation and optimization of a crystallizing process capable of efficiently purifying vancomycin in high purity and yield. In particular, we observed how the main process parameters influenced the formation of crystals, determined their morphology, and monitored purity and yield. Acetone was shown to be more effective than alcohol solvents for the crystallization of vancomycin. The optimal distilled water/acetone ratio, storage temperature, storage time, pH, conductivity, initial vancomycin concentration and stirrer velocity were shown to be 1:3.5 (v/v), 10ºC, 24 h, pH 2.5, 20 ms/cm, 0.1 g/mL, and 640 rpm, respectively. Temperature had a decisive influence on crystal formation; crystals were successfully produced at 10ºC, while at other temperatures, conglomeration, disintegration and cohesion occurred. Crystal growth developed over time and was complete at about 24 h. Vancomycin purity remained at about 97.0% irrespective of storage time while the yield increased over time, reaching a maximum of 95.0% at around 24 h, after which there was no substantial change. Crystallization occurred over a certain range of pH (2.5-3.0), but purity and yield were highest at pH 2.5. When the pH was outside this range, a conglomeration (gelation) phenomenon prevented the efficient production of crystals. Vancomycin crystals were produced irrespective of the stirrer velocity, which had no influence on purity; however, the highest yield of vancomycin was obtained at 640 rpm.

Lactic acid is an important chemical that can be used to synthesize biodegradable polymers, such as polylactic acid (PLA) and its copolymers. Precipitation was employed to recover lactic acid from a calcium lactate solution using sulfuric acid. In this study, we optimized process parameters of precipitation to obtain a high yield and purity of lactic acid from a calcium lactate solution. Also, we observed the influence of the main process parameters on precipitate formation during the course of precipitation using a video microscope. The optimal calcium lactate/sulfuric acid molar ratio and pH were 1/1 and 6.8, respectively. Also, the effect of mixing method on the efficiency of precipitation and the formation of precipitate were evaluated. When the sample was mixed continuously after adding sulfuric acid, lactic acid was obtained in high yield. By mixing the sample, we were able to significantly reduce the precipitation time, the length of which has been a problem inherent in the conventional precipitation process. The yield of recovered lactic acid was 92% with a purity of 71%.

Crystallization is a simple, energy-efficient and environmentally friendly process for purifying vancomycin from fermentation broth. However, the crystallization process has been inherently problematic due to the lengthy crystallization time that is required. An improved crystallization process could significantly reduce the crystallization time by increasing the surface area available for crystallization. Vancomycin yield was improved and the crystallization time was shortened compared to the control by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite 200), an anion exchange resin (Amberlite IRA-400), or glass beads. Most of the vancomycin could be obtained after about 12 hr of crystallization using Amberlite 200 and Amberlite IRA-400. Since high purity vancomycin can be obtained in high yield and the crystallization time can be reduced, this improved method is expected to significantly enhance the final purification process.

The atopic dermatitis (AD) is a chronic inflammatory skin illness, with remissions and exacerbations, itch. The important role of mast cells in immediate-type allergic reactions is well recognized. Mast cell activation brings about the process of degranulation that results in the fusion of the cytoplasmic granule membranes with the plasma membrane. A wild rice species, Zizania latifolia is one of the most common emergent species in East Asia and has been used heart disease, diabetes mellitus, skin disease for a long time. In this study, we investigated the effects of extract from Z. latifolia aerial parts on β-hexosaminidase release from mast cell activated by DNPBSA. Degranulation was determined by measuring the release of a granule marker, β-hexosaminidase. Rat basophilic leukemia(RBL-2H3) cells, a tumor analog of mast cells, display properties of mucosal-type mast cells. The 2H3 cells contain several hundred thousand IgE receptors on the m embrane surface, and after sensitization with mouse monoclonal IgE, the cells respond to antigen and release histamine. Z. latifolia extract significantly inhibited β-hexosaminidase release from mast cell. Inhibition of β-hexosaminidase release were 0.32, 0.23, 0.10, 0.07, and 0.08 at the treated extract concentration of 10, 25, 50, 100, and 250 ㎍/ml, respectively compared to positive control (0.02 in O.D. 405). This result may provide useful information for further discovering pharmacologically active compounds for treatment of atopic dermatitis.

Islet Transplantation is an ideal method for the treatment of type I diabetes.[1] However, there are many problems, which must be solved before entering into clinical trials. Usually, the recipient requires the islets derived from 3-4 donors in order to achieve insulin independence. Thus, secretion signal peptide linked exendin-4 gene was constructed for gene modification of islets.[2] The viability and morphology of SP-Ex-4 transduced islets weren’t significantly different from untransduced islets. Also, the amount of insulin secreted from SP-Ex-4 transduced islets was significantly higher than untransduced islets at high glucose stimulation (28 mM). Balb/c nude mice were chemically rendered diabetic and different numbers of SP-Ex-4 transduced and untransduced islets were transplanted under the kidney capsules. The median survival time (MST) of mice transplanted with 50 untransduced islet transplanted islets was 12 ± 3.75 days. However, the MST of SP-Ex-4 transduced islet recipients was >30 days. In conclusion, SP-Ex-4 transduced islets using lentiviral vectors can be used to improve the problem associated to the islet donor shortage in islet transplantation.

Bacterial transcriptional regulators belong to IclR family involved in carbon metabolism contain the DNA-binding HTH domain in the N-terminal part. The C-terminal part contains about 170 residues regulatory domain, termed Effector binding Domain (ED) as it may bind a small effector or signal molecule. The domains are named after Escherichia coli IclR (isocitrate lyase regulator), a repressor of the glyoxylate bypass operon for acetate utilization. Most IclR-type transcription regulators are repressors of specific catabolic genes in the absence of specific substrates, while excess of a specific effector triggers de-repression. Genome analysis of Streptomyces peucetius revealed a transcriptional regulator named NrpsR which belongs to the IclR family of transcriptional regulator which is located within the non-ribosomal peptide synthase (NRPS) gene cluster. Here, we report the analysis of nrpsR gene, cloning and expression into S. peucetius.

The goal of this research is to achieve development of recombinant E. coli for fatty acid synthesis (FAS). The enzyme of aceE gene converts pyruvate to acetyl-CoA and that of accA gene catalyzes the addition of CO2 to acetyl-CoA to generate malonyl-CoA. The genes were identified as homologous gene of E. coli through a metabolic pathway. The genes for pyruvate dehydrogenase (aceE) and acetyl-CoA carboxylase (accA), which are the enzymes that catalyze the first step in the synthesis of fatty acids in Escherichia coli MG1655, were cloned. That is, the E. coli strains were midterm recombinant strains to produce fatty acid. The recombinant E. coli MG1655 containing the aceE or accA, and the two genes inserted expression vector (pTrc99A) were developed. To confirm effects of each gene, the intermediate products were analyzed from in vivo metabolites and in vitro metabolites of recombinant E. coli. As a result, the three strains produced more malonic acid than its wild type E. coli. However, there was insignificant the difference between E. coli harboring a aceE gene-inserted pTrc99A vector and E. coli harboring a accA gene-inserted pTrc99A vector.

To enhance clavulanic acid (CA) production, four structural CA biosynthesis genes ceas2, bls2, cas2 and pah2 carboxyethylarginine synthase, β-lactam synthetase, clavaminate synthase and proclavaminate amidinohydrolase respectively were amplified from Streptomyces clavuligerus NRRL3585 genomic DNA and cloned first in pGEM7 subcloning vector and then integration vector pSET152 and expression vector pIBR25 containing ermE* strong promoter and generated pHN18 and pHN19 respectively. The recombinant plasmids were introduced into S. clavuligerus NRRL3585 by PEG mediated protoplast transformation method. Both recombinant strains produced more CA than the S. clavuligerus parental strain. This study showed that CA production was significantly enhanced by integration and overexpression of potential biosynthetic genes.

The purpose of this research is to produce more amount of fatty acids by improving fatty acid biosynthesis mechanism of Escherichia coli. β-Ketoacyl acyl carrier protein synthase III (fabH) is the enzymes that catalyze the first step in the synthesis of fatty acids with acetyl-CoA carboxylase (accABC) and malonyl-CoA: acyl carrier protein (fabD) acyltransferase in Escherichia coli MG1655. The fabH, accABC and fabD genes were cloned and characterized. The enzyme of fabH gene leads to increase short-chain-length fatty acids and strong preference for acetyl-CoA, and produces only straight chain fatty acids (SCFAs). β-Ketoacyl acyl carrier protein synthase III (fabH) is also thought to play a role in determining fatty acids type and compositions. The fabH, accABC and fabD genes were identified as homologous gene of E. coli MG1655 through a metabolic pathway. The recombinant E. coli MG1655 containing the fabH or accABC::fabD or accABC::fabD::fabH geneinserted expression vector (pTrc99A) were constructed. To analyze the effects of gene overexperssion, analysis of fatty acid was performed.

Dynamic changes and genetic regulation in metabolic networks are often too complicated to permit analytical solutions. So, scientists have increasingly applied mathematical models as a way to describe and better understand metabolic systems. Within a metabolic framework, appropriate parameters are related to the structure of the reaction network. Thus, the identification of the regulatory structure is related to the estimation of the appropriate set of parameters, making the parameter estimation as the most essential part in the understanding of the metabolic pathway structures. This work utilized chaotic Particle Swarm Optimization (cPSO) method for the parameter estimation in large-scale glycolytic networks of Escherichia coli metabolism. Within the modification of some of the enzyme kinetics, the model has got a better description of the network and a more fitted set of parameters. As well, the flux control coefficients (FCCs) of the metabolic control analysis were also calculated to elucidate the parameters responsible for the control of flux.

The ability to accurately determine intracellular concentrations of metabolites is of key importance in studying signaling and metabolic reaction networks and their regulation in vivo. In the last few years, much time has been invested in studying the intracellular metabolites concentration profiles of E. coli in the stationary phase. However, there are few studies related to the intracellular metabolites concentration profiles of E. coli in the exponential phase. In this study, metabolic regulation in E. coli was studied in terms of the change in intracellular metabolites (e.g., glucose-1-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl-coenzyme A, 6-phosphogluconate, ribulose-5-phosphate, xylulose-5-phosphate, erythrose-4-phosphate) concentration at two different growth phases, in batch culture. Samples were taken every 2 h after batch cultivation started. Culture sample vials (5 mL) were plunged rapidly into an equal volume of 60% aqueous methanol (-40°C). The quenched biomass was centrifuged for 10 min at 3000×g and -9°C. An extraction procedure using 100% methanol (-48°C) was performed to extract intracellular metabolites from E. coli K12 strain W3110. All measurement were carried out on an HPLC system coupled with an ion trap mass pectrometer equipped with a turbo-ion spray source.

Pradimicins, produced by Actinomadura hibisca P157-2, are potent antifungal polyketide antibiotics having an unusual dihydrobenzo [α]naphthacenequinone aglycone substituted with D-alanine and sugars. The compound is generated by type II PKS. Since acetyl-CoA is the precursor for the biosynthesis of pradimicin, we tried to enhance pradimicin production in the wild strain, by supplying excess biosynthetic precursors. So, a gene complex for acetyl-CoA carboxylase (ACCase) which catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA was overexpressed in Actinomadura hibisca P157-2 in order to maximize pradimicin production.

Bioethanol (C2H5OH) is the most attractive biofuel that can be directly applied to gasoline engines. The alcohol fuel originated from non-crop biomass has been generally produced through three steps: pretreatment, enzymatic hydrolysis and fermentation. Although many researchers have been trying to improve the conversion yields from biomass to mono-sugars through chemical/physical pretreatment and enzymatic hydrolysis processes, a large amount of organic residue has been reported to be generated as a byproduct. This residue may be reused as another feedstock in syngas platform like pyrolysis. In this study, therefore we explored the thermal decomposition characteristics of organic residue generated by a pretreatment process for bioethanol production from seaweed, which is one of the promising feedstocks. Pretreatment of a marine biomass, Ulva sp., with acid or alkaline solutions and hydrolytic enzymes resulted in organic residues as well as sugars-containing solutions. The thermal degradation patterns of the residues were examined by thermogravimetric analysis (TGA) with different thermal analysis (DTA).

Because of high contents of cellulose and hemicellulose, barley straw seems to be a potential lignocellulosic biomass for production of bioethanol. Ethanosolv applied organosolv pretreatment is more convenient method than other pretreatment. However, sulfuric acid was used as a catalyst in the ethanosolv pretreatment. The acid catalyzed treatment might have some undesirable effects such as the formation of inhibitor(furfural, HMF) and corrosion of the reaction vessels. To avoid these side effects, FeCl3 substituted for sulfuric acid. FeCl3 is less corrosive than acid and can be recycled, it is more economically. In this study, we evaluate the effects of various inorganic salts on enzymatic digestibility to select a appropriate one and investigated the effects of FeCl3 concentration and temperature on enzymatic digestibility. To confirm side effect caused FeCl3, using FeCl3 evaluate the formation of inhibitor(HMF, furfural) compared to sulfuric acid and remained FeCl3 was investigated after ethanosolv. 1. NGUYEN, Quang (2009) Dilute aicd/metal salt hydrolysis of lignocellulosics. European patent , WO 2002/012529.2. Liu, L., Sun, J. Corn stover pretreatment by inorganic salts and its effects on hemicelluloses and cellulose degradation. Bioresour. Technol. 100:5865-5871 (2009).

MITF (microphthalmia-associated transcription factor), a bHLH (basic helix-loop-helix) transcription factor with a leucine zipper, is a key regulatory protein for the pigmentation process in melanocytes. Binding of MITF to E-box caused transcription of several pigmenting genes including the tyrosinase gene. The purpose of this study is to analyze the interaction of MITF and its DNA binding site (E-box, CATGTG) by surface plasmon resonance. Label free DNA-protein interaction assay, SPR (surface plasmon resonance), coated by EDC (Ethyl-3-(3- dimethylaminopropyl)carbodiimide) / NHS (N-Hydroxysuccinimide) were used with amine coupling methods that permitted efficient immobilization of DNA on the gold surface. It is expected that SPR can be a useful method to study of MITF and E-box interactions.

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives which can adhere in aqueous conditions. Recently, we construct a functional recombinant hybrid mussel adhesive protein fp-151 that is a fusion protein comprising six-repeated fp-1 decapeptide repeats at both each terminus of fp-5 terminus and express the protein in E.coli system. However there cannot be happened post translational modification in E. coli system, so there are no 3, 4-dihydroxyphenyl-L-alanine (DOPA) and phosphorylated serine, which is known that is very important on adhesion process of mussel foot protein. For expression of originally modified fp-151 similar with natural form, we produced a recombinant fp-151 in baculovirus/insect Sf9 cell expression system and checked the DOPA and phosphor-serine formation in fp-151. Through this difference between fp-151 in inset Sf9 and E.coli, we checked expression of MAP with nearly natural form in vivo and appeared possibility of improving functionality and originality.