Earticle

Sea buckthorn (Hippophae rhamnoides) of the Elaeagnaceae family is a branched and spiny nitrogen-fixing deciduous shrub that is native to Eurasia and has been widely used for treating diseases in Tibetan and Mongolian traditional medicines[1,2]. Ultraviolet (UV) radiation, particularly UVB (290-320 nm), causes many adverse events in human including erythema, hyperplasia, hyperpigmentation, immunosuppression and photoaging[3,4]. In this study, the anti-inflammatory effect of SBSE was examined the mRNA and protein levels of interleukin-1α (IL-1α), IL-1β, IL-6, TNF-α, and COX-2 after UVB irradiation by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Also, the anti-aging effect of SBSE was investigated the mRNA and protein levels of MMP-1, type I procollagen, and laminin-5 after UVB irradiation by RT-PCR and western blot. Treatment of SBSE extract after UVB irradiation in cultured fibroblasts and keratinocytes significantly inhibited the expressions of IL-1α, IL-1β, IL-6, TNF-α, COX-2, and MMP-1 and stimulated the synthesis of type I procollagen and laminin-5 in mRNA and protein levels. These results point to the potential use of SBSE as agent for cutaneous inflammation and photo-aging response.

The potentials of medicinal plants and their miraculous properties are used for the skin health since human civilization. Intrinsic or extrinsic factors involved in the skin aging process, proceeds through MMP-1 catalyzed collagen degradation. The collagen plays a structural and functional scaffold in the extracellular matrix of skin and other tissues. The aim of the present work to screen the plant extracts of Dendropanax, Cornus, and Lycium, to search the potential inhibitor of MMP-1 expression. These plants were highly cited for their medicinal properties like anti-aging, anti-inflammatory, anti-oxidant, and immunoregulatory, etc... We extracted the plant’s active parts by various extraction procedures. The antioxidant properties of the extract were evaluated by the DPPH assay. The effect of extract on the collagen synthesis and cell viability (MTT assay) was measured to compare the inhibition in MMP-1 expression and toxicity caused, respectively. The fraction showed desirable activity was further purified by HPLC. The primitive result shows the potentials of the plant’s active compound can use as the cosmetic ingredient for minimize the aging dependent wrinkle formation.

Skin pigmentation is achieved by a series of process including melanin synthesis in melanocytes, melanosome release from melanocytes, and melanosome uptake by neighboring keratinocytes. Hyperpigmentation results from increased melanin production in the skin, and usually presents as age spots, uneven color, freckles and sometimes melasma.Tyrosinase is rate-limiting enzyme in melanogenesis, and influences on oxidation of tyrosine and DOPA. Thus, tyrosinase inhibition is the common target for skin whitening agents such as arbutin[1] and kojic acid[2]. Besides, recent studies suggest various mechanisms or approaches for developing skin whitening agents such as inhibition of melanosome transfers, acceleration of epidermal turnover and desquamation, antioxidants, and so on.In this study, we developed novel whitening agents which inhibit tyrosinase activity and antagonize melanocortin receptor 1(MC1R) at the same time. They are composed of natural origin compound region and peptide region, and prepared by solid phase synthesis [3]. Natural origin compound region having tyrosinase inhibitory activity was selected using mushroom tyrosinase assay. Through this method, caffeic acid and p-coumaric acid were screened. Also, peptide region having MC1R antagonist activity was designed by sequences originated from alpha-melanocyte stimulating hormone(α-MSH)[4] or agouti signaling protein(ASP). Whitening efficacy test of natural compounds-peptide derivatives was performed by melanin inhibitory assay using B16F1 melanoma cell line.

The pink-eyed dilution protein (P-protein or OCA2) plays a critical role in melanin synthesis in melanocytes and retinal pigment epithelium (RPE) cells. Mutation in this protein may cause complete or partial albinism. The workers claimed on bioactivities of P-protein ranges from synthesis of melanin to maturation and trafficking of the melanosome. The aim of the study is to compare the shape, size and count of melanosomes in melan-p1 and melan-a treated or not by SiRNA (siRNA for OCA2 mRNA). The above cells were examined by In vitro DOPA staining, and the melanosomes were characterized by the Transmission Electron Microscope (TEM). The results suggest significant changes in melanosomal morphology and their respective numbers. We are also trying to find out the role in trafficking of melanosome by co-culture method. The study is in progress. These results suggest that the P-protein can control the synthesis melanin as well as melanosome biology. Thus, we anticipate the role of P-protein in skin pigmentation can be exploiting for the development of new class of cosmetics as mutation or deletion of this protein does not cause any pathogenic effect.

To treat hyperpigmentating diseases such as such as melasma and lentigines, a number of melanogenesis inhibitors have been screened for their effectiveness in reducing melanin but none have shown to be completely satisfactory. Pink eyed dilution protein (P-protein) is a melanogenogenic protein and plays a critical role in cellular melanogenesis. The present study is aimed to develop an alternative molecular approach using P-protein small interfering RNA (siRNA) to control melanogenesis in the melan-a melanocyte. It has been observed the both P-protien and mRNA level were significantly lowered by the siRNA treatment. P-protein siRNA also suppressed melanin synthesis without any cytotoxicity in the melan-a melanocyte cells. These results suggest that molecular approaches using siRNA targeting P-protein may a novel approach for the control of the melanogenesis in a safer way.

Microphthalmia-associated transcription factor (MITF), a basic helixloop- helix ( bHLH) transcription factor with a leucine zipper. This is a key protein in regulation of the melanin biosynthesis in melanocytes. For the transcription initiation of the mRNAs responsible for translation of melanogenesis associated proteins, the binding of the MITF to E-box is required. The goal of this study is to evaluate the interaction of MITF with respective DNA binding site (E-box, CATGTG) by surface plasmon resonance (SPR). A Label-free gold chip based DNA-protein interaction assay, being used to evaluate the interactions. The MITF showed almost a twice higher resonance signal after binding with E-box, when compared to mutated E-Box. We have also assessed various parameters to stabilize the assay conditions, to diminish the false signals. We expect that evaluation of interaction provide a quantitative, comparative, high-throughput screening of MITF inhibitors, which will help to develop an effective methodology of screening of potential depigmenting agent.

Camellia japonica L. (Common camellia) seed oil with rich unsaturated fatty acids is a kind of functional oil. In the oriental region, the camellia seed oil has a long history of cosmetic usage as a protectant to keep health of skin and hair. Supercritical fluid extraction (SFE) is a very useful method for extracting valuable essential oil without any remaining organic solvent. Thus, this study focused on the process optimization of supercritical fluid extraction for camellia seed oil and their efficiency was compared with soxhlet extraction methods as a control experiment. The camellia seeds were sampled (5 kg), air-dried and chopped. Then, the essential oil was extracted using soxhlet and supercritical fluid extractor, respectively. And the DPPH activity and paper disc method were used for evaluating their comparative study on antioxidant and antibacterial activity. As a result, the optimal operating condition for SFE were found as 350 bar, 60 ℃. And the final yield of SFE and soxhlet extraction was found as 72.6 % and 36.8 %, respectively. The experimental results indicate that SFE extraction can be developed to be an eco-friendly process for obtaining the valuable essential oils such as Camellia japonica L. seed oil.

A sulfated polysaccharide, Fucoidan has been well studied as the anti-oxidant, anti-viral, anti- tumor and anti-coagulant, etc... We have proceeded with a aim to explore this molecule as an anti-melanogenic agent. It has been found, there was a significant reduction in melanin biosynthesis in melan-a cell, while treated with fucoidan, In vitro. The results showed that the concentration up to 100 ㎍/㎖, can reduces the melanin level approximately 40+5%, without any cell toxicity. A significant decrease in expression of cellular tyrosinase was observed. Means this molecule have some down regulatory role in expression of tyrosinase enzyme. Thus elucidation of the mechanism of action must be explored, which is poorly understood. In the present study, we have tried to evaluate the effect of fucoidan on the expression of major proteins, influencing the melanin synthesis. It is well known that melanin synthesis involves a series of reaction catalyzed by specific gene products. Therefore, we have targeted the key proteins like TYR, TRP-1, TRP-2, MITF, ERK, CREB etc. These present studies are important in terms of mechanism elucidation and for design and developing a fucoidan based safer cosmetics.

The proopiomelanocortin (POMC)-derived neuropeptide a-melanocyte stimulating hormone (a-MSH) is known to modulate some aspects of skin pigmentation. Production of the eu-melanin is controlled by a-MSH, by stimulating the melanocortin, 1 receptor (Mc1r) and the consequent reactions. Based on the computer added molecular structure prediction simulation and virtual screening the candidate molecules were selected, which may inhibit the binding of MC1R-aMSH complex formation, by intervening between them. The MC1R is a membrane-bound protein thus, In vitro binding assay with a-MSH is no possible, therefore, we have approached for the cell-based binding assay, to evaluate the binding properties of inhibitors. The human embryo kidney-293 (HEK-293) cells were immobilized in 96 well plate for study of biotin-streptavidin based fluorescence assay to measure the interaction of MC1R-aMSH complex inhibition by the inhibitor molecule. MC1R simulation causes the increase in cAMP, which has been used as a molecular marker for the interaction of inhibitor and MC1R-aMSH complex formation, thus lower the cAMP activity higher the inhibition. The results suggests, among selected molecules one was found to be better MC1R antagonist showing good binding competition. Here, we develop a level free biosensor to screen the antagonist and agonist of MC1R. We anticipate that this In vitro cell-based assay will be useful for high-throughput screening of MC1R antagonist or agonist.

Recombinant human granulocyte macrophage colony stimulating factor (rhGM -CSF), a glycoprotein induces proliferation of myeloid progenitor cells (1, 2), is produced from a variety of organisms such as Chinese hamster ovary, Saccharomyces cervisiae, E. coli, and Oryza sativa. rrhGM-CSF (rice cell origin, Oryza sativa) was recently shown to be more stable in-vivo than rhGM-CSF produced from different origins (3). However, in-vitro stability of rrhGM-CSF in liquid state has not been optimized, so the activity of rrhGM-CSF incubated at room temperature decreased more than 85% after 8 weeks. Heat treatment on rrhGM-CSF was carried out to increase stability, and rrhGM-CSF activity was evaluated by measuring proliferation rate of TF-1 cells (human pre-myeloid cell line). The activity of heat-treated rrhGM-CSF for 1, 2, 4, 6 min at 70℃ was preserved as 15%, 50%, 78%, 100% (± 20%) after 8 weeks, respectively. Protease inhibitor cocktail (Roche) also increased the stability of rrhGM-CSF. Therefore, reduction of rrhGM-CSF activity in liquid state is estimated due to plant-derived protease. The stability of heat-treated rrhGM-CSF was also affected by the degree of glycosylation. In the thermal stability of heat-treated rrhGM-CSF at 100℃, 30 min, high-glycosylated rrhGM -CSF was higher than low-glycosylated rrhGM-CSF. However, the stability was not different between high-glycosylated and low-glycosylated rrhGM-CSF in the case of heat treatment at 100℃ above 30 min. In this study, we increased the stability of rrhGM-CSF in liquid state by heat treatment, and confirmed that the thermal stability was affected by the degree of glycosylation of rrhGM-CSF.

Controlled release technologies have been applied to intracellular delivery of DNA [1]. The DNA carriers have been engineered to release an intact DNA at the optimal time and to the targeted subcellular compartment [2]. In this report, fibroin microcapsule encapsulating DNA was prepared and investigated. The property of DNA release from the fibroin microcapsules was analyzed using shear fragmented DNAs and using different core materials. Linear DNA ranging from 3 – 10 kb was released but closed circular plasmids were not released from unmodified fibroin microspheres. Spatial distribution of the DNA in fibroin microspheres was also investigated. The release of DNA molecules, its size-dependency and cellular delivery was investigated.

Cellular uptake of microcapsules has been focused on the efficiency, cellular trafficking or capabilities of cargo delivery. We investigated the effect of fibroin microcapsule on morphology and cell cycle of 3T3 cells cultured in vitro using flow cytometry. Cell shrinkage and increased granularity was observed in the cells co-incubated with various concentrations of fibroin microcapsule (up to 0.33 mg/mL), while cell viability was not affected. Because granularity can be increased by aggregated cells, the morphology change in mononuclear cells was identified by flow cytometric analysis of Propidium iodide (PI) stained cells indicated that and not caused by cell aggregates. It was also shown that cells co-incubated with 0.003 and 0.033 mg/mL of microcapsules had increased G2/M phase of the cell cycle. However, the distribution of cell cycle stages was not influenced by 0.33 mg/mL of microcapsules, suggesting cell cycle freeze. Analysis of the findings shows that cellular uptake of the fibroin microcapsule reduced cell size, increased granularity, and influenced cell cycle progression.

In this study, we prepared oil-in-water microemulsion using Tween80 and hydrophobically modified poly(vinyl alcohol) (HmPVA) as surfactant and polyethylene glycol 400 as co-surfactant. HmPVA would be able to play a role as surfactant because it has hydrophilic and hydrophobic functional groups. And epoxy propoxy coumarin (EPC) was combined with HmPVA, it has photo-dimerization characteristic at a specific wavelength UV, so that HmPVA/EPC microemulsion has photo sensitivity. HmPVA/EPC microemulsion droplet size was 100 nm and it was stable without UV irradiation, but it was unstable and droplet size increased under UV irradiation.

Photo-responsive surfactants obtained from Tween 20 and coumarin derivatives, which attached on the ends of PEO fragment of Tween 20 through ester or ether bond. The molar ratios of the coumarin residues to Tween 20 in both of these two conjugates having ester bond (TCES) and ether bond (TCET) were about 1:1.Coumarin residues of TCES and TCET could be photo-dimerized, and the photo dimerization degrees were in the order of TCET >TCES >free coumarin derivative. The air/water interfacial activity was in the order of TCET >Tween 20 >TCES, and the interfacial activities of TCES and TCET increased with the UV irradiation time. This possibly comes from the photo-dimerization of the couamrin residues on Tween 20. The decrease in the interfacial activity of TCES was slightly greater than that of TCET, probably because of the differences in the length, the polarity and the flexibility of the linker between coumarin residue and Tween 20.

Gold nanoparticles (GNPs) have unique properties which are surface plasmon resonance that make functional reagents for various biomedical applications including photothermal therapy. Most GNPs are prepared using growth-directing surfactants, such as cetyltrimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. We describe an approach to remove CTAB to a non-toxic concentration by PEGylaiton or antibody conjugation. The cytotoxicity of pristine and surface-modified GNRs was measured in human cell lines derived from thyroid cells. GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, sodium-iodide transporter (NIS), for thyroid cells. NIS antibody-conjugated GNRs specifically targeted human thyroid cells in vitro. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

The continuous spread of microbial pathogens with antibiotic-resistance has led to a growing interest in the design and development of new materials that are effective in killing bacteria. Photodynamic Therapy (PDT) is a promising approach for killing antibiotic- resistant bacterial strains. In this study, we synthesized a novel vancomycin- and photosensitizerconjugated multifunctional particle (MMPs; Fe3O4@t-PtCP&van) and characterized it as a new and effective antimicrobial reagent. The photodynamic killing of seven model organisms, including both gram-positive and gram-negative bacteria, was evaluated. The MMPs was capable of capturing these bacteria from aqueous solution. When captured microorganisms were exposed to the appropriate wavelength (510 nm), the photosensitizer closed to the bacterial surface effectively killed them with a low power density light. Gram-positive bacteria were completely killed in 6 min and gram-negative bacteria in 60 min. Antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) were also effectively killed in 6 min. The MMPs possessing magnetic and photophysical properties can be used in various medical applications, such as in magnetic resonance imaging (MRI) diagnosis, bioseparation, target delivery, and PDT.

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/RNAhydrolyzing 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 mAb, 3D8 VL, we generated a synthetic library on the yeast surface by randomizing potential base interacting residues located in surface of C, C’, F-strand, near F-strand framework 103, 105 residue. 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. Classical swine fever (CSF) or hog cholera (also sometimes called pig plague) is a highly contagious disease of pigs and wild boar. The infectious agent responsible is a virus CSFV. We isolated 3D8 VL variants with classical swine fever virus Npro and E2 genes–selective binding against 18-bp single stranded (ss)DNA substrates that substitute deoxy-uridin for thymine, selected 3D8 VL variants.

Fullerene is a form of carbon molecule that has wide application for the various fields of industry. Stable aqueous dispersions of C60 fullerene called nano-C60. We synthesized nano-C60 aggregates with different size ranges to determine their cytotoxicity in human cell lines derived from liver, lung, and skin. The nano-C60of size was controlled in the range of about 80 to 300 nm diameter.We confirmed that the differences in size distribution of C60 colloidal suspension are maintained in the presence of 10% fetal bovine serum for up to 48 hours and that their cytotoxicity is size-dependent. The cytotoxicity of nano-C60 is known that caused by reactive oxygen species (ROS) generation. Nano-C60-induced ROS reached the maximum concentrations at 6 hours in phosphate-buffered saline (PBS). The amount of ROS in PBS and the oxidative stress induced by C60exposure are also size-dependent.

The pro-apoptotic molecule death receptor 5 (DR5) is an attractive molecule for targeting agonistic monoclonal antibodies because of their inducing tumor specific cell death with little cytotoxicity in normal cell. In previously study, we obtained anti-DR5 human scFv HW1, which induces autophagic cell death in TRAIL-sensitive and –resistant cancer cells without cytotoxicity in normal cell. In this study, to improve agonistic activity of HW1 scFv without epitope change, we constructed HW1 mutant libraries by CDR randomization for affinity enhancement. Using spiked oligonucleotides, these theoretical retention rate levels of target residue 16% to 51%. Affinity maturated HW1A4 scFv increased the affinity was measured 20-fold than HW1. Also affinity maturated CDR grafted into stable human framework (VH3-Vk1), thermodynamic stability of engineered AU12 scFv was significantly improved. Affinity and stability engineered AU12 scFv from HW1 showed enhancement cell death activity in TRAIL-sensitive and –resistant cancer cell line and DR5 specific binding activity without changes of biochemical and biological specificity. These results suggest that affinity and stability improvement correlates their agonistic activity of cell death signaling through DR5.

Influenza NP is essential to viral replication, providing an ideal target for antiviral therapy. NP has been targeted at the protein level for antiviral therapy by a small molecule, but it is extremely difficult to be targeted by antibody since it is not exposed at the viral surface. Here we present a novel antibody-based antiviral strategy of targeting viral genomic RNA (vRNA) for degradation rather than neutralizing viral proteins. Based on the template of a seqeunce-nonspecific nucleic acid-hydrolyzing, single domain antibody of the light chain variable domain, 3D8 VL, we generated a synthetic library on the yeast surface by randomizing putative nucleic acid interacting residues. To target nucleocapsid protein (NP)- encoding viral genomic RNA (NP-vRNA) of H9N2 influenza virus, the library was screened against a 18- nucleotide single stranded nucleic acid substrate, dubbed asNP18, the sequence of which is unique to the NP- vRNA. We isolated a 3D8 VL variant, NP25, that had ~15-fold higher affinity (~54 nM) and ~3-fold greater selective hydrolyzing activity for the target substrate than for off targets. In contrast to 3D8 VL WT, asNP18 -selective NP25 constitutively expressed in the cytosol of human lung carcinoma A549 cells does not exhibit any signficant cytotoxicity and selectively degrades a reporter mRNA carrying the target asNP18 sequence in the stable cell lines. NP25 more potently inhibits the replication of H9N2 influenza virus than 3D8 VL WT in the stable cell lines. NP25 more selectively reduces the amount of the targeted NP-vRNA than 3D8 VL WT from the early stage of virus infection in the stable cell lines, without noticeable harmful effects on the endogenous mRNA, suggesting that NP25 indeed more specifically recognizes to hydrolyze the target NP-vRNA of H9N2 virus than off-targets. Our results provide a proof-of-concept of targeting viral genomic vRNAs by the sequence-specific nucleic acid hydrolyzing antibody for the prevention and control of influenza A virus infection in humans and animals.

Head and neck squamous cell carcinoma (HNSCC) is often resistant to conventional chemotherapy and thus requires novel treatment regimens. Here, we investigated the effects of the proteasome inhibitor MG132 in combination with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAIL receptor 1 (DR4)-specific monoclonal antibody, AY4, on sensitization of TRAIL- and AY4 -resistant human HNSCC cell lines. Combination treatment of HNSCC cells synergistically induced apoptotic cell death accompanied by caspase-8, caspase-9, and caspase-3 activation and Bid cleavage into truncated Bid (tBid). Generation and accumulation of tBid through the cooperative action of MG132 with TRAIL or AY4 and Bik accumulation through MG132-mediated proteasome inhibition are critical to the synergistic apoptosis. In HNSCC cells, Bak was constrained by Mcl-1 and Bcl-XL, but not by Bcl-2. Conversely, Bax did not interact with Mcl-1, Bcl-XL, or Bcl-2. Importantly, tBid plays a major role in Bax activation, and Bik indirectly activates Bak by displacing it from Mcl-1 and Bcl-XL, pointing to the synergistic mechanism of the combination treatment. In addition, knockdown of both Mcl-1 and Bcl-XL significantly sensitized HNSCC cells to TRAIL and AY4 as a single agent, suggesting that Bak constraint by Mcl-1 and Bcl-XL is an important resistance mechanism of TRAIL receptor-mediated apoptotic cell death. Our results provide a novel molecular mechanism for the potent synergy between MG132 proteasome inhibitor and TRAIL receptor agonists in HNSCC cells, suggesting that the combination of these agents may offer a new therapeutic strategy for HNSCC treatment.

A vital issue for a new drug carrier system based on self-assembly and polymerization of polydiacetylene amphiphiles in clinical application is analyzed. We have developed a novel polymerized vesicular carrier that composes of polydiacetylene and other types of phospholipids. Liposomes loaded with ampicillin as a model hydrophilic drug has been successfully constituted by polymerizable diacetylens into the lipid bilayer vesicle. Furthermore these liposomes were fused rapidly when the medium pH is lowered from 7 to 4. The polymerized liposomes have been characterized in terms of particle size distribution. Liposome fusion was demonstrated by Dynamic light scattering (DLS) and TEM image. Cross-linking of the diacetylene lipids prevents the drug leakage and the encapsulated drug can be instantaneously released at low pH condition. Finally, the therapeutic effect was observed by the appearance of plaques on a lawn of bacteria and fluorescent image of PDA liposomes on plaques. As a result, this research demonstrates that such novel pH-sensitive polymerized liposomes have very good prospect as a drug carrier.

Vesosomes are widely used in the field of medicine, science, and engineering as model cells. However current techniques to create vesosomes are not up to date requiring long time (up to one day) and need expertise. Vesosomes are also difficult to control diameter, resulting in broad ranges of size distribution. Therefore, we demonstrate an automated platform of multiple materials-encapsulated vesosomes using microfluidic devices integrated with two phase T-junction systems. Water soluble fluorescently labeled liposomes are encapsulated into vesosomes. The entire system is integrated with specially designed microfluidic device, enabling full automation of vesosome formation with multiple encapsulants. The principles of automated vesosome formation employed a T-junction microfluidic device with two phase system, resulting in the monolayer of lipid along the interface. Emulsions with the lipid monolayer from the T-junction is introduced to another interface of a lipid monolayer. Specially designed microfluidic system enables this entire process automatable. To demonstrate that the vesicles generated from the device are vesosomes with multiple encapsulants, we show microscopic images from fluorescent microscope and confocal laser scanning microscope along with size distribution. Our work with vesosomes with multiple encapsulants will provide simple and efficient methods and apply to various fields of research including model cell/membrane studies.

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 (DR) 5. We selected two KD variants (KD506 and KD548) which have tumoricidal activity. The anti-DR5 KD variants (KD506 and KD548) function as agonists to induce apoptotic cell death in several cancer cell lines in vitro. However, the selected KD variants have low affinity (KD = ~10-7 M) compared with TRAIL and other anti-DR5 antibodies. 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 (YSD) 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.

Granulocyte colony-stimulating factor (G-CSF) is a growth factor or that serves as a major regulator of the development of neutrophils. NEUPOGEN® (Filgrastim), a recombinant methionyl human G-CSF injected daily, was launched in 1991 for the treatment of cancer therapy-induced neutropenia and associated infections. PEGylated version of its parent molecule NEUPOGEN® is called NEULASTA® (Pegfilgrastim), which is a long-acting formulation of NEUPOGEN®. Alteogen, Inc. has developed a novel long-acting technology for extending in vivo half-lives of biodrugs using NexP fusion carrier. A CHO cell-line which stably expresses the recombinant G-CSF/NexP fusion protein was developed and after cultivation of the cell in a 10L single-use bioreactor (CELL-tainer, CELLution Biotech BV, Netherlands), we purified G-CSF/NexP fusion proteins to more than 99% purity by three chromatography steps. Rodent studies indicated that the G-CSF fused to NexP showed an increased in vivo half-life and also the duration of the long-acting activity measered by granulocyte production was substantially increased compared to that of daily G-CSF. Toxicokinetic studies of G-CSF/NexP fusion protein in rodents were alto performed.

Corni fructus was screened with anti-angiogenesis experiment for anti-obesity candidate which exerted dose-dependent inhibitory effects against angiogenesis, adipogenesis, and cell adhesion. C. fructus extract (CFE) exhibits an angiogenesis inhibitory effect superior to that of the EGCG from green tea leaves. The expression level of angiogenesis and dipogenesis-related signal molecules in the western blotting was reduced by increasing the amount of added CFE. Also, CFE was suppressed the U937 cell adhesion in a dose-dependent manner, by percentages of 50%, 112%, 231%, and 297%, respectively. The effect of CFE on lipid accumulation was measured with Oil red O staining of 3T3-L1 cell, relative to the control cells, CFE treatment reduced intracellular lipid content in a dose-dependent manner, and the most effective dosage for inhibition was found to be 25 mg/l of CFE, CFE could inhibit lipid accumulation in 3T3-L1 adipocytes in a dose-dependent manner. Moreover, a diet supplemented with CFE was deemed more effective in inducing weight loss in LB mice than a representative synthetic diet drug, orlistat, which incidently caused the side effect of denuding the mice of their hair. These results indicate that C. fructus may prove to be a useful anti-adipogenic compound, and these in vitro results may be reflected later under in vivo conditions.

Many of the known Cell penetrating pepetides (CPPs) share the common features of positively charged amino acids and amphipathicity. Unlike the other CPPs, PLAC was very short and possessed hydrophobic contents without basic amino acids. Translocation of PLAC across the cellular membrane was significantly affected by temperature, whereas ATP depletion and endocytosis inhibitors did not affect. And cellular uptake did not differ between D- and L-enantiomer of PLAC. PLAC was studied for its capacity to act as a vector molecules. The cargo protein, 120kDa β-galactosidase, was fused to the PLAC, results in translocation of the biologically active fusion protein to cytoplasm in cells. Furthermore, intraperitoneally injected PLAC fused β-galactosidase was almost delivered to each tissues in mice. More effective cell penetrating peptide (PLAC2W) was developed by amino acid substitutions of PLAC. Interestingly, uptake of PLAC-2W was inhibited by temperature, ATP depletion, and D-isomer unlike PLAC. Moreover, uptake of PLAC-2E significantly decreased by amiloride and slightly decreased by nystatin and filipin. These results indicate that cellular translocation of PLAC- 2W occur mainly through macropinocytosis pathway, while caveolae-mediated endocytosis is the minor pathway.

The cultivation of microalga is drawing attention as solution for elimination of CO2 in fuel gas from industries and plants. The microalgae have capability to reduce CO2 and to produce various pharmaceuticals, and cosmetics and biofuels. Before application of microalge to industry for reduction of CO2, the effect of fuel gas was considered. The fuel gas contains high CO2, NOx, and fine dust. In this study, Haematococcus pluvialis was cultured in outdoor condition which was maintained at 2 2℃ of temperature, 50~200 mol photon/m²s of light intensity and 25L polymer bag type of photo-bioreactor. H. pluvialis was cultivated with 20 % fuel gas and 80 % air a week for adaptation to high level CO2 and NOx at first, and then fuel gas concentration was increased up to 50 %. And because the H. pluvialis has two stage, green vegetative stage and red cyst stage, the experiments were conducted under each two stage conditions, green vegetative stage and red cyst stage. At the red cyst stage, there was no big difference between fuel gas and pure CO2. The dry cell weight was obtained almost 2 g/L and astaxanthin was 70 mg/L for 50 days. However, at the green vegetative stage, the induction of H. pluvialis was inhibited by fuel gas.

The carotenoid pigment astaxanthin is strong antioxidants that have important applications in the pharmaceutical, cosmetics, food, and feed industries. Haematococcus pluvialis is known to be the richest source of natural astaxanthin. Generally, detection methods of astaxanthin call for extraction process of astaxanthin by organic solvents, incorporating breakdown processes of cyst cells such as mechanical disruption, spray drying, enzyme lysis, have been developed for the separation of astaxanthin from the cyst cell. Our results suggest that application of the lipidic dye Nile red for detection of lipophilic compound astaxanthin. By staining cells with Nile red, we can sense the low fluorescence of astaxanthin contents. In this work, Nile red was used as an indirect measurement of cellular astaxanthin contents. This method allowed the development of cytometric method faster than the traditional techniques for detection of astaxanthin in cyst cell.

Streptomyces sp. strain BCNU 1001 was isolated from forest soil samples. Cultural, morphological, and physiological characteristics as well as 16S rDNA analysis revealed that the isolate, BCNU 1001, belonged to the genus Streptomyces. The antimicrobial activity of the ethyl acetate extract was confirmed using the broth microdilution technique. The minimum inhibitory concentration (MIC) of the BCNU 1001 ethyl acetate extract was 0.25 mg/ml for Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, and 0.125 mg/ml for Micrococcus luteus, Staphylococcus aureus, and Pseudomonas fluorescens. The MIC of the BCNU 1001 ethyl acetate extract for Aspergillus niger, Candida albicans, and Saccharomyces cerevisiae was 0.5 mg/ml, 0.125 mg/ml, and 0.25 mg/ml, respectively. BCNU 1001 was also active against dermatophytic fungi such as Trichophyton mentagrophytes and T. rubrum. Furthermore, BCNU 1001 was also found to be effective against Methicillin-resistant Staphylococcus aureus (MRSA), and its ethyl acetate extract showed MIC = 0.5 mg/ml against MRSA. The most abundant antimicrobial compound was identified as a 2-hydroxybenzyl alcohol through analysis utilizing a nuclear magnetic resonance spectroscopy. This compound was seen to be very effective against some kinds of bacteria and fungi.( This research was financially supported by the Korean Ministry of Education, Science Technology (MEST), and Korea Institute for Advancement of Technology (KIAT) through the Human Resource Training Project for Regional Innovation)