Earticle

Influenza virus hemagglutinin (HA) is expressed as a precursor protein, HA0, and then proteolytically cleaved to produce two disulfide-linked polypeptide chains, HA1 and HA2. The HA2 protein segment, an influenza virus glycoprotein, contains two membrane-interacting hydrophobic peptide sequences: an N-terminal "fusion peptide" (residues 1-22), which interacts with the target membrane, and a C-terminal transmembrane domain (residues 186-221) that passes through the viral membrane. This study aims to develop biodegradable microparticles containing both HA1 and truncated HA2 proteins for vaccination with enhanced biosafety. The HA2 ectodomain (residues 23-185) truncated with two membraneinteracting sequences was expressed in Escherichia coli. This segment of HA2 contains changes in three rare Arg codons (Arg-123, Arg-124, and Arg-127) and the mutation of Cys-137, which forms a disulfide bond with HA1 residue 14, to Ser. The full-length HA1 polypeptide segment was expressed separately in Escherichia coli with mutation of Cys-14, which forms a disulfide bond with HA2 residue 137, to Ser. Both purified HA1 and truncated HA2 segments were microencapsulated in poly(D,L-lacticco-glycolic acid) (PLGA) using a W/O/W double emulsion technique. Antigen release from microspheres was determined in vitro using a hemagglutinin-specific ELISA.

Photodynamic therapy (PDT) is a promising method for the treatment of several diseases including cancer. It involves the systemic administration of photosensitizers(PSs) such as porphyrin, followed by local photoirradiation with the light of a specific wavelength.[1] In this work, a 3rd generation dendrimer porphyrin (DP Mw ~8,000 g/mol) with 32 carboxyl groups on the periphery was used as a component to prepare a layer-by-layer (LbL) structure via an electrostatic adsorption using its anionic peripheral groups.[2] To enhance the efficacy and tumor-specificity of PDT, poly(allylamine hydro chloride) (PAH, Mw ~50,000 g/mol) / DP nanocarrier was constructed through a LbL self-assembly of PAH and DP on a removable template. It has been reported that the optimal size of drug carriers for efficient delivery of an antitumor agent to a tumor is round 100-200 nm. We chose the monodispersed polystyrene nanoparticles with a diameter of 100 nm which can be dissolved in chloroform.[3] The anticancer drug could be encapsulated into the nanocarriers and its release from hollow polyelectrolyte nanocarriers can be controlled by changes of membrane permeability, which respond to stimulus such as pH.

Atherosclerosis is a disease where vulnerable plaques build up on the inside of arteries leading to serious problems including heart attack, stroke, or even death. Current computed tomography (CT) is one of the methods to detect the atherosclerotic plaques but the CT contrast agents like iodine-based compounds have several limitations such as short imaging times due to rapid renal clearance, renal toxicity, and vascular permeation. In this study, in order to overcome current CT contrast agent limitations, we have developed liposomal contrast agents that could be taken easily by the macrophages which are abundant in the atherosclerotic plaques. The method to synthesize the liposomal contrast agents containing iodine compounds was established and the effect of cholesterol (CHOL) on the contrast effect and stability of the liposomal contrast agents was investigated. There was no significant contrast difference according to the CHOL concentrations. However, as the CHOL concentration was higher than 10%, the particle size of the liposomal contrast agents increased abruptly, which was inappropriate for the use of contrast agents. In conclusion, 8.5% of CHOL concentration was the best content for the liposomal contrast agent in respect to the particle size and stability of the liposomal contrast agents.

There are tremendous drug candidates that suffer from insolubility in water. In the present study, it is shown that Coenzyme Q10 (CoQ10), a model water-insoluble compound, can be nanoparticulated into a water-soluble form using apolipoprotein A-I (apoA-I). Similar to the way that apoA-I forms nascent discoidal high density lipoprotein (ndHDL) particles by bordering acyl chain tails of phospholipids, CoQ10 could be enclosed into the circle of a disk made of apoA-Is. The resulting nanostructure of CoQ10 and apoA-I was water-soluble with a size of _12 nm in diameter and was physically more robust than liposome. We expect that the strategy suggested in this study can be exploited to assemble nano-sized, water-soluble structures of various water-insoluble drug candidates.

Exess Ethanol intake is usually toxic in health. Chronic ethanol consumption causes oxidative damage in liver. Cytochrome P450 2E1 (CYP2E1) participates to the ethanol decomposition. Excess ethanol intake affects more microsomal enzyme CYP2E1 activity than ADH resulting in causing oxidant stress. CYP2E1 has been to be a major contributor to ethanol-induced oxidant stress. CYP2E1 induced ethanol conversion to acetaldehyde. Acetaldehyde is malignant metabolite for vasodilation substance, flushing induction, and respiratory distress induction as well as exhibiting to be intense reactive and toxic. Acetaldehyde combined with phospholipid, amino acid residues, and sulphydryl group may transform cell surface antigen and induce lipid peroxidation and oxidant stress as well as depleting antioxidant enzyme glutathione (GSH). In this study, we investigated the inhibition of ethanol-induced CYP2E1 by Curcuma longa L. Extract. The cell lines used were HepG2 E47 cells that expressed human CYP2E1 and HepG2 C34 cells which did not express CYP2E1. CYP2E1 enzymatic activity was determined in E47 and C34 cells by measuring the fluorescence and western blot. In consequence, Curcuma longa L. Extract was recognized to inhibit the ethanol-induced CYP2E1 enzymaticactivity.

Here we report a novel non-antibody protein scaffold developed based on the fold of kringle domain (KD) derived from plasminogen. Based on the sequence analysis of 39 unique KD in 31 human proteins (1), we designed KD libraries with a rationale to conserve the critical residues for the KD fold, but to introduce mutations which are exposed to the surface in the flexible loops, using synthetic shuffling technique (2). For target proteins as a proof-of-concept, we used death receptor 4 (DR4), DR5 and TNFα. Isolated KD mutants against the individual target proteins, using yeast surface display techniques combined with FACS, were solubly expressed well and specifically bind to the targeted proteins with affinity of 10-6-10-8 M, without significant cross-reactivity, assessed by ELISA and SPR. Some KD mutants selected against DR4 or DR5 induced apoptosis in several carcinoma cell lines as a single agent. Also, some KD mutants isolated against TNFα efficiently protected TNFα-induced cell death by neutralizing the cytotoxicity of TNFα. Our results suggest that a novel non-antibody scaffold based on KD fold can be developed as a specific binder to given targets to modulate the biological activity.

Poly(ethylene glycol)-polyester block copolymers have widely been used in biomedical and pharmaceutical fields because of their excellent biocompatibility, biodegradability, and amphiphilicity arising from phase-separated microstructures between a hydrophilic PEG block and a hydrophobic polyester block. The aims of this research are to prepare gemcitabine-loaded microparticles using methoxy poly(ethylene glycol)-poly(L-lactide) (mPEG-PLLA) diblock copolymer and to evaluate their release characteristics. We synthesized mPEG-PLLA diblock copolymers of different block lengths, and their molecular compositions and molecular weight were analyzed by 1H-NMR and GPC. W/O/W double emulsion solvent evaporation method was used to prepare gemcitabine-loaded mPEG-PLLA microparticles. The effects of various preparative parameters such as pH, salt concentration of external water phase, solvent composition of organic phase and ratio of internal/external water phase on the morphology and the encapsulation efficiency of the microparticles were investigated. The in vitro release profiles of gemcitabine from microparticles were also determined using HPLC.

As nucleic acids alone can’t penetrate the cell membrane, efficient carriers are needed. Therefore, we synthesized a probe for oligonucleotides and imaging using aminated APTS coated silica nanoparticles, modified with FITC-doped and cell-penetrating peptides (HIV-1 trans-activator factor (TAT)). In addition, the dye-functionalized SiNPs could make easily control of various sizes of 10nm~800nm. Here, we prepared TATfunctionalized SiNPs conjugated Cy5 labeled DNA fragments and TAT peptide for the purpose of improving intracellular delivery. By showing that SiNP-DNA probes were internalized by cells and distributed intracellularly thoughout the cytoplasm and not penetrate the nucleus. This study demonstrates the feasibility of using SiNPs as a potential carrier for therapeutic gene delivery.

Based on previous in vitro studies showing that stimulation with glutamate could increase the number of cells expressing tyrosine hydroxylase (TH) protein, we investigated whether glutamatergic mechanisms also regulate induction of the dopaminergic neuronal phenotype by treating primary cultures obtained from transgenic TH/lacZ mice and monitoring the number of β-gal-immunolabeled cells. Primary cultures were stimulated after 1 day in vitro (DIV) with glutamic acid (0.1-1 μM) added in pulses followed by a washout to prevent desensitization. Glutamic acid induced the expression of the representative dopaminergic neuronal marker TH and the second enzyme for the biosynthesis of dopamine, AADC, at a moderate level. Of interest, the expression of GTPCH, an enzyme for the biosynthesis of dopamine, was increased twofold by glutamic acid. The induction of those neuronal markers was blocked by pretreatment with kyurenic acid (100 μM), a broad spectrum excitatory amino acid antagonist. These results suggest that glutamic acid regulates dopaminergic neuronal markers in olfactory bulb cells.

pH-Responsive hydrogels based on the methacrylic acid(MAA) are widely used for biomaterial and pharmaceutical fields because of their unique swelling properties in response to the external pH changes. In order to develop a smart delivery system for cosmetic active ingredients such as arbutin and ascorbic acid, pH-responsive P(MAA-co-EGMA) hydrogels were synthesized via dispersion photopolymerization and used as a carrier. The hydrogel microparticles showed a pH-responsive behavior, at low pH a small amount of cosmetic active ingredients was released while at high pH a relatively large amount of cosmetic active ingredients was released from the hydrogels. To determine the protective ability of the P(MAA-co-EGMA) hydrogel microparticles for ascorbic acid, ascorbic acid and ascorbic acid-loaded microparticles were treated with high temperature. As a result, the hydrogel microparticles were able to protect the loaded ascorbic acid. These results indicate that the P(MAA-co-EGMA) hydrogel microparticles can be used as smart delivery carriers for cosmetic active ingredients with which the release of cosmetic active ingredients from the hydrogels can be controlled by the external pH changes.

Cocaine is recognized as the most reinforcing of all drugs of abuse. There is no available anticocaine medication. An ideal anticocaine medication would accelerate cocaine metabolism, thereby producing biologically inactive metabolites by a route similar to the primary cocaine-metabolizing pathway, that is, cocaine hydrolysis catalyzed by plasma enzyme butyrylcholinesterase (BChE). However, the native BChE has a low catalytic efficiency against naturally occurring (-)-cocaine. A novel computational method and generation of mutant butyrylcholinesterase for cocaine hydrolysis is provided. The method includes molecular modeling a possible BChE mutant and conducting molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations thereby providing a screening method of possible BChE mutants by predicting which mutant will lead to a more stable transition state for a rate determining step. Sitedirected mutagenesis, protein expression, and protein activity is conducted for mutants determined computationally as being good candidates for possible BChE mutants, i.e., ones predicted to have higher catalytic efficiency as compared with wild-type BChE. In addition, mutants A199S/A328W/Y332G, A199S/F227A/A328W/Y332G, A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/Y332G, and A199S/F227A/S287G/A328W/E441D all have enhanced catalytic efficiency for (-)-cocaine compared with wild-type BChE.

To manufacture biopharmaceutical products with highly assured and consistent quality, it should be controlled and produced under quality management system. So it is important that quality must be considered from the product design and development phases and to minimize risks and to protect consumers from it, risk management is needed over the parameters that can affect on the product quality (1-4). Manufacturing facilities that may have impact on the product quality must be qualified for their design, installation, operation and performance according to the characteristics of each equipment and facility (5-6). Validation must be conducted on the critical process parameters of each manufacturing process of biopharmaceuticals to assure high quality and continued process verification is required (7).

PGE2 is a major product derived from arachidonic acid through the cyclooxigenase pathway, being implicated in pain and inflammatory responses. However, the effects of PGE2 are multiple and sometimes seem to be functionally opposing. For example, PGE2 has been also identified as an important mediator of gastric ulcer healing, bone formation, and dermal wound healing. 15-PGDH catalyzes NAD+ dependent oxidation of 15(S)-hydroxyl group of prostaglandins and has been considered a key enzyme involved in biological inactivation of prostaglandins. Therefore, inhibitors of 15-PGDH will be valuable for the therapeutic management of diseases in which elevated PGE2 levels are needed. Structure-activity analysis of thiazolidinediones indicated that the nature of the moiety linking through ether linkage to benzylidenethiazolidine-2,4-dione plays an important role in inhibitory potency. Base on the structures of thiazolidinediones analogues and inhibitory activity, various benzylidene thiazolidinedione derivatives with different substituents on phenyl ring were synthesized using a series of reaction and test for 15-PGDH inhibitory activity. The most potent inhibitor of this series of compounds is 5-(4-(2-(thiophen-2- yl)ethoxy)benzylidene)thiazolidine-2,4-dione with a IC50 of 0.031 nM.

In previous study, it was reported that the 30K protein originating from silkworm, Bombyx mori, inhibited apoptosis in mammalian cells. In this works, we observed a novel property of 30K protein; cell-penetrating property. When recombinant 30Kc19 produced from E.coli was added to culture medium, it was shown that 30K protein was internalized into the living human cell lines. The degree of internalization was increased in a dose-dependent manner and this process was occurred within short period of time (< 1 hr). Moreover, it was examined that dextran sulfate, a kind of poly-anionic material, in the culture medium inhibited the internalization of 30K protein. To examine a cellular localization of 30K protein, immunofluorescence (IF) assay was performed using confocal microscopy. It was demonstrated that 30K protein was bound onto the cell membrane within 10 min and internalized gradually. According to these IF results, the internalization was completed within about 1 hr. Consequently, we conclude that 30K protein is a novel cell-penetrating proteins such as TAT or Antp because these properties are similar with that of the other cell-penetrating proteins. Cell-penetrating protein and its protein transduction domain (PTD) have been used to deliver macromolecules and even small molecules for their functions inside the cells. Therefore, our results support that 30K protein is anticipated to be used as new tool to deliver macromolecules into the living cells.

As Patents on the biopharmaceuticals, including many blockbusters with over $1 billion in annual sales, have expired or are about to approach expiry, development opportunity of subsequent biosimilars has emerged. And more recently, the researches on the next generation of so-called super-biosimilars are also activated. In this report, we present the optimizations of Protein A capture step for the therapeutic Fc-fusion protein, as one of the super-biosimilars, to improve the yield. The optimizing method was designed by using a design of experiment (DOE) is that statistical tool. The six decision variables used in the optimizations were the component, pH and conductivity of equilibration buffer, residence time (linear velocity), the quantity of loading samples, and pH of elution buffer. After all, an optimized procedure to obtain the therapeutic Fc-fusion proteins was developed.

There are three types of alginate lyases: the first type is specific for α-L-guluronate (G) block guluronate lyase, the second type for β-Dmannuronate (M) block mannuronate lyase, and the third type is bifunctional for G and M blocks. These enzymes depolymerize alginate through a β-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection. Alginate lyase AlyVI is specific for polyguluronate and has a molecular mass of 34 KDa. Site-directed mutagenesis was used to examine their roles in binding alginate. Several mutants were prepared, expressed in Escherichia coli, and tested by TBI method using supernatant of culture. Mutants D226K, D226A, and D226G had higher activity than the wild type. Especially, the activities of D226A and D226K were increased nearly 4- and 5-fold, respectively. A relatively conserved tryptophan residue corresponding to tryptophan 165 of alginate lyase AlyVI is proposed to be important for the functioning of the enzyme. Tryptophan 165 was individually changed to an alanine, aspartic acid, arginine, and glycine. All of these mutants were completely inactive. These results indicates that W165 is critical part for enzyme activit

Silk fibroin is an insoluble structural protein from Bombyx mori. It can be regenerated as a fibroin solution to form a variety of biomaterials, such as gels, sponges and films, for medical applications [1]. Fibroin microsphere has been suggested as a platform for controlled drug delivery [2]. In this report, submicron scale microspheres was prepared by W/O emulsion method using sorbitan monooleate (Span80), polyoxyethylene 20 sorbitan monooleate (Tween80), decane and fibroin solution. The capsules were spherical in shape as determined by SEM with diameters in the range of 200 nm to 1200 nm, depending on the homogenization rate of W/O emulsion. Hydrophilic materials can be encapsulated by dissolving them in the fibroin solution and subsequent emulsification. Dextran and FITC-dextran were efficiently encapsulated in the fibroin microcapsule. Their core-shell structure was identified by confocal microscopy. Because of its very mild conditions of fabrication, this method is potentially advantageous for the encapsulation of heat-labile drugs such as proteins.

The purpose of this study is to prepare gemcitabine-loaded poly(Llactic acid) (L-PLA) nano- and microparticles using solution enhanced dispersion by supercritical fluids (SEDS) technique. Gemcitabine is a synthetic pyrimidine nucleoside analogue with a high antitumor efficacy for ovarian, breast bladder, and pancreatic cancer. The SEDS process is a modified supercritical antisolvent (SAS) process, in which supercritical CO2 and polymer/drug solution are simultaneously introduced into a high-pressure vessel using a specially designed coaxial nozzle. The effects of temperatures, pressures, polymer molecular weight and solution concentrations on the morphology and drug entrapment efficiency of SEDS-processed particles were investigated. Our study was focused on the comparison of SEDS with ASES process.

Many studies on the composition and efficacy of essential oils have been performed for a long time. Essential oils, in general, have complex compositions containing hydrocarbons and oxygenated compounds. These compositions are closely related to their characteristic odors and flavors. Essential oils have been widely used for pharmaceutical, cosmetic, food, perfume and agricultural industries because of their antiviral, antibacterial and antifungal activities in addition to their unique odor and flavor. In the field of medicine, they have been found to have an excellent ability to treat tuberculosis, indigestion, infection, loss of appetite, insomnia, hypertension and rheumatism. In the present study, poly(ethylene glycol) (PEG) microparticles containing coriander (Coriandrum sativum) essential oil were prepared using supercritical particles from gas saturated solutions (PGSS) technique to improve the stability of coriander. The effects of temperature, pressure, nozzle geometry and molecular weight of PEG on the morphology, entrapment efficiency and crystallinity of the coriander essential oil - loaded PEG microparticles were examined.

The biopharmaceutical industry faces increasing pressure to more rapidly develop innovative products. A potential therapeutic must face many hurdles: preclinical, manufacturing, and clinical efficacy to name a few. Method of all, therapeutic sample supply is one of initial hurdles for early phase product development. Break through challenge is to pass over the hurdles in an efficient and collaborative manner during out the beginning of development.At KITECH, we established strategy of cell culture process development in the early stage of development. Overall our goal is supporting service to medium or small firms which develop biopharmaceutical product using delivery of their precious product of sample for proof of concept study. We present structural and strategic approach for early phase process development, without large resources using 6 sigma.

In this study, crosslinking of PVA based hydrogels were performed with radiation processing technology using a Co-60 gamma source and followed by freeze-thawing (FT). The influence of time, concentration and dose on hydration and gelation of the hydrogel treated in different conditions such as oven-dry (OD), room-dry (RD) and freeze-dry (FD) has been investigated. The hydration of the hydrogels increased rapidly with time for first 48 h, and then become nearly constant in the range 48–120 h. The prepared hydrogels at lower concentrations and doses exhibit higher values of hydration but they represent a fall of these values at higher concentrations and doses. These occur mainly as a result of the increase of crosslinking density in the hydrogel by gamma-ray irradiation. Futhermore, FD samples have higher hydration values as compared with OD and RD samples. Considering this, the quality control of hydrogels for biomedical applications could be achieved to some extent by employing various processing conditions.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) has received great attention in recent years as a potential treatment. Previous studies have shown that primary acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts show a variable but overall poor response to TRAIL mediated cytotoxicity. Here we reported that KD548-Fc, a novel agonistic Kringle domain against DR4/5, possessed a strong cytotoxic activity in AML and ALL cell lines. KD548-Fc induces caspase-independent cell death in TRAIL-sensitive and –resistant cell lines, whereas TRAIL induces caspase-dependent cell death in only TRAIL – sensitive cell lines. The present study demonstrates that reactive oxygen species (ROS) were generated in Molt-4 and KG-1 cells upon KD548-Fc stimulation and that ROS accumulation subsequently evoked sustained activation of c-Jun Nterminal kinase (JNK). The reducing agent, N-acetylcysteine (NAC), effectively inhibited the sustained activation of JNK. These data provide novel information of the DR4/5 –mediated cell death-signaling pathway and may shed new light on effective strategies for AML and ALL tumor therapies.

The α-MSH antagonists prevent the synthesis of melanin resulting in reducing the formation of unwanted pigmentation and making the skin bright. In this study, we have developed the intelligent delivery system for the nonapeptide (Met-Pro-D-Phe-Arg-D-Try-Phe-Lys-Pro-Val- NH2) as a α-MSH antagonist, the pH-sensitive P(MAA-co-EGMA) hydrogel microparticles were prepared and their feasibility for the intelligent delivery carries was evaluated. The loading efficiency of nonapeptide at pH 7.0 increased as the amount of MAA in the hydrogel increased. The P(MAA-co-EGMA) hydrogel microparticles showed a pH-sensitive release behavior. At low pH (pH 4.0) small amounts of nonapeptide were released from the particles while at high pH (pH 6.0) relatively large amounts of nonapeptide were released from the particles. In addition, at pH 4.0 a very little amount of nonapeptide was permeated through the skin while at pH 6.0 relatively high skin permeability was obtained. For the treatment of the nonapeptide with pepsin, the hydrogel microparticles showed the protective ability for the nonapeptide and keep the stability of the nonapeptide.

Gene silencing by targeting specific genes for degradation, particularly at the mRNA level, is an invaluable tool for gene function analysis and a powerful therapeutic strategy for human diseases, including cancer and viral infections. For degrading cytosolic RNAs is the use of protein-based RNases and DNA/RNA-hydrolyzing mAbs, which can penetrate into living cells and degrade cytosolic RNAs. However, these approaches non sequence–specificity, leading to significant cytotoxicity. Based on a cell-penetrating, nucleic acid-hydrolyzing, 3D8 VL[1], we generated a synthetic library on the yeast surface by randomizing potential base interacting residues located in surface of c,c’,f-strands. And we considered that selected randomizing residues in framework by fixing several framework residues that affect directly or indirectly to CDR and antibody stability, like upper core and lower core, charged cluster. We isolated 3D8 VL variants with classical swine fever virus Npro and E2 genes –selective binding against 18-bp single stranded (ss)-RNA substrates, selected 3D8 VL variants that had ~10-fold higher affinity and ~2-fold greater selective affinity for target ss-RNAs than for off targets.

Previously we reported novel target-specifuc binding proteins of modest potency that was derived from a synthetic kringle domain(KD) library on the yeast cell surface. We isolated KD variants that bind to anti-cancer target proteins, such as human death receptor 4(DR4) and/or DR5 and human tumor necrosis factor-a (TNFa). We selected five KD variants (KD413, KD415, KD506, KD548 and KDT26) which have specific biological activity. The anti-DR4 KD variants (KD413 and KD415) and anti-DR5 KD variants (KD506 and KD548) function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and antagonistic KD variant that bind to TNFa (KDT26) neutralizes TNFa-induced cytotoxicity. However, the selected KD variants have low affinity (KD =~10-7M) compare with TRAIL and infliximab. For affinity improvement, we engineered loops of KD variants particularly loop5 and loop6 which are selected by loop mapping by yeast surface display. Using yeast surface display system, libraries were constructed to minimize the number of non-viable structures by rational design of nucleotide mixtures which bias for the wild-type (parent mutant) residues. The high affinity mutants of the KD variants were isolated by using MACS and FACS systems. Kinetic analysis of KD variants from isolated mutants revealed that the affinity was improved. In the conference, we will present the relationship between the affinity and biological activity.

Plasmid DNA (pDNA) vaccines have potential advantages over conventional vaccines, including higher safety and ability to induce long-lived immune response. The purification process for pDNA from impurities such as RNA and endotoxins in large-scale is required. The use of monolith chromatographic support which has higher porosity and throughput than those of conventional packed-bed support has the potential to be a new and efficient pDNA purification platform. We demonstrated that the use of Cu2+-IDA exhibited hierarchical preferential capture for endotoxins, RNA and pDNA in the decreasing order1. It was also shown that this hierarchical binding of pDNA and other impurities could be harnessed for developing a simple two-step pDNA purification process2. In the present study, we investigated the dynamic binding capacity of pDNA, RNA and endotoxins to Cu2+ chelated iminodiacetic acid (IDA) monolithic column. The dynamic binding capacity of RNA and endotoxins (determined from the frontal analysis of breakthrough curves) increased with the increase in RNA or endotoxins concentration, while that for pDNA is negligible. In addition, the breakthrough curves of RNA and endotoxins are independent of the flow-rate. These make the use of monolithic column ideal for large-scale processing at high concentration and flow-rate, rendering the purification process faster and more economically viable.

Silica-coated magnetite (Fe3O4) nanoparticles were surface-modified with iminodiacetic acid (IDA) to enable metal ion chelation1. The IDA-functionalized magnetite particles of various sizes ranging from 200 to 350 nm were produced by controlling two factors (i.e. sonication time and substrate feed rate) during the modification of nanoparticles. It is believed that sonication time is likely to affect aggregation of particles and the feed rate of tetraethyl orthosilicate(TEOS) control the formation of polysiloxane on the surface. Cu2+-charged IDA2 superparamagnetic nanoparticles of various diameters were used to investigate the size-dependent protein adsorption behavior of particles using bovine serum albumin as a model protein. It was found that the size of nanoparticles is closely related to binding affinity and capacity of the adsorbent since higher maximum adsorption capacity (Qm) and lower binding affinity (i.e. larger dissociation constant (Kd)) are exhibited with the increase in particle size. This size-dependent differential protein binding behavior Cu2+-charged IDA nanoparticles will be conducive to designing nanoparticle adsorbents of tailored affinity and binding capacity most opted for target protein purification or impurity removal applications.

N-glycosylation status of purified b-haptoglobin separated from sera of patients with prostate cancer was studied in comparison to that of sera from patients with benign prostate diseases, or normal subjects. Two different approaches, as summarized below, one based on binding of lectins and antibodies to b-haptoglobin, the other on mass spectrometry of released N-linked glycans from b-haptoglobin, were performed. Some of the results were useful for distinction of prostate cancer vs. benign prostate diseases. (i) Binding of Phaseolus vulgaris-L lectin (PHA-L), defining the GlcNAcb6Mana6Man side chain present in tri- or tetra-antennary N-linked glycans, to b-haptoglobin was higher for cases of prostate cancer and high-grade prostate intraepithelial neoplasia than for benign diseases. Binding of Aleuria aurantia lectin (AAL) defining a3-, a4-, or a6FucGlcNAc, or monoclonal antibody directed to sialyl-Lex, to b-haptoglobin was also higher for some of the cancer cases than for benign diseases. Many other lectins and antibodies showed no binding to b-haptoglobin, or showed no significant difference between cancer vs. benign diseases. (ii) Mass spectrometric analysis of N-linked glycans of b-haptoglobin released by Peptide N-glycosidase-F showed enhanced expression of monosialyl tri-antennary structures in prostate cancer cases. Thus, binding of PHA-L to affinity-purified b-haptoglobin from sera of patients could lead to development of useful tools for differential diagnosis of prostate cancer vs. benign prostate diseases.

The DNA of analyses are important in many area of research. Extensive theoretical investigations for DNA focus on the base pairs guanine and cytosine in nucleotide. Using method for analysis of guanine and cytosine were HPLC (High Performance Liquid Chromatography) and their chromatograms simulated by Aspen chromatography. C18 HPLC column and water/methanol/acetic acid mixture (90/10/0.2) were applied to separation guanine and cytosine. Chromatographic parameters (selectivity, resolution and number of theoretical plate) were calculated under different flow rates and sample concentrations. Calculated numbers of theoretical plate, retention time and porosity were utilized for simulation by Aspen chromatography, and we compared the simulations with experimental data.

The development of the alternative of fossil fuel is a hot issue. Especially, bio-ethanol is produced using food crops as a renewable resource and bio-ethanol is already commercialized for fuel in South America and Europe. However, bio – ethanol production from biomass needs a complicate and expensive pretreatment procedure for saccharification of lignin, cellulose, hemicelluloses. In the view point of food resource, it causes molar problem. Therefore we tried to produce bio–ethanol using the waste from beer culture broth without the pre treatment procedure. In previous study, it took much time to produce bio-ethanol using the waste from beer culture broth at moderate temperature because the saccharification rate is too low. In this study the simultaneous saccharification and fermentation was carried at high temperature in order to improve the saccharification rate because the optimum temperature of saccharification using α,β-amylase(starch hydrolysis) was at least higher than 60 oC. As a result, fermentation time was shorter. The production of byproducts, acetic acid and acetaldehyde was also investigated.