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Sialylation, which is considered as a critical quality attribute, plays important roles in pharmacokinetics of recombinant proteins. Terminal sialic acids of glycoproteins are modulated by intracellular sialyltransferase (ST) and extracellular sialidase. Since CHO cells producing recombinant proteins can only transfer α2,3-sialic acid to a nascent oligosaccharide, inactivation of α2,6-ST results in a different composition of glycan with human proteins, which have predominantly α2,6-linkages. To overcome this problem and maximize total sialic acid contents, we used glucocorticoids (GCs) that are classified as a steroid hormone in CHO cells expressing both α2,3-ST and α2,6-ST. This study was performed to determine the effect of GCs on CHO cell cultures and sialylation of Fc-fusion protein. We observed that all cultures supplemented with GCs reduced the titer of the Fc-fusion protein, but enhanced the sialylation level, when compared to those of control. Especially, addition of corticosterone increases the levels of α2,6-sialylation as well as the total contents of sialic acid. Enhanced sialic acid contents are thought to be due to the intracellular improvement by sialylatransferase and the inhibition of sialidase. In conclusion, GCs can be utilized as an effective medium additive to improve the quality of biopharmaceuticals using CHO cell cultures.

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

Light-emitting organic semiconductors have attracted considerable attention for the nanoscale fabrication of organic-based displays and their potential application in optoelectronics, plasmonics, and photonics. In this study, core-shell hybrid nanostructures of organic rubrene coated on Ag nanoparticles (NPs) have been synthesized using a chemical reduction method. The thickness of the rubrene shell was 2.6–6.0 nm and the diameter of the Ag core was 30–70 nm. The optical and structural properties of the Ag/rubrene core-shell NPs were tuned by hydrothermal (HT) treatment at 190 °C. The Ag/rubrene core-shell NPs were characterized by high-resolution transmission electron microscopy and energy-dispersive X-ray (EDX) spectroscopy before and after the HT treatment, and their structural properties were confirmed through X-ray diffraction (XRD) analysis. XRD peaks related to an orthorhombic phase were observed along with the original triclinic crystal structure of the rubrene shell, and the triclinic crystal domain size increased from 28.2 nm to 30.8 nm owing to the HT treatment. Interestingly, the green light emission (λem = 550 nm) of the Ag/rubrene core-shell NPs changed to blue light emission (λem = 425 nm), increasing in intensity through the HT treatment. This is caused by the crystal change with H-type aggregation and enhanced energy transfer from a surface plasmon resonance.

Pulse-echo laser ultrasonic propagation imaging is a nondestructive testing technique developed for composite materials and aluminum alloys used in aerospace. Although this method has been in usage for a considerable time, information of the detectable depth and the relationship between ultrasonic frequencies and the acoustic properties of metals is not readily available. Therefore, we investigate the A-scan and C-scan ultrasonic testing data of aluminum alloy, hot rolled steel, stainless steel, and copper alloy, which are used in aircraft bodies, frameworks, and gas pipelines. Experiments are performed with the pulse-width and excitation laser power fixed at 32 ns and approximately 4 W, respectively. The metal specimens include 24 artificial cylindrical defects with a diameter of 5 mm, located at depths of 1–12 mm on the front surface. The A-scan and C-scan data obtained at room temperature indicate the detectable depth for metals via the pulse-echo laser ultrasonic propagation imaging technique.

We developed a flow distributor for high-performance chromatographic processes during the purification of biopharmaceuticals. Twelve distributor candidates are provided, including four types of distributors with different-shaped holes, and three sub-types with different number of holes. We evaluated the flow distributor candidates by spreading bromophenol blue dye on the membrane , and selected the most suitable distributor Arc 45 deg. type. Using 10 ml columns with three different length-to-diamter (L/D) ratios (4.33, 0.92, and 0.34) of column, we measured peak asymmetry (As) values. The 10 ml column with 0.92 L/D ratio showed best As value. We also carried out flow experiments on the three sub-types of Arc 45 deg. distributors on the 0.92 L/D ratio 50 ml column and Arc 45 deg. 1:8 distributor showed best performance. The As values of this optimal flow distributor are 1.2 or less. In summary, this study provides optimal distributor values for chromatographic purification of biopharmaceuticals.

Protein arginine methyltransferases (PRMTs) are enzymes that regulate the evolutionarily conserved process of arginine methylation. It has been reported that PRMTs are involved in many metabolic regulatory pathways. However, until now, their roles in adipocyte function, especially browning and thermogenesis, have not been evaluated. Even though Prmt1 adipocyte-specific–deleted mice (Prmt1fl/flAQcre) appeared normal at basal level, following cold exposure or β-adrenergic stimulation, impaired induction of the thermogenic program was observed in both the interscapular brown adipose tissue and inguinal white adipose tissue of Prmt1fl/flAQcre mice compared with littermate controls. Different splicing variants of Prmt1 have been reported. Among them, PRMT1 variant 1 and PRMT1 variant 2 (PRMT1V2) are well conserved between humans and mice. Both variants contribute to the activation of thermogenic fat, with PRMT1V2 playing a more dominant role. Mechanistic studies using cultured murine and human adipocytes revealed that PRMT1V2 mediates thermogenic fat activation through PGC1α, a transcriptional coactivator that has been shown to play a key role in mitochondrial biogenesis. To our knowledge, our data are the first to demonstrate that PRMT1 plays a regulatory role in thermogenic fat function. These findings suggest that modulating PRMT1 activity may represent new avenues to regulate thermogenic fat and mediate energy homeostasis. This function is conserved in human primary adipocytes, suggesting that further investigation of this pathway may ultimately lead to therapeutic strategies against human obesity and associated metabolic disorders.

Although plant cell cultures produce low yields of recombinant proteins compared to other production systems, a dramatic increase of the heterologous protein production in transgenic rice cells was achieved with the alpha-amylase isozyme 3D (RAmy3D) promoter system. However, this expression system has inherent limitations in that gene expression is initiated by sucrose/glucose deprivation, concurrently triggering starvation-derived autophagy and rapid cell death. Decreased viability and culture longevity subsequently prevent further increment of production. In this study, we introduced autophagy inducers and inhibitors in the rrhGAA-producing transgenic rice cell cultures in order to explore their effects on production controlled by the RAmy3D promoter. The autophagy inducers rapamycin and CCI-779 increased autophagosome and autolysosome while concanamycin A1 and bafilomycin A successfully decreased autolysosome. Interestingly, autophagy inhibitors improved viability, DCW loss, and rrhGAA production, while autophagy inducers deteriorated these profiles compared to the control. As the production conditions under the death phase may facilitate protein degradation, and subsequently exacerbate functional activity, the size variant distribution and enzyme activity of the purified rrhGAAs were evaluated. However, no significant difference in rrhGAA degradation as well as GAA activity was observed compared to the control condition, thus indicating that the autophagy regulation is an efficient approach to increase protein yield in rice cell culture system for rrhGAA production.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the commercial production of therapeutic proteins. Fed-batch culture is widely used to produce therapeutic proteins, including monoclonal antibodies, because of its operational simplicity and high product titer. Despite technical advances in the development of culture media and cell cultures, it is still challenging to maintain high productivity in fed-batch cultures while also ensuring good product quality. In this review, factors that affect the quality attributes of therapeutic proteins in recombinant CHO (rCHO) cell culture, such as glycosylation, charge variation, aggregation, and degradation, are summarized and categorized into three groups: culture environments, chemical additives, and host cell proteins accumulated in culture supernatants. Understanding the factors that influence the therapeutic protein quality in rCHO cell culture will facilitate the development of large-scale, high-yield fed-batch culture processes for the production of high-quality therapeutic proteins.

Melatonin, a pineal gland hormone, has been suggested to treat postmenopausal osteoporosis due to its inhibitory effect on osteoclast differentiation. We previously reported that protein arginine methyltransferase 1 (PRMT1) was an important mediator of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. However, the relationship between melatonin and PRMT1 in osteoclast differentiation and estrogen deficiency–induced osteoporosis is unclear. In this study, we investigated the inhibitory mechanisms of melatonin in vitro and in vivo by focusing on PRMT1. Melatonin treatment effectively blocked RANKL-induced osteoclastogenesis by inhibiting PRMT1 and asymmetric dimethylarginine (ADMA) expression. RANKL-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) and the phosphorylation of JNK were also suppressed by melatonin, and TRAF6 siRNA attenuated RANKL-induced p-JNK and PRMT1 production. Melatonin inhibited the transcriptional activity of NF-κB by interfering with the binding of PRMT1 and NF-κB subunit p65 in RANKL-treated bone marrow–derived macrophages. Our results also revealed that melatonin inhibits RANKL-induced PRMT1 expression through receptors-independent pathway. Thus, the anti-osteoclastogenic effect of melatonin was mediated by a cascade of inhibition of RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling in melatonin receptors-independent pathway. In vivo, ovariectomy caused significant decreases in bone mineral density, but melatonin treatment alleviated the ovariectomized (OVX)-induced bone loss by inhibiting bone resorption. Furthermore, the expression PRMT1 and TRAP mRNA was upregulated in OVX-femurs, but effectively suppressed by melatonin injection. These findings suggest that melatonin inhibited osteoclast differentiation and estrogen deficiency–induced osteoporosis by suppressing RANKL-induced TRAF6, JNK, PRMT1, and NF-κB signaling cascades in melatonin receptors-independent pathway.

Background BaelanChagsangBang (BCB), a herbal formulation consisting of eleven herbs, may be prescribed as a reproductive functional supplement to improve ovulation and implantation during the treatment of infertility and recurrent abortion in Korean Medicine. This study aimed to investigate the effects and action mechanisms of water-extracted BCB on endometrial receptivity and blastocyst implantation under normal conditions and in a mifepristone (RU486)-induced implantation failure murine model. Methods In vitro, the antioxidant potentials of BCB were evaluated using DPPH and superoxide anion radical scavenging assays and a DCFH-DA assay, and the cytotoxic and cytoprotective effects of BCB were confirmed using an MTT assay. In vivo, C57BL/6 female mice (n = 6 per group) orally received BCB (300 mg/kg/day), a dose similar to that used clinically, from 7 days before pregnancy until the end of the experiment. On day 4 of pregnancy, RU486 (4 mg/kg) was injected subcutaneously to induce implantation failure. The effect of BCB on embryo implantation was evaluated by implantation rate analysis, histological examination, and western blotting of uterus tissues. Results BCB water extract showed strong anti-oxidative and cytoprotective effects in vitro. In vivo administration of BCB water extract increased the number of newborn pups in BCB-treated mice versus sham-treated mice under normal conditions and improved the number of implantation sites in pregnant mice despite RU486 injection. BCB increased the protein levels of cyclooxygenase-2 and inducible nitric oxide synthase through IκB activation. Moreover, the expression levels of matrix metalloproteinases at uterus implantation sites were up-regulated in the BCB-treated group as compared with those in the RU486-treated group. Conclusion These results show BCB improved embryo implantation through IκB activation in our mouse model and suggest that BCB has therapeutic potential in the context of poor endometrial receptivity.

Recent studies have revealed that protein arginine methyltransferases (PRMTs) are responsible for diverse neurodegenerative diseases. However, their pathophysiological role in dopaminergic neuronal death in Parkinson's disease (PD) has not been evaluated. In this study, we demonstrated that 1-Methyl-4-phenylpyridinium iodide (MPP+), rotenone and paraquat, which cause dopaminergic neuronal cell death, increased PRMT1 expression in dopaminergic cell line. Dopaminergic neuronal cell death was increased by PRMT1 overexpression. MPP+-induced cell death was attenuated by PRMT1 knockdown. Poly (ADP-ribose) polymerase-1 (PARP1) expression and activity, poly-ADP-ribosylation (PARylation), were elevated by MPP+. Moreover, we found that PRMT1 positively regulates nuclear translocation of apoptosis-inducing factor (AIF). Elevated PRMT1 expression was observed in the substantia nigra pars compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected mice. Furthermore, MPTP-induced dopaminergic neuronal death was reduced in PRMT1 haploinsufficient (prmt1+/-) mice. These data suggest that PRMT1 is implicated in PARP1/AIF-mediated dopaminergic neuronal cell death, which might be involved in the pathology of PD. Therefore, our results propose PRMT1 as a new target to develop a potential treatment of PD.

Green tea contains bioactive compounds, such as polyphenols, responsible for its health-promoting effects, including antiobesity and antidiabetic effects. We previously reported that ultra-sonication extraction (UE) could efficiently increase the extraction yield of green tea compounds. In the present study, we found that the extract obtained using UE contained higher phenolic and flavonoid contents than that obtained using the conventional method. We therefore considered the extract as a bioactive metabolite-rich functional green tea extract (BMF-GTE), and tested its glucose-lowering effect by generating an adipocyte cell line stably expressing 7myc-GLUT4-GFP. We found that BMF-GTE treatment increased GLUT4 translocation to the plasma membrane. Moreover, BMF-GTE administration attenuated weight gain in mice fed a high-fat diet (HFD). Importantly, HFD-induced glucose tolerance was ameliorated in the mice receiving BMF-GTE. Therefore, we conclude that BMF-GTE worked against obesity and diabetes, at least partially, by enhancing GLUT4 translocation in adipocytes. PRACTICAL APPLICATIONS: As green tea is one of the most consumed beverages worldwide, its health effects have been widely tested. In our previous studies, we found that ultra-sonication extraction (UE) has the potential to increase the aqueous extraction yield of green tea compounds compared to conventional extraction techniques. In this study, we examined the biological effect of bioactive metabolite-rich functional green tea extract (BMF-GTE) obtained using UE; we observed that administering BMF-GTE lowered the body weight and increased insulin sensitivity in mice fed a high-fat diet, potentially by facilitating the membrane translocation of GLUT4 in adipocytes. Therefore, this study suggests that the extract obtained with UE had antiobesity and antidiabetic properties, indicative of a potential application of UE in maximizing the beneficial effects of green tea on human health.

We previously reported the involvement of protein arginine methyltransferase 1 (PRMT1) in adipocyte thermogenesis. Here, we investigate the effects of PRMT1 inhibitors on thermogenesis. Unexpectedly, we find that the PRMT1 inhibitor TC-E 5003 (TC-E) induces the thermogenic properties of primary murine and human subcutaneous adipocytes. TC-E treatment upregulates the expression of Ucp1 and Fgf21 significantly and activates protein kinase A signaling and lipolysis in primary subcutaneous adipocytes from both mouse and humans. We further find that the thermogenic effects of TC-E are independent of PRMT1 and beta-adrenergic receptors. Our data indicate that TC-E exerts strong effects on murine and human subcutaneous adipocytes by activating beige adipocytes via PKA signaling.

Selaginella Herba is the dried, aerial part of Selaginella tamariscina (P.Beauv.) Spring and has been used to treat amenorrhea, abdominal pain, headaches, and hematuria in Korea. However, scientific evidence regarding the anti-inflammatory activity and action mechanism of Selaginella tamariscina is lacking. Thus, the present study was performed to investigate the anti-inflammatory and antioxidant activities of Selaginella tamariscina ethanol extract (STE) against lipopolysaccharide (LPS)-induced inflammatory responses and identify the molecular mechanism responsible. STE was prepared by heating in 70% ethanol and its quality was confirmed by HPLC. STE dose-dependently inhibited the productions of inflammatory mediators (NO and PGE2) and proinflammatory cytokines (IL-1β and IL-6) in LPS-stimulated RAW 264.7 cells. STE markedly suppressed the phosphorylations of MAPKs, IκB-α, and NF-κB and the nuclear translocation of NF-κB induced by LPS stimulation. In addition, STE exhibited good free radical scavenging activity and prevented ROS generation by LPS. STE also upregulated the expression of Nrf2 and HO-1 and promoted the nuclear translocation of Nrf2. Taken together, STE was found to have anti-inflammatory and antioxidant effects on RAW 264.7 macrophages and the mechanism appeared to involve the MAPK, NF-κB, and Nrf2/HO-1 signaling pathways. These results suggest that STE might be useful for preventing or treating inflammatory diseases and provide scientific evidence that supports the developments of herbal prescriptions or novel natural products.

Protein arginine methylation is a novel form of posttranslational modification mediated by protein arginine methyltransferase (PRMTs). PRMT1, a major isoform of the PRMT family, is responsible for various biological functions, including cellular differentiation. Although the important function that PRMT1 plays in various tissues is being increasingly recognized, its role in receptor activation of NF-κB ligand (RANKL)-induced osteoclastogenesis or osteoporosis has not yet been described. Here, we show that PRMT1 is essential for RANKL-induced osteoclastogenesis in vitro and for bone loss in vivo. RANKL treatment increased the expression of PRMT1 and its nuclear localization in bone marrow-derived macrophages (BMDMs) in a c-Jun N-terminal kinase (JNK)-dependent manner. Silencing PRMT1 attenuated RANKL-induced osteoclastogenesis by decreasing tartrate-resistant acid phosphatase (TRAP)-positive cells and inhibiting F-actin ring formation and bone resorption, which was confirmed in a separate experiment using haploinsufficient cells from PRMT1+/- mice. Our results also revealed that PRMT1 regulates the transcription activity of NF-κB by directly interacting with it in RANKL-treated BMDMs. An in vivo study showed that the haploinsufficiency of PRMT1 reduced the enzyme activity of TRAP and increased the bone mineral density in the metaphysis of ovariectomized (OVX) mice. Finally, treatment with estrogen (E2) downregulated the RANKL-induced expression of PRMT1, suggesting that estrogen may exert an inhibitory effect on osteoclastogenesis by suppressing PRMT1 expression. Our results suggest that PRMT1 plays an important role in the progression of osteoporosis and that it might be a good therapeutic target for postmenopausal osteoporosis., Osteoporosis: protein trigger for postmenopausal bone loss identified A protein that helps trigger bone loss in postmenopausal osteoporosis could be a potential therapeutic target. After the menopause, decreases in estrogen hormone levels can lead to bone diseases including osteoporosis. Osteoporosis occurs when the bone remodeling process breaks down, and bone resorption by cells called osteoclasts outweighs bone formation. In a mouse model of postmenopausal osteoporosis, Jong-Hwan Park at Chonnam National University, Gwangju, South Korea and co-workers identified key players in the progression of the disease. The team focused on factors influencing the RANKL protein, a known controller of bone remodeling. They found that RANKL triggers the formation of osteoclasts via interaction with another protein, PRMT1. Suppression of PRMT1 by estrogen appears to inhibit excessive osteoclast formation, suggesting it could be a potential therapeutic target for treating osteoporosis.

It has been reported that class I histone deacetylase (HDAC) inhibition increases thermogenesis in fat, but adipocyte-specific Hdac3 deletions have presented inconsistent results. In this study, we observed that HDAC3 protein levels were lower in brown fat compared with inguinal subcutaneous adipose tissue, and they decreased in both fat depots upon cold exposure. PR domain–containing 16 (PRDM16) physically interacted with HDAC3, and treatment with HDAC3-selective inhibitor RGFP966 induced thermogenic gene expression in murine and human fat cultures. This induction was blunted in the absence of PRDM16. Our results provide evidence that HDAC3 is involved in thermogenesis, suggesting selective inhibition of HDAC3 in brown and beige fat might hold therapeutic potential for counteracting human obesity and metabolic disorders.

Chinese hamster ovary (CHO) cells have been widely used for the production of glycoproteins due to various advantages such as high productivity, ease of genetic manipulation, adaptability to serum-free media, and human-like glycosylation. Glycosylation, which occurs sequentially using nucleotide sugars as donor substrates, can affect the therapeutic function of glycoproteins. Terminal sialic acid residues of N-glycans play an important role in determining in vivo half-life of glycoproteins. Therefore, various strategies have been developed to enhance sialylation of glycoproteins. In this study, a nucleotide sugar precursor feeding strategy was optimized to increase sialic acid content of albumin-erythropoietin (Alb-EPO). Glucosamine, galactose, and N-acetylmannosamine were added to CHO cell cultures in lag (day 0) or exponential phase (day 3). Central composite design and response surface methodology were used to evaluate the effects of nucleotide sugar precursors on cell growth, titer, and sialic acid content. For the exponential feeding strategy, culture performance was not altered by addition of optimal precursor concentrations, whereas sialic acid contents increased 1.43-fold compared to the control. Consequently, our nucleotide sugar precursor feeding strategy can provide an efficient culture process for production of highly sialylated glycans on Alb-EPO.

BACKGROUND/OBJECTIVES Thermogenic fat is present in humans and emerging evidence indicates that increasing the content and activity of these adipocytes may lead to weight loss and improved metabolic health. Multiple reporter systems have been developed to assay thermogenic fat activity based on the transcriptional and translational activation of Ucp1, the key molecule that mediates nonshivering thermogenesis. Our study aims to develop a much-needed tool to monitor thermogenic fat activity through a mechanism independent of Ucp1 regulation, therefore effectively assaying not only canonical β-adrenergic activation but also various non-UCP1-mediated thermogenic pathways that have been increasingly appreciated. METHODS We detected increased luciferase activity upon thermogenic activation in interscapular brown and inguinal subcutaneous fat in ODD-Luc mice, a hypoxia reporter mouse model. We then developed an OLTAM (ODD-Luc based Thermogenic Activity Measurement) system to assay thermogenic fat cell activity. RESULTS In both primary murine and human adipocytes and an immortalized adipose cell line that were transduced with the OLTAM system, luciferase activity can be readily measured and visualized by bioluminescence imaging in response to a variety of stimuli, including UCP1-independent thermogenic signaling. This system can offer a convenient method to assay thermogenic activity for both basic and translational research. CONCLUSIONS The OLTAM system offers a convenient way to measure of the activation of thermogenic fat and presents opportunities to discover novel signaling pathways and unknown compounds targeting metabolically active adipocytes to counteract human obesity.

Cinnamaldehyde (CA) is a food compound that has previously been observed to be protective against obesity and hyperglycemia in mouse models. In this study, we aimed to elucidate the mechanisms behind this protective effect by assessing the cell-autonomous response of primary adipocytes to CA treatment.Primary murine adipocytes were treated with CA and thermogenic and metabolic responses were assessed after both acute and chronic treatments. Human adipose stem cells were differentiated and treated with CA to assess whether the CA-mediated signaling is conserved in humans.CA significantly activated PKA signaling, increased expression levels of thermogenic genes and induced phosphorylation of HSL and PLIN1 in murine primary adipocytes. Inhibition of PKA or p38 MAPK enzymatic activity markedly inhibited the CA-induced thermogenic response. In addition, chronic CA treatment regulates metabolic reprogramming, which was partially diminished in FGF21KO adipocytes. Importantly, both acute and chronic effects of CA were observed in human adipose stem cells isolated from multiple donors of different ethnicities and ages and with a variety of body mass indexes (BMI).CA activates thermogenic and metabolic responses in mouse and human primary subcutaneous adipocytes in a cell-autonomous manner, giving a mechanistic explanation for the anti-obesity effects of CA observed previously and further supporting its potential metabolic benefits on humans. Given the wide usage of cinnamon in the food industry, the notion that this popular food additive, instead of a drug, may activate thermogenesis, could ultimately lead to therapeutic strategies against obesity that are much better adhered to by participants.

This study investigated the antioxidant and antidiabetic activities of Stachys sieboldii Miq. extracts by solvents (water, ethanol, butanol, chloroform, and hexane). The contents of total polyphenols (7.18-37.25 mg/g) and flavonoids (0.21-5.21 mg/g) in extracts from Stachys sieboldii Miq. showed a significant difference dependent on the extraction solvents, butanol > ethanol > water > chloroform > hexane. Antioxidant activities by DPPH and ABTS radical scavenging were increased in a dose-dependent manner. These activity trends associated with the extraction solvent were different at each concentration, but resembled phenolic compound contents trend, generally. FRAP value increased in a dose-dependent manner, but there was a difference in radical scavenging activities when comparing between extraction solvents by butanol > ethanol > hexane > chloroform > water on all concentrations. The trend of α-amylase inhibition of extracts from 1,000 μg/mL to 2,000 μg/mL was not affected as enzyme activity is promoted and not inhibited. The inhibition of α-glucosidase was increased in a dose-dependent manner without water extracts, the activity on hexane extracts was higher than others per the extraction solvent. α-Glucosidase inhibition of hexane extracts showed 57.76% at 250 μg/mL, which is 2.8 times higher than the second highest chloroform extract (20.65%). From these results, we presume that the active ingredients of Stachys sieboldii Miq. is different according to the extraction solvent and also the activity is different by these major functional groups.

Various polymers were used as transfection factors for small interfering RNA (siRNA) to effectively suppress human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene in transgenic rice cells. Five kinds of polymers (PEI, PVA, PVP, and 8 and 20 kDa PEGs) were applied for delivery of siRNA with lipofectamine used as a control. In the cytotoxicity test, all polymers except 8 kDa PEG showed nontoxicity in relation to cell viability. For transfection efficiency, polyplexes composed of siRNA and PEG (20 kDa) did not significantly reduce production of intracellular hCTLA4Ig. On the other hand, siRNA + PEI polyplexes showed the most effective suppression efficiency with regards to production of intracellular hCTLA4Ig among all other polyplexes (PVA, PVP, and PEG (8 kDa)). Effects of molecular weight ratios of siRNA:PEI were investigated to obtain optimal transfection efficiency and avoid excessive damage to cells. PEI-based polyplexes with a 1:10 ratio of siRNA:PEI reduced production of intracellular hCTLA4Ig up to 70.6% without alteration of cell viability. These results demonstrate that PEI-based polyplexes are easy to prepare, inexpensive, non-toxic, and effective to deliver siRNA to transgenic plant cell cultures.

Terminal sialic acids on N-glycan of recombinant human erythropoietin are very important for in vivo half-life, as this glycoprotein has three N-glycosylation sites. N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) catalyze the formation of a glycan antennary structure. These enzymes display different reaction kinetics for a common substrate and generally show low expression in Chinese hamster ovary (CHO) cells. Therefore, genetic control of Mgat expression is an effective method to increase sialic acid contents by enhancing glycan antennarity. To produce highly sialylated albumin-erythropoietin (Alb-EPO), we co-overexpressed the Mgat1 and Mgat4 genes in CHO cells and determined the optimal ratio of Mgat1:Mgat4 gene expression. All transfected cell lines showed increased gene expression of Mgat4, including Mgat1 overexpressing cell line. Sialic acid content of Alb-EPO was highest in co-transfected cells with excess Mgat4 gene, and these cells showed a higher tri- and tetra-antennary structure than control cells. Based on these results, we suggest that co-transfection of the Mgat1 and Mgat4 genes at a ratio of 2:8 is optimal for extension of antennary structures. Also, regulation of Mgat gene expression in the glycan biosynthesis pathway can be a novel approach to increase the terminal sialic acids of N-glycans.

To examine the effects of suramin in CHO cell cultures in terms of the cell culture performance and quality of the Fc-fusion protein. Suramin had positive effects on the CHO cell cultures. The addition of suramin caused an increase in the viable cell density, cell viability, and titer of the Fc-fusion protein. Moreover, suramin had no impact on protein aggregation and enhanced the sialic acid contents of Fc-fusion protein by 1.18-fold. The enhanced sialylation was not caused by the increased nucleotide sugar level but by the inhibition of sialidase activity. The results showed that suramin inhibited apoptosis and had positive impacts on the productivity and quality of Fc-fusion protein. The addition of suramin increased the production of Fc-fusion protein and enhanced sialylation when added as a supplement to the media component in CHO cell cultures. This study suggested that suramin could be a beneficial additive during the biological production in terms of the productivity and quality of Fc-fusion protein.

To evaluate the characteristics of a novel human cell line, F2N78, including growth performance, physicochemical properties, and biological activity via direct comparison with CHO cells. The culture performance and physicochemical properties of antibodies produced from F2N78 and CHO cells were compared. For charge variants, antibodies produced from F2N78 cells contained a greater acidic charge variants than CHO cells. Regarding main glycoforms, degree of galactosylation was 52% in CT-A produced from F2N78 cells compared to CHO cells (37%). For sialic acid forms, α-2,6-linked sialic acid and N-acetylneuraminic acid (NANA) residues were observed in antibodies produced from F2N78 cells. In contrast, only α-2,3 linked sialic acid forms were detected in antibodies produced from CHO cells, and NANA and N-glycolylneuraminic acid were detected. Hybrid structure and bisecting structure were only observed in F2N78 cells. F2N78 cells stably produced antibodies with human specific N-glycan. The novel expression system based on human cells may facilitate the development of an alternative host cell for production of recombinant proteins.

In glycoprotein, Terminal sialic acid residues of Nlinked glycan are imperative things because they prevent the recognition from asialoglycoprotein-receptor that affect the half-life of glycoproteins. So establishment of culture process for enhancing sialic acid is important to maximize sialic acid contents of glycoprotein. In this study, we investigated effects of biphasic culture of Chinese hamster ovary (CHO) cells producing albumin-erythropoietin to increase sialylation. Biphasic cultures were performed with shift of CO₂ concentrations and temperatures at day 5 when viable cell density was decreased and sialidase was started to be released by cell lysis. The examined temperature set points were 33, 35 and 37℃ respectively and the CO₂ concentration was 1, 5, 10 and 15%. We confirmed that sialidase activity was the lowest in biphasic culture that was shifted from normal culture condition to 1% of CO₂ and 33℃ on day 5. However, the temperature and concentration of CO₂ have little effect on activity of α2,3-sialyltransferase. Also, sialic acid contents were enhanced 1.13-fold higher than that in control culture. In conclusion, Biphasic cultivation in CHO cells led to inhibition of sialidase activity and increases of sialylated glycan.

Ethnopharmacological relevance Evodiae Fructus (EF) is the dried, unripe fruit of Evodia rutaecarpa Benth., and one of the main components of traditional herbal prescriptions issued for the treatment of sterility caused by irregular menstruation in Korea. However, scientific evidence regarding the efficacy and action mechanism of EF is lacking. Aim of the study In this study, the authors established an in vitro screening tool to identify promising new drug candidates in herbal medicines for the prevention and treatment of premature ovarian failure. The protective effects of EF extracts against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity were investigated and the molecular mechanism responsible was sought. Material and Methods EF extract was prepared by boiling EF in water and its quality was confirmed by high performance liquid chromatography. CHO-K1 (Chinese hamster ovary cells) and COV434 (human ovarian granulosa cells) cells were plated, pretreated with EF extract for 2 h and then treated with 1.5 mM or 0.5 mM VCD for 24 h, respectively. Cell viabilities were measured using an MTT assay, and protein levels were determined by western blotting. Results VCD significantly suppressed the viability of both CHO-K1 and COV434 cells in a dose-dependent manner and induced the apoptosis of CHO-K1 cells at 1.5 mM. EF extract dose-dependently blocked the ovotoxicity induced by treatment with VCD. Furthermore, EF extract significantly activated Akt and downstream effectors such as mTOR and GSK-3β in CHO-K1 cells. The ability of EF extract to prevent cytotoxicity by VCD was antagonized by pretreatment of LY294002, a PI3K/Akt inhibitor. Conclusion EF has the ability to protect ovary cells against VCD-induced ovotoxicity, probably via Akt activation. These results suggest that the beneficial effects of EF might be useful for preventing premature ovarian failure or unexplained infertility caused by environmental factors.

Asymmetric dimethylarginine (ADMA), a high risk factor for endothelial dysfunction and cardiovascular disease (CVD), has been reported to promote cellular dysfunction via endoplasmic reticulum (ER) stress activation in various cells. Additionally, increased serum ADMA levels have been observed in incipient kidney diseases. Previously, we reported that activated ER stress is associated with mesangial cell apoptosis, observed mainly in overt nephropathy or chronic kidney disease (CKD). However, the effect of ADMA on mesangial cell apoptosis is unknown. Thus, we investigated the effects of ADMA on mesangial cell apoptosis and ER stress signaling. ADMA treatment increased caspase-3 activity and activated three branches of ER stress signaling (PERK, IRE1, and ATF6) that induce mesangial cell apoptosis. Pharmacological inhibitors of ER stress (inhibitors of PERK, IRE1, and S1P) attenuated ADMA-induced cleavage of caspase-3 and induced a decrease in the mitochondrial membrane potential. Furthermore, these inhibitors diminished the number of apoptotic cells induced by ADMA treatment. Taken together, our results indicated that ADMA treatment induces mesangial cell apoptosis via ER stress signaling. These results suggest that ADMA-induced mesangial cell apoptosis could contribute to the progression of overt nephropathy and CKD.

A series of Cu(II) complexes, [LnCuCl2] (Ln = LA–LF), supported by N′-aromatic-group-substituted N,N-bis((3,5-dimethyl-1H-pyrazol-1-yl)methylamine ligands have been synthesized. Variations of substituents at the ortho position of the aniline moiety influenced the solid-state structures of these complexes. The X-ray structures of [LnCuCl2] (Ln = LA–LC and LF) revealed that ligands are coordinated in a N,N,N-tridentate fashion to Cu(II) center and adopted a distorted square-pyramidal geometry, while [LDCuCl2] with N,N-bidentate coordination adopts distorted tetrahedral geometry. These complexes were capable of polymerizing methyl methacrylate (MMA) in the presence of modified methylaluminoxane (MMAO), with [LFCuCl2] displaying the highest activity (2.81 × 104 g PMMA (mol Cu)−1h−1). Regardless of ligand architecture, syndio-enriched PMMAs have been furnished with a slightly broader polydispersity index (PDI). Additionally, the in situ generated dimethyl derivatives, polymerized rac-LA and furnished PLA with mediocre heterotacticities at room temperature. Importantly, the catalytic efficiencies of the Cu(II) complexes studied have been found to be influenced by the steric and electronic properties of ligand.

Recombinant human acid alpha-glucosidase (rhGAA) from Chinese hamster ovary cells is the only approved treatment for patients with Pompe disease. In this study, rhGAAs were produced in transgenic rice cell suspension cultures under eight different conditions; untreated, 5 μM of 2-fluoro- l -fucose (2-FF), 50 μM of 2-FF, 100 μM of 2-FF, 100 μM of 2-FF + 0.5% Pluronic F-68 (PF-68), 100 μM of 2-FF + 0.05% Tween 20 (Tw 20), 0.5% PF-68, and 0.05% Tw 20. The N-glycans of eight rhGAAs were analyzed using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry. The relative quantity (%) of each glycan was obtained from the corresponding UPLC peak area per the sum (100%) of individual UPLC peak area. Fifteen N-glycans, comprising seven core-fucosylated glycans (71.5%, sum of each relative quantities) that have immunogenicity-inducing potential, three de-core-fucosylated glycans (15.4%), and five non-core-fucosylated glycans (13.1%), were characterized with high mass accuracy and glycan-generated fragment ions. The increases or decreases of relative quantities of each glycan from seven rhGAAs were compared with those of untreated control. The percentages of the sum of the relative quantities of core-fucosylated glycans divided by the sums of those of de-core- (core-fucose removed) and non-core-fucosylated glycans were calculated, and the lowest percentage was obtained in 100 μM of 2-FF combined with 0.5% PF-68. These results indicate that the relative quantity of each glycan of rhGAA produced in rice cell suspension cultures is significantly affected by their culture condition. This study performed the comparison of the N-glycan profiles of rice cell-derived rhGAA to identify the core-fucosylated glycans using UPLC and tandem mass spectrometry.

Gaucher disease is an inherited metabolic disease caused by genetic acid β -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.

Secreted Wnts play crucial roles in synaptogenesis and synapse maintenance, but endogenous factors promoting synapse elimination in central neurons remain unknown. Here we show that proline-rich 7 (PRR7) induces specific removal of excitatory synapses and acts as a Wnt inhibitor. Remarkably, transmembrane protein PRR7 is activity-dependently released by neurons via exosomes. Exosomal PRR7 is uptaken by neurons through membrane fusion and eliminates excitatory synapses in neighboring neurons. Conversely, PRR7 knockdown in sparse neurons greatly increases excitatory synapse numbers in all surrounding neurons. These non-cell autonomous effects of PRR7 are effectively negated by augmentation or blockade of Wnt signaling. PRR7 exerts its effect by blocking the exosomal secretion of Wnts, activation of GSK3β, and promoting proteasomal degradation of PSD proteins. These data uncover a proximity-dependent, reciprocal mechanism for the regulation of excitatory synapse numbers in local neurons and demonstrate the significance of exosomes in inter-neuronal signaling in the vertebrate brain., Wnts are important for synapse formation and maintenance. Here, the authors show that proline-rich 7 (PRR7) is a Wnt inhibitor that is secreted via exosomes to regulate excitatory synapse numbers.

Ethnopharmacological relevance Dipsaci Radix, which is the dried root of Dipsacus asperoides C. Y. Cheng and T. M. Ai (Dipsacaceae), is used to treat back pain and blood stasis syndrome in Korean traditional medicine. Aim of the study To understand the mechanisms responsible for the pharmacological activities of D. asperoides, we investigated the inhibitory effect of D. asperoides on lipopolysaccharide (LPS)-induced inflammation in mouse macrophages RAW 264.7 cells. Materials and methods Aqueous extract of D. asperoides (AEDA) was prepared by boiling D. asperoides in water and then administered to LPS treated RAW 264.7 cells. Cell viabilities were measured using an MTT assay, and protein levels were determined by western blotting. The ROS scavenging activity of AEDA was measured using a DCFH-DA assay and levels of nitric oxide (NO) were determined using a NO assay. The nuclear translocations of NF-κB and Nrf2 were investigated immunocytochemically, and pro-inflammatory cytokines in supernatant were evaluated by ELISA. Results Treatment with AEDA suppressed the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. AEDA also reduced ROS, pro-inflammatory cytokine (IL-6 and IL-1β) levels, and iNOS-derived NO and COX-2-derived prostaglandin E2 release to medium, and suppressed the phosphorylation and degradation of IκB and the activation of NF-κB in macrophages. Furthermore, treatment with AEDA inhibited the ERK1/2 pathway but not the JNK or p38 MAPK pathways. In addition, AEDA significantly promoted Nrf2 translocation from cytoplasm to nucleus and up-regulated the expression of HO-1. Conclusion These results suggest that AEDA has anti-inflammatory and anti-oxidative effects through the inhibition of NF-κB and ERK1/2 and the activation of Nrf2/HO-1 in macrophages.

In current semiconductor manufacturing, as the feature size of integrated circuit (IC) devices continuously shrinks, detecting endpoint in plasma etching process is more difficult than before. For endpoint detection, various kinds of sensors are installed in semiconductor manufacturing equipments, and sensor data are gathered with predefined sampling rate. Generally, detecting endpoint is performed using OES data of by-product. In this study, OES data of both by-product and etchant gas are used to improve reliability of endpoint detection. For the OES data pre-processing, a combination of Signal to Noise Ratio (SNR) and Principal Component Analysis (PCA),are used. Polynomial Regression and Expanded Hidden Markov model (eHMM) technique are applied to pre-processed OES data to detect endpoint.

Streptozotocin (70 mg/kg B.W., i.p.)으로 유발된 당뇨쥐에서 돼지감자(Helianthus tuberosus L.) 추출액의 혈당강하 및 혈중 지질대사 개선효과를 알아보고자, SD 랫드(200∼220 g) 수컷을 3군(NC, DC, DJ)으로 나누어 돼지감자 추출액을 4주간 경구투여 하였다. 본 연구 결과 음수량과 식이량은 당뇨군에서 유의한 증가를 보였으며, 체중은 유의한 감소를 보였다(p＜0.05). 그러나 당뇨돼지 감자군에서 체중감소를 줄이고 식이효율을 다소 개선하는 것으로 나타났다. 혈청 중 AST 및 ALT 농도는 당뇨대조군(DC)에 비해 당뇨 돼지감자군(DJ)은 각 14.3%, 27.3% 유의한 감소를 보였다(p＜0.05). 혈청 HDL-C의 농도는 DC군에 비해 DJ군은 31.8% 유의한 증가를 보였다(p＜0.05). 혈청 TG, LDL-C 및 glucose 농도는 DC군에 비해 DJ군에서 각 25.4%, 56.6% 및 33.0% 유의한 감소를 나타내었다 (p＜0.05). 혈중 포도당 농도를 측정한 결과 당뇨 유발 후 3주 및 4주 후 DC군에 비해 DJ군은 각 31.0%, 31.6% 유의하게 감소를 보였다(p＜0.05). 이로서 돼지감자는 당뇨로 인한 체중 저하를 방지하고 혈당을 강하시키며 지방대사를 개선하는 것으로 판단된다.

The auxiliary beta subunit plays an important role in the regulation of voltage-gated calcium (Ca-V) channels. Recently, it was revealed that beta 2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of beta-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the beta 2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the beta 2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated beta 2e subunit on inactivation kinetics and regulation of CaV channels by PIP2. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of beta 2e (K2A/W5A) increased the PIP2 sensitivity of Ca(V)2.2 and Ca(V)1.3 channels by similar to 3-fold compared with wild-type beta 2e subunit. Together, our results suggest that membrane targeting of the beta 2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-beta 2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.

Background: It is well known that obesity increases the risk of metabolic syndrome (MS); however, the associations between walking and MS risk factors among Korean adults still need to be elucidated. The purpose of this study was to examine the relationships of body mass index (BMI) and walking with MS risk factors among Korean adults from the fifth Korea national health and nutrition examination survey. Methods: A total of 17,019 (7,334 males, 9,685 females) Korean adults participated in the cross-sectional study. We measured walking, BMI, and MS risk factors including waist-circumference (WC), glucose, triglyceride (TG), high density lipoprotein cholesterol (HDL-C), systolic blood pressure (SBP), and diastolic blood pressure (DBP). Results: Results showed that 1) subjects with high BMI (23 kg/m 2 ≤ BMI) had significantly increased MS risk factors compared to subjects with low BMI (BMI < 23 kg/m 2 ), 2) subjects who participated in walking had significantly decreased WC, TG and increased HDL-C compared to subjects who didn’t participate in walking, and 3) when subjects were divided into four groups according to walking and BMI levels, subjects with nonparticipation in walking and high BMI showed the worst profile of metabolic syndrome risk factors. Moreover, subjects with nonparticipation in walking and high BMI had 7.42 times higher the prevalence of MS compared to subjects with participation in walking and low BMI after adjusted for age, sex, BMI, and smoking. Conclusion: Our study showed that improvement in walking and reduction in BMI are important factors for the prevention of metabolic syndrome in Korean adults.

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

A first-generation calix-dendrimer 1 suitable as a multidentate ligand for metal-ion binding was prepared from 1,3,5-tris(aminomethyl)-2,4,6-trimethylbenzene as the dendrimer core and p-tert-butylcalix [4] arene monomethyl ester as a repetitive branch, and characterized by spectroscopic techniques.

Activation of adiponectin receptors (AdipoRs) by its natural ligand, adiponectin has been known to be involved in modulating critical metabolic processes such as glucose metabolism and fatty acid oxidation as demonstrated by a number of in vitro and in vivo studies over last two decades. These findings suggest that AdipoRs' agonists could be developed into a potential therapeutic agent for metabolic diseases, such as diabetes mellitus, especially for type II diabetes, a long-term metabolic disorder characterized by high blood sugar, insulin resistance, and relative lack of insulin. Because of limitations in production of biologically active adiponectin, adiponectin-mimetic AdipoRs' agonists have been suggested as alternative ways to expand the opportunity to develop anti-diabetic agents. Based on crystal structure of AdipoR1, we designed AdipoR1's peptide agonists using protein-peptide docking simulation and screened their receptor binding abilities and biological functions via surface plasmon resonance (SPR) and biological analysis. Three candidate peptides, BHD1028, BHD43, and BHD44 were selected and confirmed to activate AdipoR1-mediated signal pathways. In order to enhance the stability and solubility of peptide agonists, candidate peptides were PEGylated. PEGylated BHD1028 exhibited its biological activity at nano-molar concentration and could be a potential therapeutic agent for the treatment of diabetes. Also, SPR and virtual screening techniques utilized in this study may potentially be applied to other peptide-drug screening processes against membrane receptor proteins.

Effects of Fermented Soybean on Body Weight, Body Fat and Serum Lipid in Obese WomenEun-Young Nam 1 , Dong-Il Kim 2 , Min-Sun Choi 2 , Hyung-Jun Kim 11 Dept of Korean Gynecology, College of Korean Medicine Se-Myung University 2 Dept of Korean Gynecology, College of Korean Medicine Dong-Guk UniversityObjectives: The purpose of this study was to examine the effects of fermented soybean on body weight, body fat, serum lipid profiles in obese women, especially specific to menopausal woman.Methods: Sixty healthy obese volunteers who visited ⃝⃝ University Oriental Hospital from May 20th, 2014 to September 25th, 2014 took part in clinical trial. They divided into 2 groups, 30 volunteers allocated to fermente d soybean and other 30 to placebo group. Body weight, BMI, waist and hip ratio, ser um lipid were measured 3 times, and fat percentage, leptin, adiponectin were evaluated 2 times.Results: All 60 volunteers completed 12-week trial. 5 men were excluded , and 2 women against the clinical decision rule were excluded. In the end, 53 women were studied as clinical subjects. After 12 weeks intervention, there was no effects in comparison of group by time interaction. Without considering time interaction, there was a significant difference in triglyceride level betwee n soybean group and placebo group (p=0.044). Treatment group were dividing by age 4 0, a group in age 40 or over 40, and other group aged below 40. There was a signi ficant difference in group by time interaction of total cholesterol level, and witho ut considering time interaction, there was a significant change in waist-hip ratio between groups.Conclusions: There were no effects on weight and body fat decrease in 12-we ek trial using fermented soybean as a supplement. But there were s ignificant differences in triglyceride change between the treatment and placebo groups , also cholesterol and waist and hip ratio in soybean group divided by age 40. It seems that fermented soybean is effected on improving serum lipid profiles. Key Words: Obesity, Women, Fermented Soybean, Cheonggukjang, Serum lipid