Earticle

Melanogenesis is a multistage process involving melanin synthesis, melanin transport and melanosome release. Melanin synthesis is stimulated by various effectors, including α-melanocyte-stimulating hormone (α-MSH), cyclic AMP (cAMP)-elevating agents (forskolin, isobutylmethylxantine, and glycyrrhizin) and ultraviolet light. In this study, the water extract from mangosteen (garcinia mangostana) leaves was assessed with regard to its effects on melanogenesis in B16F1 melanoma cells line. The mangosteen leaves extract was shown to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner without any significant effects on cell proliferation. Formation of melanin from cultured B16F1 melanoma induced by mangosteen leaves extract treatment was estimated using spectrophotometer. In the range of non-cytotoxic concentrations at 7.8x10-4 % ~ 6.2x10-3 % (w/v) mangosteen leaves extract strongly increased melanogenesis. The results obtained clearly shows that mangosteen leaves extract increase melanogenesis almost 2.5 folds compare to the α-MSH control group. In order to clarify the further mechanism of tyrosinase activation by mangosteen leaves extract, the level of tyrosinase expression in B16F1 cells were examine by intracellular tyrosinase assay and tyrosinase zymography. The result showed that tyrosinase activity was markedly enhanced from mangosteen treated cells. These results suggested that mangosteen leaves extract might be one of the promising candidates in order to treat hypo pigmentation disorder and useful for self tanning cosmetic products.

As a herbal medicine, seaweed has been used in traditional cosmetics, in treatments for cough, boils, goiters, stomach ailments, and urinary diseases, and for reducing the incidence of tumors, ulcers, and headaches. Despite the fact that seaweeds are frequently used in the practice of human health, little is known about the role of seaweed in the context of inflammation. This study was aimed to investigate the influence of Jeju endemic seaweed on RAW 264.7 cells under the stimulation of LPS. In this study, ethyl acetate extracts obtained from 14 different kinds of Jeju seaweeds were screened for inhibitory effects on pro-inflammatory mediators. Our results reveal that 5 seaweeds, Laurencia okamurae, Grateloupia elliptica, Sargassum thunbergii, Gloiopeltis furcata, and Hizikia fusiformis, were potent inhibitors of the production of pro-inflammatory mediators such as NO, PGE2, IL-6, and TNF-a. Based on these results, the anti-inflammatory effects of these seaweeds, together with their low cell toxicity, suggest potential therapeutic applications in the regulation of the inflammatory response.

Various natural herbal sources provide a defensive mechanisms against various skin diseases especially allergic skin diseases. Now days, most of them are caused by contaminated environmental pollution and related reactive oxygen species. Based on DPPH assay, three oriental herbal sources, Ledebouriella seseloides root, Scutellaria baicalensis root, and Bupleurum falcatum root showed strong anti-oxidant activities among 28 oriental herbal sources. We found that specific ratio among three herbal sources could show more increased anti-oxidant effect and anti-atopic effect with lowered cellular toxicities. We conducted the anti-atopic dermatitis effects on two pathological phenomena. One is expression of defensin peptide and the other is expression of atopic dermatitis-mediated cytokine. On natural human skin, there are defensin peptides with a major role in the barrier function and the innate host immunity against bacteria. These antimicrobial peptides are small, cationic, amphiphilic peptides of around 50 to 70 amino acids with distinct three set of disulfide bonds. So far, isoform 3 (human beta-defensin 3, hBD-3) is suggested as key defensin in human keratinocyte. The specifically formulated herbal extract showed an increase of hBD3 and decrease of atopic dermatitis-mediated cytokine.

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. Much effort has been paid to develop new therapeutic agents against pigmentation abnormalities, especially by using novel biologically active compounds from natural plants. To develop a novel skin depigmenting agent from natural sources, the inhibition of melanogenesis by Chinese plant, N. glandulifrea, was evaluated. The methanol extract of this plant showed significantly down-regulated melanin synthesis in a dose dependent manner at a non-toxic concentration in cultured B16F10 mouse melanoma cells. This extract was further fractionated by using bioactivity-guided fractionation and identified as glycerol monolinoleate by means of NMR, IR and HPLC. This compound showed significant depigmenting activity both in B16F10 mouse melanoma cells and melan-a cells. Compared with arbutin (IC50 = 0.74 mM) as a positive control, the depigmentation IC50 value for this compound was 9.1 μM. Its inhibitory effect on cellular tyrosinase, the key enzyme of melanogenesis, was observed. These results suggest that glycerol monolinolate isolated from N. glandulifera is a promising compound that could be useful for treating hyperpigmentation as a skin-whitening agent.

Isoflavones, a family of the major groups of phytoestrogens, are known to have anticarcinogenic, antioxidant and photoprotective effects. Although they have beneficial bioactivities for human health, low bioavailability and poor solubility have been problems in applications. To address this problems, we first have optimized large-scale conditions for enzymatic bio-conversion of crude isoflavone glycosides(≤20%) from soybean into aglycones by using a β-glucosidase. Further purification processes enabled us to obtain more than 90% purity of isoflavone aglycones analyzed by HPLC. The purified isoflavone showed to a have strong anti-inflammation activity. UVB irradiation of HaCaT cells induced tumor necrotic factor-α (TNF-α)production by 3 folds but the treatment of the purified isoflavone reduced TNF-α production back to the normal level. In order to make derivatives of isoflavone, which might have novel bioactivities with better solubility, acylation reactions of the purified isoflavone were first performed and the resulting isoflavone derivatives were purified for further analyses. Especially, mono-palmitoyl isoflavone not only reduced TNF-α production at much lower concentration than that of isoflavone aglycone, but showed increased solubility by 10 folds compared with isoflavone aglycone. Our results show that mono-palmitoyl isoflavone can potentially enhance bioavailability of isoflavone due to increased solubility and bioactivity.

Vitiligo is an acquired disorder of pigmentation in which depigmentation of skin and hairs occurs due to loss of functional melanocytes from the epidermis. It is known that the existence of inactive melanoblasts in the hair root follicles provide a melanocyte source for repigmentation in vitiligo. Migration of these melanocyte precursors from the outer root sheath of hair follicles into clinically depigmented epidermis is crucial for repigmentation in vitiligo therapy. An effective treatment has been proposed that would stimulate these inactive melanoblasts to migrate from the hair follicle to the nearby epidermis. Although the current phototherapies may induce varying degrees of repigmentation in vitiligo lesions, treatment challenges persist, as either not all patients respond to available therapies and the treatment duration is too long or sometimes a plateau effect is seen with the treatment becoming less effective after an initial response. Lowpower lasers and light-emitting diodes (LED) are well-accepted therapeutic tools in the treatment of infected, ischemic, and hypoxic wounds, along with other soft tissue injuries. To evaluate the potential of LED on melanoblasts, a Melb-a cell line was subjected to LED irradiation and its effect on differentiation, proliferation and migration of melanoblasts were investigated. The findings of the present study may be important in developing new strategies for vitiligo treatment.

Skin color is determined by the amount of melanin present. Many enzymes have a relationship in melanin synthesis. Especially, tyrosinase is well known as key enzyme for melanin synthesis. MITF (Microphthalmia-associated transcription factor) belongs to the basic helix-loop-helix-zip family of transcription factors and is the major regulator of tyrosinase and other related enzymes (TRPs) by binding E-box (CATGTG). To screen for MITF – E-box binding inhibitor, EMSA (Electrophoretic Mobility Shift Assay) was performed. Based on the computer-simulated structure, 27 chemicals were selected and were investigated as MITF – E-box binding inhibitors. To check the depigmenting activity, cell tests were performed by using melan-a cells. Among them, compound #18 was found to show the most potent inhibitory activity against MITF – E-box binding. As a result of EMSA, intensities of MITF – E-box bands were reduced by compound #18 in a dose-dependent manner. In addition, unlabelled Tyr-p, mutated Tyr-p and random oligomer were used for EMSA to investigate the competitive binding inhibitory effect. As a result, excess unlabelled Tyr-p was an effective binding competitor. Conversely, mutated Tyr-p and random oligomer did not compete with MITF. To check the protein level of MITF, tyrosinase, and TRP-1, western blot analysis was performed by using melan-a cells. The protein level of tyrosinase was reduced by compound #18, but MITF and TRP-1 were not reduced. Therefore, compound #18 could be used as a specific MITF – E-box binding inhibitor on melanocytes.

Melanin is a pigment that plays an important role in providing coloration and protecting human skin from the harmful effects of UV light radiation. However, the overproduction and accumulation of melanin in the skin could lead to a serious skin disorders. 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 are completely satisfactory. M uch effort has been paid to develop new therapeutic agents against pigmentation abnormalities, especially using novel biologically active compounds from natural plants. To develop new and safe skin whitening agents from micro algae, several species of Chlorella have been evaluated. From our screening results, Chlorella kessleri culture broth extract can inhibit melanin synthesis of murine B16F10 melanoma cells in a dose dependent manner and exhibited no cytotoxicity up to a concentration of 200 ug/ml. Inhibitory activity on tyrosinase, the key enzyme of melanogenesis, and antioxidant activity were further investigated. This culture broth extract exhibited no inhibitory effects. Based on DPPH assay, this culture medium extract showed minimal antioxidant activity. These results suggest that Chlorella kessleri culture broth extract could be a high potential candidate as a skin depigmenting agent. To isolate the active components in this plant, solvent-solvent partition was performed. The chloroform fraction showed significant inhibition properties on melanin synthesis with low toxicity. For further isolating the effective compound, silica open column chromatography and high performance liquid chromatography were used.

Phytochemicals from oriental herbs have been reported to exhibit useful functions as anti-viral, skin and hair care. Since cyclodextrins have hydrophobic cavities, they are easy to assemble with small hydrophobic molecules. The encapsulation into cyclodextrin will affect slow degradation of useful chemical compounds like phytochemicals. Phytochemicals encapsulation products by cyclodextrin, such as shampoo, hair tonic, body wash and cream, were carried out to investigate the functional changes for toxicity, hair growth, antimicrobial activity and hydroxyproline content. We prepared the cosmetics containing phytochemical supramolecules by mixing the surfactants, fragrances and the crude extracts of eight oriental herbs with cyclodextrin. And we have tested the animal system for the hair growth and hydroxyproline content and toxicity. We used three microorganisms for test to antimicrobial activity. In these studies, we suggest the supramolecular encapsulation of phytochemicals can give the benefits of advantageous changes for the increasing of antibiotic activity and hair growth and delaying the aging of skin.

The anti-aging and whitening effects of Aloe extract were studied using proteome analysis. Aloe extracts were prepared by hot water, ethanol, and supercritical fluid extraction. supercritical fluid extraction was performed at pressure of 200~350 bar and temperature of 40~70℃. The effect of extract was measured antioxidant activity [DPPH (1,1-diphenyl- 2-picryl hydrazyl) radical scavenging assay] and skin whitening activity (Tyrosinase inhibition assay). Use proteome analysis (2D-PAGE), expression level changes of the main proteins related to skin aging inhibition and skin whitening by Aloe extracts were observed. The human fibroblasts and mouse B16F1 melanoma cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with or without extracts. Isoelectric focusing (IEF) was carried out using commercial immobiline dry strips 24cm. SDS-PAGE was performed using 25 x 21cm, 10~12.5% polyacrylamide gel. Gels were stained with silver nitrate. Following proteome analysis could explain the mechanism and expression pattern of skin aging and whitening related proteins.

Paraben preservatives are popular in many cosmetic products but they causes a lot of skin troubles and other harmful effects on human health. In order to replace this paraben compounds, much researches on natural and safe preservatives have been done. In this study, novel organoclays were tested for cosmetic preservatives. The microbial challenge test was performed followed by USP XXIV method, and CTFA Method M-3 and M-4. The strains used are as follows: E. coli KCTC 1682, Staphylococcus aureus KCTC 1927, Candida albicans IFO No 1594, P. aeruginosa KCTC 2513, Aspergillus niger ATCC 16404, Enteric bacilli, and S. aureus. Organoclays inhibit the bacterial growth from 1% concentration in pH dependent manner. For fungi, there have been no growth inhibition activity. This study shows that 99.99% death of bacteria was observed in 7 days. Thus Mg- and Ca-organoclay could be used as antibiotic alternatives. Further investigation should be performed to be used as preservation ingredients in cosmetics.

Edible and medicinal mushrooms have been immensely popular in Asia for millennia. There are primarily three ways to extract the medicinal compounds from these mushrooms. In this study, hot water extraction methods were used to make soap, cosmetic face pack and drinking pouch products for the application to the skin with atopic dermatitis and acne symptoms by Propionibacterium acne. The complex mushroom extracts are made of the following seven edible and medicinal mushrooms; Tremella fuciformis, Cordyceps militaris, Ganoderma lucidum, Lentinus edodes, Agaricus blazei Muril, Phellinus linteus, and Inonotus obliquus. Preliminary clinical trials were progressed and the results were recorded for 4 weeks. According to photos taken before and after the treatment and questionnaire results, considerable improvements in skin conditions are observed in the patients with atopic dermatitis and acne. For atopic patients, erythema and edema have been improved but the degree of effect was dependent on the individual’s constitution. Concerning acne, the effect of coating of the extracts was prominent for first week and the degree decreased with time till 4 weeks. The complex mushrooms extracts were efficient in soothing rash and maturation. Side effects such as a scar were not detected during the application and treatment.

In recent years the introduction of computer systems for data handling in the pharmaceutical, biotechnology and medical device industries has increased. Computer systems introduced in GMP-areas of pharmaceutical companies have to be validated. But Computer System Validation (CSV) activities may consume much cost and time. If it is possible to use Risk Based Lean Compliance Methodology (RBLCM), it will be more effective in validating standard or customized IT systems than traditional ones. The RBLCM consists of 4 CSV phases; validation planning, specification definition, protocol design and qualification phases. Also it is defined 2 risk assessment activities; GMP impact assessment and user requirements assessment for systems. And while performing CSV projects, it referred the vendor’s documents; function design, database design, unit test reports and integration test reports. It is expected that this methodology can improve compliance with regulatory expectation, reduce the cost and time consumed by duplicated works and eliminate the need for expensive retrospective validation.

Breast cancer is the most common cancer in women and second most deadly. The onventional treatment involves a non-specific systemic delivery of therapeutic agent and this often results in unwarranted side effects which greatly decrease the patient’s quality of life. This work aims to identify aptamers capable of specifically targeting the breast cancer cell surface protein to develop: 1) multifunctional magnetic nanoparticles for in vivo breast cancer detection and targeted therapy; 2) biosensor for the detection of tumor biomarker. Human epidermal growth factor receptor 2 (HER2) is a transmembrane protein overexpressed in 20-30% of breast cancers. HER2 positive cancer is notorious for higher fatality and recurrence rates primarily due to strong drug resistance of HER2 positive cancer cells. Herceptin, a monoclonal antibody cognitive of HER2 extracellular domain (ECD), is currently used in the tr atment of HER2 positive cancer. Aptamer has advantages over monoclonal antibody in targeting HER2 in terms of lower cost, equal or greater targeting specificity, lower immunogenicity, more hydrophilic (hence less likely to cause particles aggregation in an aqueous environment once it is immobilized onto the particles), greater stability, and reproducible synthesis. We characterized the binding affinity and specificity of single-stranded DNAs, screened via 10 rounds of CE-based SELEX method, against HER2 ECD. The percentage of ssDNA eluted from the nitrocellulose membrane filter binding assay, measured for the fractionated DNA pool following each round of CE-based SELEX screening increased with selection rounds, indicating successful enrichment of aptamers with enhanced affinity to the target protein. The affinity of the selected aptamers to HER2/Fc protein after the 10th round was also tested by capillary electrophoresis. After incubation of HER2/Fc protein with the selected aptamers at 1:1 ratio, a significant reduction (ca. 46%) of free aptamer peak during voltage separation (as compared to the aptamer peak in the absence of protein) was found, further confirming that 10 rounds of CE SELEX screening led to a successful enrichment of affinity aptamers to HER2/Fc. The specificity of the screened aptamers toward HER2 positive breast cancer cell line (SkBr3) is under investigation with the use of breast cancer cell line (MDA-MB-231) lacking HER2 ECD as a control. Following the confirmation of specificity of the screened aptamers, their targeting efficacy (through fluorescence microscopy observation), cell ytotoxicity, and cell growth inhibition (via MTT assay) will be further tested to judge the suitability of harnessing the screened aptamers for targeted aptamer-based imaging, therapy and diagnostics of breast cancer.

The present study was investigated that astaxanthin improves stem cell potency via an increase in the proliferation of neural progenitor cells (NPCs). Treatment of astaxanthin (1, 5, and 10 ng/ml) for 3 days significantly increased the proliferation of NPCs. Especially, 10 ng/ml astaxanthin applied for 3 days showed the highest proliferation of NPCs. A clonogenic (CFU) assay was also performed to estimate the proliferation efficiency of the astaxanthin-treated NPCs. In CFU assay, 10 ng/ml astaxanthin-treated NPCs had an approximately 2-fold increase in colony formation. To identify the possible activated signaling molecules involved in active cell proliferation occurring after astaxanthin treatment, the total protein levels of several proliferation-related proteins and expression levels of proliferation-related transcription factors were assessed in NPCs by Western blot analysis and RT-PCR. In Western blot analysis, astaxanthin induced significant activation of PI3K and its downstream mediators, p-Rac, p-c-Raf, p-MEK, p-ERK, p-Akt, p-mTOR, and p-Stat3 in a time dependent manner. The results of RT-PCR analysis showed upregulation of proliferation-related transcription factors (Rex1, CDK1, and CDK2) and stemness genes (OCT4, SOX2, Nanog, and KLF4). To estimate the relevance of PI3K and MEK signaling pathways in cell growth of astaxanthin–treated NPCs, inhibition assays were performed with LY294002 (10 mM, a specific inhibitor of PI3K) and PD98059 (10 mM, a specific inhibitor of MEK). These results clearly showed that astaxanthin induces the proliferation of NPCs via the activation of PI3K and MEK signaling pathways and improves stem cell potency via stemness acting signals, including OCT4, SOX2, Nanog, and KLF4.

Synthesis and characterization of nanocrystalline trivalent silver polydiguanide complex was described. The antibacterial activity of the synthesized nanoparticles was evaluated against burn wound pathogens. Synthesis of essentially monodispersed nanoparticles of higher valent metal complex was accomplished by oxidation of the monovalent silver, followed by stabilization of the oxidized higher-valent metal through complexation with chlorhexidine, a polydiguanide ligand in a reverse microemulsion at room temperature. Their antibacterial potencies were assessed invitroby determining the MICs and MBCs against four Gram-positive and four Gram-negative bacteria using agar dilution and microdilution methods. The synthesized nanoparticles showed strong antibacterial activity against the tested Gram (+)/(-) and methicillinresistant Stahylococcusaureus (MRSA) strains. The minimal inhibitory concentrations (MICs) of the synthesized complex were much lower than those of the ligand, AgNO3, and the gold standard, silver sulfadiazine. These nanoparticles may serve as a new generation antibacterial metallopharmaceutical in wound care.

Gemcitabine (2,2-difluorodeoxycytidine, dFdC) is an effective antitumor agent in the treatment of several solid tumors. However, it has been reported that gemcitabine has problems such as short plasma half-life, rapid metabolism, and low selectivity towards tumor tissue. In this study, gemcitabine-loaded biodegradable poly(L-lactic acid) (L-PLA) microparticles were prepared by a supercritical fluid process, called aerosol solvent extraction system (ASES). ASES is a supercritical technique based on solvent extraction using supercritical carbon dioxide for the preparation of microparticles and microspheres. The influence of operating parameters such as temperature, pressure, concentration, CO2 flow rate and solution flow rate on the morphology and characteristics of the microparticles was studied in detail. The drug entrapment efficiency and loading efficiency were determined by HPLC assays. The entrapment efficiency increased with increasing temperature, concentration and CO2 flow rate. The ASES-processed drug-loaded microparticles showed a relatively high initial burst due to the presence of pores of the microparticles and the poor affinity between gemcitabine and L-PLA.

Protein function based on its folding state is very interesting. Alpha-lactalbumin (α-LA) can be a good example. Major function of α-LA is to synthesize lactose as a coenzyme but it gains a more interesting function, tumor selective cytotoxicity, once it forms a stable folding intermediate (apo-state) complexed with oleic acid [1, 2]. Many researches focused on characterizing the structural properties of α-LA and its complex have been done using far/near UV circular dichroism [3] and heteronuclear NMR spectroscopy [4]. Fast et al. reported the thermodynamic studies on the stability of α-LA and oleic acid complex using differential scanning calorimetry [5]. Isothermal titration calorimetry (ITC) directly measures the heat exchange between binding partners without labeling or immobilization. Thermodynamic parameters such as stoichiometry, affinity constant, enthalpy change are measured from ITC experiment. In this work, ITC was evaluated as a tool for determining the stoichiometry in complexation of bovine α-LA with oleic acid. Ca++, ANS (8-anilino-1-naphathalene sulfonic acid) fluorophore, and sodium oleate were titrated into apo bovine α-LA. Enthalpy change (ΔH), entropy change (ΔS), Gibbs free energy (ΔG), affinity constant (Ka) were obtained for each condition. Also the ANS and intrinsic fluorescence experiments confirmed the apo state of α-LA after Ca++ were detached from the protein. The stoichiometry for Ca2+ binding was ranged from 0.3 - 0.7, that for ANS was from 5.0 - 6.0. Also the stoichiometry for sodium oleate was ca. 6.0 but in the presence of chelating agent (5 mM EDTA) it increased to 8.0 - 9.0. The ΔG for Ca2+ binding at 45℃ was -37.0 kJ/mol and that for ANS at 40℃ was -20.6 kJ/mol indicating that calcium binding was stronger than ANS. This work demonstrated how the ITC analysis could contribute to understanding of the hermodynamics underlying the protein folding and binding phenomenon.

Recently porcine islet is emerging as an alternative source for clinical islet transplantation regardless of unaddressed barriers. The purpose of this study was to utilize PEGylation (PEG-SCM/PEG-lipid 1% wt/vol) in combination with a subtheraputic dose of FK506 (0.1mg/kg, IM) in an attempt figuring out an efficient apparatus for islet transplantation. The imaging data showed that islets were well covered with PEG-lipid; furthermore, modified islets had no significant difference compared to control islets in terms of functionality and viability as evidenced by apoptosis rate, insulin secretion, and OCR/DNA. M ore importantly, PEGylated islets revealed the PEG protective effect on islets as shown by CFSE-base assay. As in combination with FK506, the histological data suggested that engineered islets stayed intact, less infiltrated macrophages and somehow more generated vascular compared to the control group. Taken together, our findings indicated that PEG-lipid and FK506 would be an effective regime to have islets resisted against immunological system.

In this study, gemcitabine-loaded poly(L-lactic acid) (L-PLA) nano- and microparticles were prepared 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 coaxial nozzle. The effects of CO2 flowrate, solution flowrate, temperature, pressure, polymer molecular weight and solution concentration on the morphology and drug entrapment efficiency of SEDS-processed particles were investigated. The entrapment efficiency and in vitro release profiles of gemcitabine from microparticles were also determined using HPLC. Our study was focused on the comparison of SEDS with ASES process.

Prostaglandins have a short life in vivo because they are metabolized rapidly by oxidation to 15-ketoprostaglandins catalyzed by a cytosolic enzyme known as NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Previously, CT-8, a thiazolidinedione analogue, was found to be a potent inhibitor of 15-PGDH. Structure-activity analysis indicated that the N-methylation of thiazolidine-2,4-dione, CT-8, abolished the inhibitory activity, whereas the introduction of an ethyl hydroxyl group at amine in CT-8 still had a good inhibitory effect. Based on the structures of the thiazolidinediones analogues and inhibitory activity, a range of benzylidene thiazolidinedione derivatives were synthesized with different substituents on the phenyl ring and their inhibitory activity was evaluated. Replacement of the cyclohexylethyl group of CT-8 with the hetero 5-member ring increased the inhibitory potency. However, replacement of the cyclohexylethyl group with a hetero 6-member ring decreased the inhibitory potency significantly. It was found that compound 2(5-(4-(2-(thiophen-2-yl)ethoxy)benzylidene)thiazolidine-2,4-dione) was the most potent inhibitor that was effective in the nanomolar range.

Research multi-target-multi-drug approaches need to be accelerated in the view of designing efficient therapeutic strategies against diseases bearing alterations in multiple cellular targets. Expression of untargeted protein leading desired or undesired off-target effects is one of the major concerns in performing multi-target-multi-drug approaches. In this study, we investigated the possibility of off-target effects of 4-phenylbutyrate and Iressa (Gefitinib) on the expression of vascular endothelial growth factor (VEGF) and histone deacetylase (HDAC), respectively. Treatment with 10 μM Iressa at the time of co-transfection or 48h after co-transfection of RFP-HDAC/YFP-VEGF plasmids in HEK 293 cells resulted in off-target effects on HDAC protein. These interesting results indicate possible applications of Iressa in the treatment of disease (e.g., Mantle cell lymphoma; MCL) wherein both the HDAC and VEGF protein needs be inhibited. 4-Phenylbutyrate (2.0 mM) did not show any off-target effects on VEGF protein. The developed techniques based on live/single cellular cotransfection imaging can be employed as a higher accuracy multi-target-multi-drug analysis tool at single cellular level to select better drug combinations.

Zebrafish was a very useful animal model for anti-angiogenesis. Zebrafish is very useful for animal model because it's very easy for getting its eggs which is very fast for germination and maturation within 48 hrs. Control and DMSO treated Zebrafish was not so changed in its blood vessel for 6 days. While, 4 kinds of regional special natural products such as Anthrisci Radix, Psoraleae Semen, Siegesbeckiae Herba and Corni Fructus treated Zebrafishs were remarkably decreased their blood vessel for same duration. This zebrafish model is very convenient for anti-angiogenesis and anti-obesity model including zebrafish egg model.

4 kinds of RSNPs(Regional Special Natural Products), Anthrisci Radix, Psoraleae Semen, Siegesbeckiae Herba, Corni Fructus were selected as a anti-obesity subatance through anti-angiogenesis effect. Each natural product inhibited signal pathway such as VEGFR2, or PI3K, or β-catenin or VE-cadherin. And adipogenesis was reduced by 7.5%, 14.4%, 18.3% and 30% at different concentration of Anthrisci Radix when performed Oil red O staining. Also, it was reduced by 2%, 4.9%, 9.3% and 38% at different concentration of Psoraleae Semen. When treated with Siegesbeckiae Herba, it was inhibited by 1.4%, 6.4%, 16.4% and 30.1%, respectively. And Corni Fructus was also showed by 0.9%, 6.3%, 13.7% and 33% at same concentration of Siegesbeckiae Herba. Also, weight was remarkably reduced by each TSNPs based on reducing triglyceride caused on anti-angiogenesis.

Pancreatic islet transplantation is now been used as a new strategy to treat Type I diabetes. One of the major obstacles in islet transplantation is the difficulty to preserve islets cells, which result in loss of cell viability during isolation and culture. Gene transfer into islet cell clusters could improve islet functions and viability via enhancing anti-apoptotic effect and insulin sensitivity to glucose. We investigated whether the genetically modified ICC can successfully be used for transplantation. Rat islets were dispersed and treated with SP-Ex-4 lentivirus for 5 h. After reaggregation, islet 500 and 1000 IEQ of ICC was implanted into diabetic Balb/c nude mice. The graft function over a 30-days period was observed with regard to glucose and insulin level, glucose tolerance tests, and graft insulin content. Our study reveals that reaggregated ICC have better viability rate and gene transduction efficiency than intact islets. High gene transduction of dispersed islets is attributable to the greater number of cells becoming exposed to the lentivirus. SP-Ex-4 transduced ICC enhanced the responsiveness to glucose, thus an effective reverse hyperglycaemia can be achieved. Therefore, Exendin-4 transduction into ICC is needed for successful islet transplantation.

Bacilli are widely used as functional probiotics, feed and food additives. In this work, Bacillus sp. LS 1-2, was isolated from Korean transitional soybean paste foods and had similar 16S rRNA of Bacillus sp. about 98%. LS 1-2 was cultured in citrus-processing-waste containing 5% solid as a culture medium and produced an antimicrobial agent against both of the gram-positives and negatives at optimal culture conditions (pH, Temperature, Culture period, etc). An antimicrobial agent produced by LS 1-2 was purified by alcohol precipitation, C-18 cartridge, YM-10, silica gel open column, prep TLC and prep HPLC. The antimicrobial activity was detected in the fermentation supernatant of LS 1-2. The active substance exhibited more activity through the each purification step. The molecular weight of an antimicrobial agent was found to be lower than 10,000Da, and was a hydrophilic compound. The active substance was not sensitive to lipase, proteinase K, amylase, catalase but was affected by pronase E. Thus this antimicrobial agent was expected to be a bacteriocin-like-substance. In order to evaluate its commercial value, the minimum inhibitory concentration of, the purified antimicrobial agent from Bacillus sp. LS 1-2 was compared with commercialized antimicrobial agents (Colistin, Chlortetracycline, Ampicillin) against Staphylococcus aureus and Escherichia coli O-157.

Gloshedobin is one of promising therapeutic thrombin like enzymes, potentially useful for the treatment of blood clotting disorders through their anti-coagulant function. However, it is difficult to obtain a sufficient amount of the enzymes from the nature, so we use Escherichia coli to offer a route for the rapid and economical production of recombinant proteins. However, over-expression of recombinant gloshedobin in E.coli led to intracellular accumulation as solid aggregates known as inclusion bodies (IBs). We would like to develop novel refolding cocktail based refolding strategies by harnessing in vitro realization of a molecular chaperone network for renaturation of solubilized but denatured gloshedobin in a manner to mimic cellular folding machinery.[1] In order to redesign the traditional batch dilution refolding schemes with the use of refolding cocktail containing recyclable molecular chaperones,[2] two types of beads functionalized with various molecular chaperones (e.g. DnaK, DnaJ, GrpE, ClpB, TF, GroEL, GroES) are prepared : 1) metal chelating beads loaded with in-house cloned His-tagged molecular chaperones, and 2) CNBr-activated Sepharose 4B covalently conjugated with chaperones. To address any plausible alteration on the chaperoning activity of each chaperone following the bead coupling, each chaperone is cloned to harbor the 6xHis-tag at either N- or C-terminus. Following exploration of chaperoning efficacy of individual bead attached chaperones on the refolding of gloshedobin, a recyclable refolding cocktail comprising essential chaperones at an optimal stoichiometry will be prepared to compare the efficiency of cocktail assisted refolding strategy with that of the conventional batch dilution refolding strategy.

Potential advantages of plasmid DNA (pDNA) vaccine over the conventional vaccines include higher stability and life-long immunity against multiple diseases in a single inoculation. In order to develop a scalable intensified process capable of selectively purifying pDNA from its major, difficult-to-remove contaminants such as RNA and endotoxin, we compared two chromatography platforms based on packed-bed column (PBC) and monolith column (MC). We first investigated the dynamic binding capacity (DBC) of RNA, a major contaminant following selective removal of endotoxin, to Cu2+-chelated IDA in PBC and MC using pure RNA solution at various concentrations (0.05 – 8 mg-RNA/ml) and flowrates (0.5 – 3 ml/min for PBC and 0.5 –7 ml/min for MC). RNA exhibited largely concentration-dependent increasing DBC profiles prior to reaching a plateau (max DBC of 1.7 and 1.4 mg-RNA/ml for PBC and MC, respectively) above 0.5 mg/ml of RNA in the flowrate ranges investigated. It was confirmed that the DBC of RNA remained unchanged with the use of feedstock (partially purified from alkaline cell lysate) containing RNA and pDNA. Since PBC or MC has its own merit in view of binding capacity (i.e. higher maximum DBC for PBC) or throughput (i.e. higher flowrate for MC), the overall efficiency of each PBC- or MC-based process is determined by productivity defined as processed feedstock volume per unit resin volume per unit time. For several feedstocks with varying concentrations of pDNA and RNA, the productivity changed in an RNA concentration dependent manner, but generally MC showed higher productivities than PBC. This study proves that the use of monolith chromatographic support which has lower mass transfer limitation and higher throughput than those of conventional packed-bed supports has the benefits in processing macromolecules such as RNA and pDNA, thereby providing a more economically viable platform for pDNA purification.

The expression of recombinant proteins in microorganisms frequently leads to the formation of insoluble aggregates, inclusion bodies (IBs). Thus, an additional protein unfolding and refolding process is required to convert the IBs to the water-soluble active proteins. Various techniques, such as batch-dilution, dialysis and chromatography-based refolding processes, have been reported for this. In addition, numerous additives such as amino acids (cysteine, arginine, lysine and proline), plolyamine (putrescine, spermidine and spermine) and oligosaccharides have been suggested to enhance the refolding efficiency. Especially, in the case of refolding of disulfide bond-containing proteins, the redox reagents (cysteine, glutathione and cysteamine) are added to recover disulfide bonds. Room temperature ionic liquids (RTILs) are organic salts which do not crystallize at room temperature. Tunable hydrophobicity and polarity of RTILs leads the expansion of its applications in chemical and biological processes. Recently, RTILs were recommended as a promising additive of protein refolding. In this study, the effect of hydrophobicity and sulfur residue in RTILs combined with redox system on the dilution-based refolding of protein was investigated. The RTILs which has MS cation was more effective than BF4. The refolding yield was proportionally decreased with increased hydrophobicity, which has longer alkyl chain length, of RTILs. And without redox system, there are no significant additive effect on lysozyme refolding efficiency.

Biochemical characterization of lectin isolated on Sephadex G-200 was studied in Chungkukjang soybean treated with and without heat. Hemagglutination activity of lectin by rabbit erythrocyte digested with trypsin was detected in soybean treated without heat, whereas not detected in soybean treated with heat. The optimal temperature for lectin activity of soybean seed (SS), fermented soybean (FS), and fermented and freeze dried soybean (FDS) were 50℃, 30℃, and 10℃, respectively. This lectin was relatively stable to heat at 20-40℃ in SS, at 20-60℃ in FS, and at 20-60℃ in FDS. The maximal lectin activity was determined at pH 6.2 in SS, at pH 8.0-9.1 in FS, and at pH 7.2 in FDS. This activity of lectin was dropped by urea, thio-urea, and guanidine-HCl as denaturants.