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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.

Beige adipocytes, which consume energy mainly in an uncoupling protein 1 (UCP1)-dependent manner, are risen in white adipose tissue (WAT) depots. Since beige adipocyte development is gaining attention as a potential strategy for conquering obesity, worldwide researchers are making efforts to study its biological aspects. However, assessing UCP1 protein levels in beige adipocytes is challenging because of the high level of lipid contaminants in WAT. This study showed that an acetone precipitation method had advantages over conventional methods for eliminating lipid contaminants, achieving clear Western blot bands for WAT proteins, especially UCP1. Our results suggest that the acetone precipitation cleaning method could be useful for the clear analysis and precise evaluation of WAT proteins.

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.

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.

International audience; Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is an autoimmune disorder which affects small-and, to a lesser degree, medium-sized vessels. ANCA-associated vasculitis encompasses three disease phenotypes: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). This classification is largely based on clinical presentations and has several limitations. Recent research provided evidence that genetic background, risk of relapse, prognosis, and co-morbidities are more closely related to the ANCA serotype, proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA, compared to the disease phenotypes GPA or MPA. This finding has been extended to the investigation of biomarkers predicting disease activity, which again more closely relate to the ANCA serotype. Discoveries related to the immunopathogenesis translated into clinical practice as targeted therapies are on the rise. This review will summarize the current understanding of the immunopathogenesis of ANCA-associated vasculitis and the interplay between ANCA serotype and proposed disease biomarkers and illustrate how the extending knowledge of the immunopathogenesis will likely translate into development of a personalized medicine approach in the management of ANCA-associated vasculitis.

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.

Beige adipocytes have recently been shown to regulate energy dissipation when activated and help organisms defend against hypothermia and obesity. Prior reports indicate that beige-like adipocytes exist in adult humans and that they may present novel opportunities to curb the global epidemic in obesity and metabolic illnesses. In an effort to identify unique features of activated beige adipocytes, we found that expression of the cholinergic receptor nicotinic alpha 2 subunit (Chrna2) was induced in subcutaneous fat during the activation of these cells and that acetylcholine-producing immune cells within this tissue regulated this signaling pathway via paracrine mechanisms. CHRNA2 functioned selectively in uncoupling protein 1 (Ucp1)-positive beige adipocytes, increasing thermogenesis through a cAMP- and protein kinase A-dependent pathway. Furthermore, this signaling via CHRNA2 was conserved and present in human subcutaneous adipocytes. Inactivation of Chrna2 in mice compromised the cold-induced thermogenic response selectively in subcutaneous fat and exacerbated high-fat diet-induced obesity and associated metabolic disorders, indicating that even partial loss of beige fat regulation in vivo had detrimental consequences. Our results reveal a beige-selective immune-adipose interaction mediated through CHRNA2 and identify a novel function of nicotinic acetylcholine receptors in energy metabolism. These findings may lead to identification of therapeutic targets to counteract human obesity.

A reproducible method for cryopreservation of transgenic rice cells (Oryza sativa L. cv. Dongjin) producing recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) has been established. Here, we assessed recovery media and investigated recombinant protein homogeneity after long-term preservation. For recovery of cryopreserved transgenic rice cells, AA medium was suitable in terms of both morphology and production of hCTLA4Ig. There were no differences in cell growth, sugar consumption, and hCTLA4Ig production between non-cryopreserved and cryopreserved cells for up to 1 month. hCTLA4Ig produced from cryopreserved cells was identical that of hCTLA4Ig from non-cryopreserved cells, as determined by analysis of its molecular weight and isoforms. For long-term preservation, cell viability was stably maintained at 61% for 26 months. In conclusion, these results demonstrate the possibility for reproducible cryocell-banking of transgenic rice cells without changes in the characteristics of cells and target proteins.

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 The aim of this study was to investigate the effect of a six-week-long exercise program using outdoor exercise equipment on fitness, insulin resistance and adipocytokines among Korean elderly. Methods A total of 47 participants were randomized into one of the following three groups; control, resistance exercise or combined exercise (aerobic and resistance exercise). The resistance exercise group completed three resistance types of exercise. The combined exercise group completed five exercises, including three resistance types of exercise and two aerobic types of exercise. Participants’ body composition, fitness level, homeostasis model assessment of insulin resistance (HOMA-IR), and adipocytokines were measured at baseline and at the end of six weeks. Results After six weeks of exercise training, participants in the combined exercise group exhibited significant reduction in insulin, HOMA-IR and chemerin levels, while significant reduction was observed in HOMA-IR only in the resistance exercise group compared with the control group. Meanwhile, six weeks of exercise training, whether resistance exercise alone or combined, significantly improved upper body muscular strength/endurance and physical function compared to the control group. Conclusions Six weeks of combined exercise using outdoor exercise equipment was effective in improving fitness, HOMA-IR, circulating chemerin levels, and other known risk factors of chronic diseases.

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.

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 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 this research, recombinant human cytotoxic Tlymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced by transgenic rice cells. RAmy3D promoter was used for overcome the limitation of low expression level in transgenic plant cells, and the secretion of target protein was accomplished by signal peptide. However, the RAmy3D promoter system which can be induced only by sugar starvation causes the decrease of cell viability. As a result, cell death promotes the release of protease which degrades the target proteins. The protein stability and productivity can be significantly influenced by proteolysis activity. Therefore, development of new strategies are necessary for the in situ recovery of target proteins from cell culture media. In this study, in situ recovery was performed by various strategies. Direct addition of Protein A resin with nylon bag leads to cell death by increased shear stress and decrease in production of hCTLA4Ig by protease. Medium exchange through modified flask could recover hCTLA4Ig with high cell viability and low protease activity, on the other hand, the productivity was lower than that of control. When in situ recovery was conducted at day 7 after induction in air-lift bioreactor, 1.94-fold of hCTLA4Ig could be recovered compared to control culture without in situ recovery. Consequently, in situ recovery of hCTLA4Ig from transgenic rice cell culture could enhance productivity significantly and prevent degradation of target proteins effectively.

In this system, rice cells were genetically modified to express human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) using RAmy3D promoter induced by sugar depletion. Even though the target protein fused with signal sequence peptide, plant cell wall can be a barrier against secretion of recombinant proteins. Therefore, hCTLA4Ig can be trapped inside cell wall or remained in intracellular space. In this study, to enhance the secretion of hCTLA4Ig from cytoplasm and cell walls into the medium, permeabilizing agents, such as dimethyl sulfoxide (DMSO), Triton X-100 and Tween 20, were applied in transgenic rice cell cultures. When 0.5% (v/v) of DMSO was added in sugar-free medium, intracellullar hCTLA4Ig was increased, on the other hand, the secreted extracellular hCTLA4Ig was lower than that of control. DMSO did not give permeable effects on transgenic rice cell cultures. And Triton X-100 was toxic to rice cells and also did not give enhancing permeability of cells. When 0.05% (v/v) Tween 20 was added in rice cell cultures, however, intracellular hCTLA-4Ig was lower than that of control cultures. And the maximum 44.76 mg/L hCTLA4Ig was produced for 10 days after induction, which was 1.4-fold increase compared to that of control cultures. Especially, Tween 20 at 0.05% (v/v) showed the positive effect on the secretion of hCTLA4Ig though the decrease of intracellular hCTLA4Ig. Also, Tween 20 as a non-toxic surfactant did not affect the cell growth, cell viability and protease activity. In conclusion, secretion of hCTLA4Ig could be increased by enhancing permeability of cells regardless of the cell growth, cell viability and protease activity.

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.

A human hybrid cell line, F2N78, was developed by somatic fusion of HEK293 and Namalwa cells for the production recombinant biopharmaceutical proteins. In this study, we performed perfusion culture to verify its potential in culture process used for human cell expression platform. Cell viability could be maintained over 90% and high viable cell density was obtained at higher than 1.0 × 10(7) cells/mL by bleeding process in perfusion culture. The cells were adapted well in both culture modes, but there were apparent differences in protein quality. Compared to fed-batch culture, degalactosylated forms such as G0F and G0 as well as Man5 showed no significant increases in perfusion culture. In terms of charge variants, acidic peaks increased, whereas main peaks constantly decreased according to the length of culture period in both methods.

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.

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.

The dopamine transporter (DAT) protein, a component of the dopamine system, undergoes adaptive neurobiological changes from drug abuse. Prevention of relapse and reduction of withdrawal symptoms are still the major limitations in the current pharmacological treatments of drug addiction. The present study aimed to investigate the effects of essential oils extracted from Elsholtzia ciliata, Shinchim, Angelicae gigantis Radix, and Eugenia caryophyllata, well-known traditional Korean medicines for addiction, on the modulation of dopamine system in amphetamine-treated cells and to explore the possible mechanism underlying its therapeutic effect. The potential cytotoxic effect of essential oils was evaluated in PC12 rat pheochromocytoma cells using cell viability assays. Quantification of DAT, p-CREB, p-MAPK, and p-Akt was done by immunoblotting. DAT was significantly reduced in cells treated with 50 μM of amphetamine in a time-dependent manner. No significant toxicity of essential oils from Elsholtzia ciliata and Shinchim was observed at doses of 10, 25, and 50 μg/mL. However, essential oils from A. gigantis Radix at a dose of 100 μg/mL and E. caryophyllata at doses of 50 and 100 μg/mL showed cytotoxicity. Treatment with GBR 12909, a highly selective DAT inhibitor, significantly increased DAT expression compared with that of amphetamine only by enhancing phosphorylation of mitogen-activated protein kinases (MAPK) and Akt. In addition, essential oils effectively induced hyperphosphorylation of cyclic-AMP response element-binding protein (CREB), MAPK, and Akt, which resulted in DAT upregulation. Our study implies that the essential oils may rehabilitate brain dopamine function through increased DAT availability in abstinent former drug users.

The present study examined the preventive effect of osteoporosis in ovariectomized (OVX) mice by feeding of phytate-reduced brown rice inoculated with the selected Lactobacillus sakei Wikim001 having high phytase activity. Contrary to 5% BR-WK diet, OVX mice with normal diet containing 10% brown rice manufactured with L. sakei Wikim001 (10 % BR-WK diet) exhibited the increase of trabecular bone volume/tissue volume, bone surface/tissue volume, trabecular number, and bone mineral density in femur as compared with the control OVX mice. Moreover, OVX mice fed with 10% BR-WK diet decreased bone turnover markers, including osteocalcin and CTX-1. These results suggest that BR-WK diet has a bone sparing effect in OVX-induced trabecular bone loss and prevents deterioration of bone microarchitecture by suppressing bone turnover rate. Therefore, BR-WK diet may be useful for preserving bone mass and structure against osteoporosis.

Membrane targeting of the β2e subunit is dynamically regulated by M1 muscarinic receptor signaling to promote fast inactivation of CaV2.2., High voltage-activated Ca2+ (CaV) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating CaV channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on CaV2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed CaV2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of CaV2.2(β2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of CaV2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in CaV channel gating. The phospholipid–protein interaction observed here provides structural insight into mechanisms of membrane–protein association and the role of phospholipids in ion channel regulation.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for the commercial production of recombinant proteins. However, they show relatively low yields of recombinant proteins in comparison with microbial cells. Various strategies have been tried to overcome this drawback. The acetyl moieties are attached to the N-terminus of histone by histone acetyltransferase (HAT) while histone deacetylase (HDAC) removes histone-bound acetyl groups. HDAC inhibitor (HDACi), such as sodium butyrate, sodium propionate and valproic acid, can enhance specific productivity of CHO cells. Human albumin-erythropoietin (Alb-EPO) is a novel 105 kDa protein comprising recombinant human EPO fused to human albumin. In this study, we examined the effects of HDACi on the production of Alb-EPO in CHO cells with various concentrations in the range of 0-1 mM. The results showed that sodium butyrate was found to be the best HDACi for enhancing productivity. It enhanced not only the production of Alb-EPO but also the apoptosis of recombinant CHO cells.

Glucagon-like peptide-1 receptor (GLP-1R) is closely associated with the onset of diabetes and its complications. However, its roles in diabetic retinopathy are unknown. Retinal pigment epithelial (RPE) cells are a crucial component of the outer blood-retina barrier and their death is related to the progression of diabetic retinopathy. Thus, we examined the pathophysiological role of GLP-1R in RPE cell apoptosis. We found that GLP-1R expression was lower in the isolated neuroretina and RPE cells of streptozotocin-treated rats than in vehicle-treated rats. High-glucose treatment also decreased GLP-1R expression in a human RPE cell line (ARPE-19 cells). GLP-1R was silenced in ARPE-19 cells, in order to elucidate the pathophysiological roles of GLP-1R. This increased intracellular reactive oxygen species (ROS) generation and activated p53-mediated Bax promoter and endoplasmic reticulum (ER) stress signaling. We also found that GLP-1R knockdown-mediated p53 expression was regulated by ER stress. Interestingly, antioxidant treatment and peroxiredoxin 1 (Prx1) overexpression attenuated GLP-1R knockdown-induced ER stress signaling and p53 expression. Finally, to confirm that GLP-1R activation has protective effects, ARPE-19 cells were treated with exendin-4, a synthetic GLP-1R agonist. This attenuated high-glucose-induced ROS generation, ER stress signaling, and p53 expression. Collectively, these results indicated that hyperglycemia decreases GLP-1R expression in RPE cells. Such a decrease generates intracellular ROS, which increases ER stress-mediated p53 expression, and subsequently causes apoptosis by increasing Bax promoter activity. Our data suggested that regulation of GLP-1R expression is a promising approach for the treatment of diabetic retinopathy.

Wnt signaling controls critical developmental processes including tissue/body patterning. Here we report the identification of a novel regulator of Wnt signaling, OTTOGI (OTG), isolated from a large-scale expression screening of human cDNAs in zebrafish embryos. Overexpression of OTG in zebrafish embryos caused dorso-anteriorized phenotype, inhibited the expression of Wnt target genes, and prevented nuclear accumulation of β-catenin. Conversely, knockdown of zebrafish otg using specific antisense morpholino promoted nuclear accumulation of β-catenin and caused ventralization. However, OTG failed to rescue headless-like phenotype induced by inhibition of GSK-3β activity, suggesting that OTG acts upstream of GSK-3β. OTG bound specifically to Frizzled8 (Fz8) receptor and caused retention of Fz8 in the endoplasmic reticulum possibly by preventing N-linked glycosylation of Fz8. Taken together, our data indicate that OTG functions as a novel negative regulator of Wnt signaling during development by the modulation of cell surface expression of Fz receptor.

Background & Aims Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and type 2 diabetes. Thioredoxin-interacting protein (TXNIP) regulates the cellular redox state and metabolism and has been linked to many diseases, including diabetes. Therefore, we examined the role of TXNIP in hepatic steatosis in vitro and in vivo . Methods Lipogenic and inflammatory proteins produced by hepatocytes treated with palmitic acid (PA) or transfected with TXNIP or Txnip siRNA were measured by Western blotting. Lipid accumulation was assessed using Oil Red O staining. Protein interactions were assessed by immunoprecipitation and proximity ligation assay. Hepatic protein levels were measured by Western blotting from wild type or Txnip −/− mice fed a high-fat diet (HFD) or chow diet. Livers from NAFLD patients were compared with normal liver by immunohistochemistry. Results PA increased TXNIP, and inflammatory and lipogenic proteins in both AML12 and H4IIE cells. It also increased the peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α), which mediated the expression of lipogenic markers and lipid accumulation. In addition, PA increased protein arginine methyltransferase-1 (PRMT1) and PRMT1 siRNA abolished the increase in lipogenic markers with PGC-1α. Furthermore, TXNIP interacted with PRMT1 in PA-treated hepatocytes. In vivo , levels of lipogenic proteins, inflammatory molecules, PGC-1α, and PRMT1 were increased in the livers of HFD mice compared with those fed a chow diet, and were ameliorated in HFD Txnip −/− mice. Moreover, TXNIP, PRMT1, and PGC-1α were elevated in the livers of human NAFLD patients. Conclusions TXNIP mediates hepatic lipogenesis via PRMT1 and PGC-1α regulation and inflammation in vitro and in vivo , implying that targeting TXNIP and PRMT1 is a potential therapeutic approach for treatment of NAFLD.

Remsima® (infliximab) was recently approved as the world's first biosimilar monoclonal antibody (mAb) in both the European Union and Korea. To achieve this, extensive physicochemical characterization of Remsima® in relation to Remicade® was conducted in order to demonstrate the highly similar properties between the two molecules. A multitude of state-of-the-art analyses revealed that Remsima® has identical primary as well as indistinguishable higher order structures compared with the original product. Monomer and aggregate contents of Remsima® were also found to be comparable with those of Remicade®. In terms of charge isoforms, although Remsima® was observed to contain slightly less basic variants than the original antibody, the difference was shown to be largely due to the presence of C-terminal lysine. On the other hand, this lysine was found to be rapidly clipped inside serum in vitro and in vivo, suggesting it has no effect on the biological potency or safety of the drug. Analysis of the glycan contents of the antibodies showed comparable glycan types and distributions. Recent results of clinical studies have further confirmed that the two antibody products are highly similar to each other. Based on this research as well as previous clinical and non-clinical comparability studies, Remsima® can be considered as a highly similar molecule to Remicade® in terms of physicochemical properties, efficacy, and safety for its final approval as a biosimilar product to Remicade®.

Hyperglycemia-induced apoptosis of retinal pigment epithelial (RPE) cells is considered to be involved in the progression of diabetic retinopathy. Histone arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) has emerged as an important histone modification involved in gene regulation. However, the role of PRMTs in diabetic retinopathy has not been elucidated. Here, we found that expression of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) was increased in the high-glucose treated human RPE cell line ARPE-19 and in the RPE layer of streptozotocin-treated rats. In addition, high-glucose induced apoptosis in ARPE-19 cells. To determine the function of CARM1 on RPE cell apoptosis, we performed gain- and loss-of-function studies. CARM1 overexpression increased apoptosis of RPE cells. In contrast, silencing of CARM1 expression by siRNA and pharmacological inhibition of CARM1 activity abolished high-glucose-induced RPE cell apoptosis. Furthermore, we found that inhibition of histone 3 arginine 17 (H3R17) asymmetric dimethylation attenuates both CARM1- and high-glucose-induced apoptosis in RPE cells. Together, these results show that high-glucose-induced CARM1 expression increases RPE cell apoptosis via H3R17 asymmetric dimethylation. Strategies to reduce CARM1 expression or enzymatic activity could be used to prevent apoptosis of RPE cells in the progression of diabetic retinopathy.

It has been reported that safflower seeds have preventive effects against osteoporosis. Recently, safflower buds (SB) were found to have more useful functional ingredients than safflower seeds. In the current study, we evaluated the anti-osteoporosis effects of SB diet in ovariectomized (OVX) rats. The rats were divided into five groups; sham operated group, OVX alone group, OVX plus 17β-estradiol (E2 10 μg/kg, i.p.) and OVX plus SB diet feeding group (0.3% or 1%). Feeding of SB diet (0.3% or 1%) to OVX rats markedly increased bone mineral density (BMD) of femurs, compared to the OVX group. The OVX rats exhibited a marked increase in trabecular separation (Tb.Sp) and this change was inhibited by the feeding of SB diet, similar to that seen with OVX+E2 group. Moreover, feeding of SB diet to OVX rats decreased the markers of bone turnover, including osteocalcin and alkaline phosphatase (ALP). These results suggest that SB extract has a bone sparing effect in OVX-induced trabecular bone loss and prevents deterioration of bone microarchitecture by suppressing the rate of bone turnover. Therefore, SB may be useful for preserving bone mass and structure in estrogen deficient women with a potential role in reducing postmenopausal osteoporosis.

Arginine methylation is responsible for diverse biological functions and is mediated by protein arginine methyltransferases (PRMTs). Nonalcoholic fatty liver disease (NAFLD) is accompanied by excessive hepatic lipogenesis via liver X receptor α (LXRα). Thus we examined the pathophysiological role of PRMTs in NAFLD and their relationship with LXRα. In this study, palmitic acid (PA) treatment increased PRMT3, which is correlated with the elevation of hepatic lipogenic proteins. The expression of lipogenic proteins was increased by PRMT3 overexpression, but decreased by PRMT3 silencing and use of the PRMT3 knockout (KO) mouse embryonic fibroblast cell line. PRMT3 also increased the transcriptional activity of LXRα by directly binding with LXRα in a methylation-independent manner. In addition, PA treatment translocated PRMT3 to the nucleus. In animal models, a high-fat diet increased the LXRα and PRMT3 expressions and binding, which was not observed in LXRα KO mice. Furthermore, increased PRMT3 expression and its binding with LXRα were observed in NAFLD patients. Taken together, LXRα and PRMT3 expression was increased in cellular and mouse models of NAFLD and human patients, and PRMT3 translocated into the nucleus bound with LXRα as a transcriptional cofactor, which induced lipogenesis. In conclusion, PRMT3 translocation by PA is coupled to the binding of LXRα, which is responsible for the onset of fatty liver.

The clinical use of therapeutic antibodies has increased sharply because of their many advantages over conventional small molecule drugs, particularly with respect to their affinity, specificity, and serum stability. Tumor or infected cells are removed by the binding of antibody Fc regions to Fc gamma receptors (FcγRs), which stimulate the activation of immune effector cells. Aglycosylated full-length IgG antibodies expressed in bacteria have different Fc conformations compared to their glycosylated counterparts produced in mammalian cells. As a result, they are unable to bind FcγRs, resulting in little to no activation of immune effector cells. In this study, we created a combinatorial library randomized at the upper CH2 loops of an aglycosylated Fc variant (Fc5: E382V/M428) and used a high-throughput flow cytometry library screening method, combined with bacterial display of homodimeric Fc domains for enhanced FcγR binding affinity. The trastuzumab Fc variant containing the identified mutations (Q295R, L328W, A330V, P331A, I332Y, E382V, M428I) not only exhibited over 120 fold higher affinity of specific binding to FcγRI than wild type aglycosylated Fc, but also retained pH-dependent FcRn binding. These results show that an aglycosylated antibody expressed in bacteria can be evolved for novel FcγR affinity and specificity.

Mesenchymal stem cells (MSCs) are must be cultured in chemically defined, xeno-free culture system for cell-based therapies. One major overcome for their clinical use is the unsafety of fetal bovine serum (FBS), which is a crucial part of all media currently used for the culture of MSCs. Previously, we developed novel methods the production of silkworm gland hydrolysate (SGH). Also, we showed the effect of the SGH on the cell growth promotion. We demonstrated whether the characteristics of MSCs were maintained in the serum-free media containing SGH during long-term cultivation. The results of microscopic observation showed that the morphology of stem cells were not different between the media containing 10% FBS and 1 mg/mL SGH. After the long-term culture of MSCs in serum-free media containing SGH, the rate of MSCs growth was well maintained. The analysis of anti-apoptotic effect of SGH showed that early apoptosis of MSCs was reduced by 3.7-fold. Furthermore, MSCs specific surface markers and stemness gene expression (Nanog, Sox2) were preserved as that of MSCs in the media containing 10% FBS. In conclusion, we demonstrate that the SGH can be used as useful supplement for MSCs cultivation in serumfree media.

Decursin is a major biological active component of Angelica gigas Nakai and is known to induce apoptosis of metastatic prostatic cancer cells. Recently, other reports have been commissioned to examine the anticancer activities of this plant. In this study, we evaluated the inhibitory activity and related mechanism of action of decursin against glioblastoma cell line. Decursin demonstrated cytotoxic effects on U87 and C6 glioma cells in a dose-dependent manner but not in primary glial cells. Additionally, decursin increased apoptotic bodies and phosphorylated JNK and p38 in U87 cells. Decursin also down-regulated Bcl-2 as well as cell cycle dependent proteins, CDK-4 and cyclin D1. Furthermore, decursin-induced apoptosis was dependent on the caspase activation in U87 cells. Taken together, our data provide the evidence that decursin induces apoptosis in glioblastoma cells, making it a potential candidate as a chemotherapeutic drug against brain tumor.

Formaldehyde (FA) is toxic to the respiratory system, and nitric oxide (NO) dysfunction stimulates the onset of respiratory diseases. The involvement of dimethylarginine dimethylaminohydrolase (DDAH), the l-arginine analogue asymmetric dimethylarginine (ADMA) degrading enzyme, in FA-induced cell death in lung epithelial cells has not been investigated. In this study, we assessed the effect of FA on DDAH expression and endoplasmic reticulum (ER) stress in A549 cells. We also investigated the preventive effect of DDAH overexpression on ER stress and apoptosis in FA-induced cell death. FA decreased viability in A549 cells and decreased DDAH1 and DDAH2 mRNA and protein expression in a time-dependent manner (>4h). This coincided with increased phosphorylation of the ER stress proteins IRE1α, PERK, and eIF-2α, as well as increased expression of pro-apoptotic proteins such as Bax, C/EPB homologous protein (CHOP), cleaved PARP, and cleaved caspase-3, but decreased expression of the anti-apoptotic protein Bcl-2. ADMA treatment mimicked the effect of FA. Overexpression of DDAH1, but not DDAH2, prevented FA-induced decreases in cell viability, phosphorylation of IRE1α, PERK, and eIF2α, and expression of CHOP. Effects of DDAH1 overexpression, but not DDAH2 overexpression, restored FA-induced increases in Bax, CHOP, cleaved PARP, cleaved caspase-3 and decreases in Bcl-2. In conclusion, FA induces apoptosis of lung epithelial cells via a decrease of DDAH1 through ER stress.

Recent studies comparing the insulin-producing cell (IPC) differentiation capacity of mesenchymal stem cells (MSCs) derived from four different sources (bone marrow, Wharton’s jelly, adipose tissue, and the periosteum) demonstrated that IPC differentiation of periosteum-derived progenitor cells (PDPCs) progressed faster than any other MSCs within 7 days, indicating that PDPCsare most suited to IPC differentiation. Here, two different cell culture methods, adhesion and cluster culture, were assessed for their ability to support in vitro IPC differentiation. The induction of IPC differentiation was confirmed by RTqPCR analysis of insulin gene expression levels and immunofluorescence analysis of insulin protein. An enzyme-linked immunosorbent assay was used to quantify secreted insulin. PDPC-derived IPCs from cluster cultures demonstrated a significantly increased expression of insulin and an enhanced secretion of insulin of insulin protein in response to glucose compared to IPCs derived from adhesion cultures. Thus, pancreatic islet-like cluster cultures appear to provide the optimal conditions such as cluster culture for IPC differentiation of PDPCs.

Transgenic plant cell cultures are an attractive expression system for the production of industrial and pharmaceutical proteins because of their advantages in safety and low production cost. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced and secreted when sugar was depleted in culture medium by transgenic rice cell lines (Oryza sativa L.) using RAmy3D promoter. Due to the production of the target protein by sugar depletion, concomitant occurrence of cell death is inevitable. For that reason, inhibition of cell death for enhancing productivity was necessary for the production period without energy sources. Supplementation of 0.1 mM sodium nitroprusside improved cell viability by 1.4-fold and maximum hCTLA4Ig production by 1.3-fold compared to those of control. Addition of 1 and 10 mM glutathione, N-acetylcysteine (NAC), and nicotinamide inhibited apoptotic-like programmed cell death by decreasing the activity of reactive oxygen species. Production hCTLA4Ig was enhanced 1.4-, 1.25-, and 1.15-fold with 10 mM NAC, 1 mM NAC, and 1 mM glutathione, respectively. In addition, it was found that the supplementation of NAC enhanced the cell viability.

Chemerin is a recently discovered adipocytokine, associated with adiposity and insulin sensitivity. The current study investigated the effects of lifestyle intervention on circulating chemerin level and its association with insulin resistance and adiponectin in human.Forty male and 20 female obese adults (mean age: 29.7±5.7 y, mean BMI: 29.3±4.5 kg/m(2)) completed an 8-week lifestyle intervention program, which consisted of a home-based diet and exercise program. Anthropometric measurements and biomarkers were assessed at the baseline and at the end of the study.Eight weeks of lifestyle intervention reduced body weight, visceral fat and subcutaneous fat by 3.8%, 15.3% and 11.5%, respectively. The lifestyle intervention further reduced fasting insulin (10.9±6.6 vs. 7.6±5.3 μU/ml, p0.001) and homeostasis assessment of insulin resistance (HOMA-IR) (2.3±1.5 vs. 1.6±1.2, p0.001), chemerin (103.3±20.7 vs. 96.5±19.5 ng/ml, p0.001) and hs-CRP levels (1.3±1.8 vs. 0.2±0.2 mg/dl, p0.001) while it increased fasting pentraxin (PTX) 3 (0.6±0.7 vs. 0.7±0.4 ng/ml, p=0.049) level. The Δ chemerin levels correlated with Δ insulin (r=0.349, p=0.024) and HOMA-IR (r=0.333, p=0.36) even after adjusting for age and gender.The lifestyle intervention reduced circulating chemerin levels independent of visceral fat mass and adiponectin. Chemerin levels are associated with insulin resistance at the baseline and after the lifestyle intervention.

This study was to provide the base data on the status of vegetations and soils in green roofs by analyzing the soil and vegetation characteristics of 4 green roofs in Dongguk University in September 2012. Sanglokwon(SW), Dongguk Hall(DH), University Library(UL), and Information and Culture Hall P(IC) were established in 2005, 2008, 2009, and 2010, respectively. The areas of green roofs were , , , and in SW, DH, UL, and IC respectively. The investigated floras of vascular plants were 26 families, 55 genera, 65 species in Sanglokwon(SW), 53 families, 99 genera, 112 species in Dongguk Hall(DH), 43 families, 77 genera, 84 species in University Library(UL), and 41 families, 71 genera, 75 species in Information and Culture Hall P(IC), respectively. A positive correlation is shown between the number of plant species and planting area. Total nitrogen, organic matter, and potassium in soil have positive correlation with the number of plant species. The number of plant species was proportional to area and increased more than twice after planting. About a quarter of the invaded plants (including native and naturalized species) were naturalized plants. The total soil depths including vegetation soil and drainage soil at SW, DH, UL, and IC were 20cm, 10cm, 10cm, and 8cm, respectively. The depths of vegetation soil at SW, DH, UL, and IC were , , , and at SW, DH, UL, and IC, respectively.

Background: In this study, the anti-melanogenesis efficacy of clinically used herbal prescription LASAP-C, which consists of four herbal medicines-Rehmanniae Radix Crudus, Lycii Fructus, Scutellariae Radix, and Angelicae Dahuricae Radix, was investigated. Methods: The chemical profile of LASAP-C was established by conducting ultra-performance liquid chromatography-electrospray ionization-mass spectrometry. Anti-melanogenic efficacy was evaluated by tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 expression in B16F10 melanoma cells. In vivo evaluation was performed by using zebrafish model. Results: Molecular evidences suggested that melanin synthesis was inhibited via the down-regulation of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 expression in B16F10 melanoma cells treated with LASAP-C. The anti-melanogenesis efficacy was also confirmed in vivo by using the zebrafish model. Conclusion: The results of this study provide strong evidences that LASAP-C can be used as an active component in cosmeceutical products for reducing excess pigmentation in the human skin.