Young-Kug Choo

On the mechanism of internalization of α‐synuclein into microglia: roles of ganglioside GM1 and lipid raft

α‐Synuclein (α‐syn) has been known to be a key player of the pathogenesis of Parkinson’s disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of α‐syn into cells has also been observed. In this study, we observed that dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric α‐syn into BV‐2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of α‐syn internalization in dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol‐treated BV‐2 cells. Furthermore, inhibition of clathrin‐, caveolae‐, and dynamin‐dependent endocytosis did not prevent the internalization of α‐syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of α‐syn. In addition, we further confirmed these observations by co‐culture system of BV‐2 cells and α‐syn‐over‐expressing SH‐SY5Y cells. These findings suggest that extracellular α‐syn is internalized into microglia via GM1 as well as hitherto‐unknown protein receptors in clathrin‐, caveolae‐, and dynamin‐independent, but lipid raft‐dependent manner. Elucidation of the mechanism involved in internalization of α‐syn should be greatly helpful in the development of new treatments of α‐syn‐related neurodegenerative diseases.

Role of genetic factors and environmental conditions in recombinant protein production for molecular farming.

Plants are generally considered to represent a promising heterologous expression system for the production of valuable recombinant proteins. Minimal upstream plant production cost is a salient feature driving the development of plant expression systems used for the synthesis of recombinant proteins. For such a plant expression system to be fully effective, it is first essential to improve plant productivity by plant biomass after inserting genes of interest into a suitable plant. Plant productivity is related closely to its growth and development, both of which are affected directly by environmental factors. These environmental factors that affect the cultivation conditions mainly include temperature, light, salinity, drought, nutrition, insects and pests. In addition, genetic factors that affect gene expression at the transcriptional, translational, and post-translational levels are considered to be important factors related to gene expression in plants. Thus, these factors influence both the quality and quantity of recombinant protein produced in transgenic plants. Among the genetic factors, the post-translational process is of particular interest as it influences subcellular localization, protein glycosylation, assembly and folding of therapeutic proteins, consequently affecting both protein quantity and biological quality. In this review, we discuss the effects of cultivation condition and genetic factors on recombinant protein production in transgenic plants.

Gangliosides are involved in neural differentiation of human dental pulp-derived stem cells

Human dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the neural differentiation of hDPSCs. When hDPSCs were cultured under neural differentiation conditions, expression of neural cell marker genes such as Nestin, MAP-2, and NeuN was detected. Immunostaining and high-performance thin-layer chromatography analysis showed that an increase in ganglioside biosynthesis was associated with neural differentiation of hDPSCs. Specifically, a significant increase in GD3 and GD1a expression was observed during neural differentiation. To confirm the role of gangliosides in neural differentiation, ganglioside biosynthesis was inhibited in hDPSCs by knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg), which prevented differentiation into neural cells. These results suggest that gangliosides may play a role in the neural differentiation process of hDPSCs.

Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptors (EGFR). These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in osteoblastogenesis is not yet clearly understood, therefore, in this study, the relationship between gangliosides and EGFR activation was investigated during osteoblast differentiation in human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GM3 expression was decreased, whereas ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. In addition, an increase in the activation of alkaline phosphatase (ALP) was observed in response to treatment with EGF (5 ng/ml) and GD1a (1 microM) (p<0.05). The activation of ALP was significantly elevated in response to treatment of ganglioside GD1a with EGF when compared to control cells (p<0.01). However, treatment with GM3 (1muM) resulted in decreased ALP activation (p<0.01), and treatment of hMSCs with a chemical inhibitor of EGFR, AG1478, removed the differential effect of the two gangliosides. Moreover, incubation of the differentiating cells with GD1a enhanced the phosphorylation of EGFR, whereas treatment with GM3 reduced the EGFR phosphorylation. However, AG1478 treatment inhibited the effect of ganglioside GD1a elicitation on EGFR phosphorylation. Taken together, these results indicate that GD1a promotes osteoblast differentiation through the enhancement of EGFR phosphorylation, but that GM3 inhibits osteoblast differentiation through reduced EGFR phosphorylation, suggesting that GM3 and GD1a are essential molecules for regulating osteoblast differentiation in hMSCs.

Developmental expression patterns of alpha1H T-type Ca2+ channels during spermatogenesis and organogenesis in mice.

The objectives of the present study were to investigate the expression patterns of T‐type Ca2+ channel mRNA during spermatogenesis and organogenesis in mice. Reverse transcription–polymerase chain reaction (RT–PCR) was performed to identify the subtypes of calcium channels present in the round spermatids isolated from mouse testes by flow cytometry. Transcripts of L‐type (α1D), non‐L‐type (α1E) and T‐type Ca2+ channels were detected in round spermatids. Analysis of PCR products of T‐type Ca2+ channels indicated that only α1H subunits were detected in round spermatids. The appearance and differential distribution of α1H T‐type Ca2+ channel mRNA during mouse spermatogenesis and postimplantation embryogenesis (embryonic (E) days E9, E12, E15) were investigated by in situ hybridization with digoxigenin‐labeled RNA probes coupled with alkaline phosphatase detection. In testes from adult and immature mice (postnatal 2 and 3 weeks), α1H T‐type Ca2+ channel mRNA was expressed in all developing germ cells and sertoli cells. On E9 and E12, tissues of the central nervous system, such as the telencephalon, expressed α1H T‐type Ca2+ channel mRNA. On E15, signals were detected throughout all organs of the embryo. These findings indicate that the expression of α1H T‐type Ca2+ channels is spatio‐temporally regulated during spermatogenesis and organogenesis.

Inhibition of ganglioside GD1a synthesis suppresses the differentiation of human mesenchymal stem cells into osteoblasts.

In this study, we investigated the regulatory role of ganglioside GD1a in the differentiation of osteoblasts from human mesenchymal stem cells (hMSCs) by using lentivirus‐containing short hairpin (sh)RNA to knockdown ST3 β‐galactoside α‐2, 3‐sialyltransferase 2 (ST3Gal II) mRNA expression. After hMSCs were infected for 72u2003h with the lentivirus constructed with ST3Gal II shRNAs, the puromycin‐resistant cells were selected and subcultured to produce hMSCs with ST3Gal II mRNA knockdown. The hMSCs established from human dental papilla abundantly expressed CD44 and CD105, but not CD45 and CD117. Osteoblasts that differentiated from normal hMSCs showed a significant increase in alkaline phosphatase (ALP) activity and ganglioside GD1a expression level compared with those in hMSCs. Lentiviral infection of hMSCs successfully induced a marked inhibition of ST3Gal II mRNA expression and caused a significant decrease in ALP activity and ganglioside GD1a expression. During osteoblastic differentiation, the increased ALP activity remarkably reduced by suppression of ganglioside GD1a expression by ST3Gal II shRNA. Ganglioside GD1a and ALP were mainly expressed in the cell body of hMSCs and osteoblasts with colocalization. The phosphorylation of extracellular signal‐regulated kinases (ERK) 1/2 mitogen‐activated protein (MAP) kinase and epidermal growth factor receptor (EGFR) was significantly reduced in the osteoblasts that had differentiated from the hMSCs with ST3Gal II mRNA knockdown. These results suggest that ganglioside GD1a plays an important role in the regulation of osteoblastic differentiation of hMSCs through the activation of ERK 1/2 MAP kinase and EGFR.

Effects of endocrine disrupting chemicals on expression of phospholipid hydroperoxide glutathione peroxidase mRNA in rat testes

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), an antioxidative selenoprotein, is modulated by estrogen in the testis and oviduct. To examine whether potential endocrine disrupting chemicals (EDCs) affect the microenvironment of the testes, the expression patterns of PHGPx mRNA and histological changes were analyzed in 5-week-old Sprague-Dawley male rats exposed to several EDCs such as an androgenic compound [testosterone (50, 200, and 1,000 µg/kg)], anti-androgenic compounds [flutamide (1, 5, and 25 mg/kg), ketoconazole (0.2 and 1 mg/kg), and diethylhexyl phthalate (10, 50, and 250 mg/kg)], and estrogenic compounds [nonylphenol (10, 50, 100, and 250 mg/kg), octylphenol (10, 50, and 250 mg/kg), and diethylstilbestrol (10, 20, and 40 µg/kg)] daily for 3 weeks via oral administration. Mild proliferation of germ cells and hyperplasia of interstitial cells were observed in the testes of the flutamide-treated group and deletion of the germinal epithelium and sloughing of germ cells were observed in testes of the diethylstilbestrol-treated group. Treatment with testosterone was shown to slightly decrease PHGPx mRNA levels in testes by the reverse transcriptionpolymerase chain reaction. However, anti-androgenic compounds (flutamide, ketoconazole, and diethylhexyl phthalate) and estrogenic compounds (nonylphenol, octylphenol, and diethylstilbestrol) significantly upregulated PHGPx mRNA in the testes (p < 0.05). These findings indicate that the EDCs might have a detrimental effect on spermatogenesis via abnormal enhancement of PHGPx expression in testes and that PHGPx is useful as a biomarker for toxicity screening of estrogenic or antiandrogenic EDCs in testes.

Isolation and analysis of natural compounds from silkworm pupae and effect of its extracts on alcohol detoxification

Silkworm pupae have much potential and many applications as a natural medicine to promote human health. However, their chemical components have not been fully characterized or understood. HPLC analysis was conducted to determine the content ratio (%) of individual amino acids in total protein of the pupae. It showed that glutamic acid (18.3%), histidine (14.6%) and alanine (10.2%) are the most common amino acids in silkworm pupae. Fatty acid composition of silkworm pupae oil was revealed by high‐pressure liquid chromatography and gas chromatography – mass spectroscopy analyses. They contain a high ratio of essential fatty acids, [α‐linolenic acid (ω‐3 fatty acid]+ linoleic acid) (49.0%), and also contain non‐essential fatty acids, oleic acid (19.9%), palmitoleic acid (2.5%), palmitic acid (19.7%), stearic acid (8.6%), and eicosapentaenoic acid (EPA) (0.3%). In addition, they also contain antioxidants, quercetin diglucoside and nutritionally important riboflavin (vitamin B2). This study suggests that silkworm pupae are a nutritionally valuable food product and are applicable as cosmetic components with essential amino acids, essential fatty acids, antioxidants and vitamins. The animal experiment showed that alcohol dehydrogenase (ADH) activity was significantly higher in the liver of mice orally administered with 0.5u2003mg/mL of silkworm extract and alcohol than with commercial Dawn808™ and alcohol, indicating that silkworm pupae extracts have alcohol detoxification activity.

Expression of recombinant anti‐breast cancer immunotherapeutic monoclonal antibody in baculovirus–insect cell system

The anti‐breast cancer monoclonal antibody (mAb) BR55 was expressed in the baculovirus–insect cell expression system, which is advantageous because of its high production capacity, cell culture flexibility and glycosylation capability. The baculovirus–insect cell expression system was successfully established for production of mAb BR55 and mAb BR55 fused with the KDEL (Lys–Asp–Glu–Leu) endoplasmic reticulum (ER) retention signal (mAb BR55K). The heavy chain (HC) and light chain (LC) genes of mAb BR55 were cloned under the control of the polyhedrin (PPH) and P10 promoters, respectively, in the pFastBacDual vector. The antibody gene‐expression cassettes carrying both the HC and LC genes were transferred into a bacmid in Escherichia coli (DH10Bac). The bacmid carrying the expression cassettes was transfected into Sf9 insect cells to generate baculovirus expressing mAb BR55 and BR55K. Western blot analysis confirmed the expression of mAb BR55 and BR55K in baculovirus‐infected insect cells. Cell direct enzyme linked immunosorbent assay (ELISA) showed that both mAbs from insect cell lysates or cell culture medium bound to MCF‐7 human breast cancer cells. Both mAb BR55 and BR55K were successfully purified using a Protein A affinity column. Collectively, these results suggest that the anti‐breast cancer mAb BR55 can be expressed, properly assembled and purified from the baculovirus expression system, which can serve as an alternative system for antibody production.

Pathophysiological implication of ganglioside GM3 in early mouse embryonic development through apoptosis.

Apoptosis may occur in early embryos where the execution of essential developmental events has failed, and gangliosides, sialic acid-conjugated glycosphingolipids, are proposed to regulate cell differentiation and growth. To evaluate the regulatory roles of ganglioside GM3 in early embryonic development, this study examined its expressional patterns in apoptotic cells during early embryonic development in mice. Pre-implanted embryos were obtained byin vitro fertilization, which were treated at the 4-cell stage with three the apoptosis inducers, actinomycin D, camptothecin and cycloheximide, for 15 h. All three inducers significantly increased the percentage of apoptotic cells, as measured using a TUNEL method, but remarkably reduced the total cell numbers. The numbers of morula and blastocyst stages were significantly decreased by treatment of the embryos with the three apoptosis inducers compared with the control, with a similar result also observed in the number of blastomeres. Staining of early embryos with Hoechst 33342 revealed a significant percentage of apoptotic nuclei. Prominent immunofluorescence microscopy revealed a significant difference in the ganglioside GM3 expression in apoptotic embryos compared with the control, and RT-PCR also demonstrated a dramatic increase in ganglioside GM3 synthase mRNA in the apoptotic embryos. These results suggest that ganglioside GM3 may be pathophysiologically implicated in the regulation of early embryonic development through an apoptotic mechanism.