Ik Hwan Kim

Characteristic immunostimulation by angelan isolated from Angelica gigas Nakai.

The immunopharmacological characteristics of angelan, a polysaccharide purified from Angelica gigas Nakai, were investigated in relation to the specificity to immune cells. The treatment of angelan increased the expression of IL-2, IL-4, IL-6, and IFN-gamma. The expression of IL-6 and IFN-gamma was rapidly augmented but that of IL-2 responded later. In the case of IL-4, angelan stimulated at early time after exposure but down-regulated thereafter. These results suggested that macrophages and natural killer cells involved in nonspecific immunity were primarily activated and helper T cells were secondarily affected by angelan. Angelan also had lympho-proliferative potential to B cells, specifically. The specificity of angelan was also elucidated in a cell fractionation experiment. The activated B cells by angelan also increased antibody production. The direct activation of B cells, macrophages, and accessory cells and the indirect activation of helper T cells coordinately increased immune functions such as in vitro and in vivo T-dependent immunization and antibody production. The experiment of host resistance to syngeneic tumors also showed that angelan potentiated the immune functions. In conclusion, angelan, a purified polysaccharide from an oriental herbal drug, showed characteristic immunostimulation, which was different from clinically used polysaccharides such as lentinan and PSK.

Fabrication of core-shell microcapsules using PLGA and alginate for dual growth factor delivery system.

To effectively harness the great potential of stem cells, we designed a dual growth factor delivery system for the application toward stem cell differentiation into specific lineages. This system carries a core-shell structure within microcapsules made of poly(L-lactide-co-glycolide) (PLGA) and alginate, which were fabricated using a coaxial electro-dropping method. Both PLGA and alginate were supplied from the inner and outer nozzles, respectively. The size and shape of microcapsules were greatly varying depending on the variables: nozzle size, applied voltage, volumetric feeding ratio (PLGA:alginate), feeding rate, and polymer concentrations. Once proper conditions were met, single or multi PLGA cores were found settled within the microcapsules. From the microscopic images, wrinkled surfaces of microcapsules were observed, along with the PLGA cores inside the alginate domain. When two different microcapsules were made, switching the position of bone morphogenetic protein (BMP)-2 and dexamethasone (Dex) for either core or shell domain, their release profiles were very unique on a temporal basis, based on their location in the microcapsules. An initial burst of biomolecules was highly suppressed when either biomolecule was loaded in the PLGA core. It was clear that the osteogenic biomolecules encapsulated in the microcapsule could be released together and their concentrations were disparate at each time point. Meanwhile as the hydrogel constructs including rat bone marrow stromal cells (BMSCs) and osteogenic factor-loaded microcapsules were cultured for up to 4 weeks, the gene expressions levels of osteopontin, type I collagen, and osteocalcin were significantly upregulated as compared to the control group. The present coaxial system was very effective in manufacturing PLGA core-alginate shell microcapsules and in encapsulating multiple biomolecules essential for stem cell differentiation.

The correlation between human adipose-derived stem cells differentiation and cell adhesion mechanism.

In recent years, research in the areas of stem cells has dramatically increased, including studies of cellular adhesion to a substrate. We sought to determine the adhesive properties of human adipose-derived stem cells (hASCs) for extracellular matrix proteins. The adhesion of hASCs to collagens and laminin was completely inhibited by a monoclonal antibody, Mab 2253, which binds to the beta1 integrin subunit. These data indicate that hASC adhesion to collagens and laminin was exclusively mediated by an integrin. Cell adhesion on fibronectin (Fn) was inhibited by the heparin-binding peptide (HBP) in the presence of Mab 2253, but not by either Mab 2253 or HBP alone. These results indicate that both the beta1 subunit and the heparan sulfate proteoglycan participated in the cell adhesion to Fn. Microscopic views showed extensive spreading of hASCs cultured on Fn, whereas the cells maintained a round shape when cultured on a heparin-binding domain (HBD) substrate. hASCs differentiated into adipocytes, which stained positive for lipid vacuoles by Oil Red-O analysis, more readily on HBD substrate than on FN substrate. These results suggest that hASCs have an adhesion mechanism for the HBD of Fn and hASC morphology is controlled by the adhesion mechanism and strongly correlated with adipogenic differentiation.

Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media

Ammonium ion accumulation in mammalian cell culture media causes toxicity which inhibits cell growth and productivity. To reduce the level of the accumulated ammonium ion, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC) were used, which catalyze the first and second steps of the urea cycle in the liver. To examine the effects of overexpressed CPS I and OTC genes on the concentration of the ammonium ion in culture media, the two genes were introduced into Chinese hamster ovary (CHO) dhfr-cells. The CPS I expressing cell lines (CPS I-CHO) and both CPS I and OTC expressing cell lines (CPS I/OTC-CHO) were confirmed at the mRNA level and analyzed in terms of the cell growth and the accumulation of ammonium ion in culture media. The accumulation of ammonium ion was approximately 25-33% less in CPS I/OTC-CHO than in either CPS I-CHO or the vector-control cell lines. Interestingly however, the cell growth was approximately 15-30% faster in both CPS I-CHO and CPS I/OTC-CHO than in the control cell lines. Forced expression of urea cycle enzymes in the CHO cells revealed that both the expression of CPS I and OTC can reduce the accumulation of ammonium ion in the culture media.

Prohibitin interacts with RNF2 and regulates E2F1 function via dual pathways

Prohibitin, a tumor suppresser protein, plays an important role in the transcriptional regulation of various genes involved in cell-cycle control and proliferation. Recent studies have reported that the growth-suppressive property of the prohibitin protein is exhibited in its physical interaction with E2F family proteins and its subsequent repression of their transcriptional activity. Herein, we report that prohibitin interacts with RING finger protein 2 (RNF2), a member of the PcG (polycomb-group) family of proteins, and that the two proteins regulate the activity of E2F1 via dual pathways: the direct, prohibitin-mediated pathway and the indirect, p16-mediated pathway of E2F1 transcriptional regulation. Co-immunoprecipitation experiments showed that endogenous prohibitin interacts with endogenous RNF2. Interestingly, the expressed amounts of RNF2 and prohibitin were interdependently affected at the post-translational level. Furthermore, the depletion of either endogenous RNF2 or prohibitin using the RNA interference technique increased the level of p16 protein expression, resulting in a decrease in the transcriptional activity of E2F1 via the p16–CDK4–Rb pathway. In addition, chromatin immunoprecipitation assays showed that RNF2 was recruited to E2F1-response promoters along with prohibitin to inhibit the transcriptional activity of E2F1. Cell proliferation was also regulated by the prohibitin–RNF2 interaction. These results suggest that the RNF2–prohibitin complex regulates the activity of E2F1 via dual pathways.

Dual Growth Factor Delivery Using Biocompatible Core–Shell Microcapsules for Angiogenesis

An optimized electrodropping system produces homogeneous core-shell microcapsules (C-S MCs) by using poly(L-lactic-co-glycolic acid) (PLGA) and alginate. Fluorescence imaging clearly shows the C-S domain in the MC. For release control, the use of high-molecular-weight PLGA (HMW 270 000) restrains the initial burst release of protein compared to that of low-MW PLGA (LMW 40 000). Layer-by-layer (LBL) assembly of chitosan and alginate on MCs is also useful in controlling the release profile of biomolecules. LBL (7-layer) treatment is effective in suppressing the initial burst release of protein compared to no LBL (0-layer). The difference of cumulative albumin release between HMW (7-layer LBL) and LMW (0-layer LBL) PLGA is determined to be more than 40% on day 5. When dual angiogenic growth factors (GFs), such as platelet-derived GF (PDGF) and vascular endothelial GF (VEGF), are encapsulated separately in the core and shell domains, respectively, the VEGF release rate is much greater than that of PDGF, and the difference of the cumulative release percentage between the two GFs is about 30% on day 7 with LMW core PLGA and more than 45% with HMW core PLGA. As for the angiogenic potential of MC GFs with human umbilical vein endothelial cells (HUVECs), the fluorescence signal of CD31+ suggests that the angiogenic sprout of ECs is more active in MC-mediated GF delivery than conventional GF delivery, and this difference is significant, based on the number of capillary branches in the unit area. This study demonstrates that the fabrication of biocompatible C-S MCs is possible, and that the release control of biomolecules is adjustable. Furthermore, MC-mediated GFs remain in an active form and can upregulate the angiogenic activity of ECs.

Effects of lactate dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on cellular metabolism

AbstactIn order to conduct a physiological functional study of lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (GPDH), we engineered a CHO dhfr− cell, by overexpressing either the anti-sense LDH-A RNA (anti-LDH cells) or GPDH (GP3 cells), or both (GP3/anti-LDH cells). LDH activity in the cell cytosol, and lactate content and pHe change in the growth media were found to decrease according to the order: cell lines GP3/anti-LDH − anti−LDH − GP3 − CHO. Intracellular ATP contents, representing the extent of respiration rate, also decreased, according to a rank order as follows: GP3 − CHO − GP3/anti−LDH − anti−LDH. We also attempted to identify and characterize any physiological changes occurring in the cells which harbored diverse metabolic pathways. First, anti-LDH cells with heightened respiration rates were found to display a higher degree of sensitivity to the prooxidant tert-butyl hydroperoxide (tBOOH), and the mitochondrial complex III inhibitor, antimycin A, than the GPDH-expressing cells (GP3 and GP3/anti-LDH), which have a lower respiration rate. Second, the anti-sense LDH-A RNA-expressing cells (anti-LDH and GP3/anti-LDH) evidenced a higher degree of resistance to apoptosis by cell-cell contact inhibition, and a faster doubling time (∼19 h compared with ∼26 h) than the CHO and GP3 cells. Additionally, cell growth in an extended culture under HCO3−-free conditions to induce a steep acidification could be maintained with the anti-sense LDH-A RNA-expressing cells, but could not be maintained with the CHO and GP3 cells. Third, we observed that the most appropriate cell line for the optical production of a certain therapeutic protein (Tissue-Plasminogen Activator) was the GP3/anti-LDH cells. Collectively, our data indicate a variety of physiological roles for LDH and GPDH, including cellular acidosis, oxidoresistance, apoptosis by both acidosis and cell-cell contact inhibition, cell growth, and the generation of recombinant proteins.

Synthesis and characterization of novel thermo-responsive F68 block copolymers with cell-adhesive RGD peptide

Thermosensitive poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer, Pluronic F68, containing a hydrophobic unit, oligo-(lactic acid)(oligo-LA) or oligo-caprolactone (oligo-CL), 2-META and RGD as side groups was successfully synthesized and characterized by (1)H NMR, FTIR, and elemental analysis. Their aqueous solution displayed special gel-sol-gel phase transition behavior with increasing temperature from 10 to 70°C, when the polymer concentration was above critical micelle concentration (CMC). The gel-sol phase diagram was investigated using tube inversion method, rheological measurement, and dynamic light scattering. Based on these results, the gelation properties of modified F68 were affected by several factors such as the composition of the substituents, chain length of oligo L-LA or oligo ε-CL, and the concentration of the polymer solutions. The unique phase transition behavior with temperature was observed by modified F68 triblock copolymer, composed of the PPO blocks core and the PEO blocks shell in aqueous solution. This phenomenon was elucidated using (1)H NMR data; the alteration of hydrophobic interaction and chain mobility led to the formation of transparent gel, coexistence of gel-sol, and opaque gel. These hydrogels may be useful in drug delivery and tissue engineering.

Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli.

L‐amino acid deaminases catalyze the deamination of natural L‐amino acids. Two types of L‐amino acid deaminase have been identified in Proteus species. One exhibits high levels of activity toward a wide range of aliphatic and aromatic L‐amino acids, typically L‐phenylalanine, whereas the other acts on a relatively narrow range of basic L‐amino acids, typically L‐histidine. In this study, we cloned, expressed, and characterized a second amino acid deaminase, termed Pm1, from P. mirabilis KCTC 2566. Homology alignment of the deduced amino acid sequence of Pm1 demonstrated that the greatest similarity (96%) was with the L‐amino acid deaminase (LAD) of P. vulgaris, and that homology with Pma was relatively low (72%). Also, similar to LAD, Pm1 was most active on L‐histidine, indicating that Pm1 belongs to the second type of amino acid deaminase. In agreement with this conclusion, the Vmax and Km values of Pm1 were 119.7 (μg phenylpyruvic acid/mg/min) and 31.55 mM phenylalanine, respectively, values lower than those of Pma. The Pml deaminase will be very useful industrially in the preparation of commercially valuable materials including urocanic acid and α ‐oxoglutarate. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Upregulation of the cysteine protease inhibitor, cystatin SN, contributes to cell proliferation and cathepsin inhibition in gastric cancer.

BACKGROUND Cysteine proteases like cathepsins are widely distributed proteolytic enzymes and form tight equimolar complexes with cystatins at their active sites. Among cystatins, CST1, encoding cystatin SN, is a member of the type 2 salivary cystatin family found in a variety of fluids and secretions, including plasma, tears, and saliva. CST1 was identified as an upregulated gene in gastric cancer tissues compared to noncancerous regions using our Affymetrix GeneChip microarray. METHODS The upregulation of CST1 in gastric cancer was analyzed using RT-PCR (n=15), immnohistochemistry, and clinicopathological (n=77) analysis. CST1-siRNA was used for the suppression of CST1 gene expression and cathepsin proteolytic activity was assayed. RESULTS CST1 was upregulated in cancerous lesions of gastric cancer tissues compared to noncancerous regions and clinicopathological analysis showed a significant correlation between high expression of CST1 and pTNM stage (p=0.044). In CST1-siRNA transfected cells, cell proliferation was reduced and the proteolytic activity of cathepsins was increased. CONCLUSIONS CST1 might be highly involved in gastric tumorigenesis and regulate the proteolytic activity of cysteine proteases.