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Malignant melanoma is one of the most progressive skin cancers, with a poor prognosis, various side effects, and high resistance to conventional treatment. Recently, 7,8-dihydroxyflavone (7,8-DHF), a polyphenolic flavonoid derived from certain plants, has been studied for its anti-carcinogenic, anti-inflammatory, antioxidant, and pharmacological effects in several types of cancer. Ganglioside, a modulator of diverse cell signals on the microdomain of the surface of the cell membrane, is closely involved in various cancers. However, the correlation between ganglioside expression and the anti-cancer effects of 7,8-DHF in malignant melanoma remains unclear. In this study, 7,8-DHF showed potential as an anti-cancer agent through specific anti-proliferation, anti-oxidant, anti-migration, pro-apoptotic, and G2/M phase cell cycle arrest effects on SK-MEL-2 and G-361 melanoma cells. In contrast, 7,8-DHF did not induce cytotoxicity in non-tumoral epidermal HaCaT cells. Additionally, we confirmed for the first time that 7,8-DHF significantly reduces the expression levels of ganglioside GD3, which is closely involved in carcinogenesis, in both melanoma cancer cell lines using high-performance thin-layer chromatography analysis. Taken together, our findings suggest that 7,8-DHF might be a potent anticancer drug candidate for the treatment of malignant melanoma.

Ganglioside GT1b is well-known for its role in cytokine production and in activating epidermal growth factor receptor (EGFR)-mediated signaling pathways in cancer cells. However, there are no reports that clearly elucidate the role of GT1b in EGFR-mediated signaling pathways in porcine oocytes during the process of in vitro maturation (IVM). In this study, we investigated the role of GT1b in EGFR-mediated activation of the ERK1/2 pathway in porcine cumulus-oocyte complexes (COCs) at 44 h of IVM. Our data show that expression of the ST3GAL2 protein significantly increased in porcine COCs at 44 h irrespective of treatment with EGF. Meiotic maturation and mRNA levels of factors (HAS2, TNFAIP6, and PTX3) related to cumulus cell expansion significantly increased in COCs treated with 2 μM GT1b during IVM in the absence of EGF. They also increased in COCs treated with EGF/GT1b as compared to that in the other groups. Interestingly, protein levels of EGFR, phospho-EGFR, ERK1/2, and phospho-ERK1/2 dramatically increased in COCs treated with EGF/GT1b. Moreover, the rate of fertilization and the developmental competence of blastocyst were significantly higher in EGF/GT1b-treated COCs. Taken together, these results suggest that exogenous GT1b improves meiotic maturation and cumulus cell expansion in porcine COCs via activation of EGFR-mediated ERK1/2 signaling.

Rice cultivation needs extensive amounts of water. Moreover, increased frequency of droughts and water scarcity has become a global concern for rice cultivation. Hence, optimization of water use is crucial for sustainable agriculture. Here, we characterized

This study aimed to investigate the effects of the human macrophage (MP) secretome in cellular xenograft rejection. The role of human nucleoside diphosphate kinase A (hNME1), from the secretome of MPs involved in the neuronal differentiation of miniature pig adipose tissue-derived mesenchymal stem cells (mp AD-MSCs), was evaluated by proteomic analysis. Herein, we first demonstrate that hNME1 strongly binds to porcine ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 (pST8SIA1), which is a ganglioside GD3 synthase. When hNME1 binds with pST8SIA1, it induces degradation of pST8SIA1 in mp AD-MSCs, thereby inhibiting the expression of ganglioside GD3 followed by decreased neuronal differentiation of mp AD-MSCs. Therefore, we produced nanobodies (NBs) named NB-hNME1 that bind to hNME1 specifically, and the inhibitory effect of NB-hNME1 was evaluated for blocking the binding between hNME1 and pST8SIA1. Consequently, NB-hNME1 effectively blocked the binding of hNME1 to pST8SIA1, thereby recovering the expression of ganglioside GD3 and neuronal differentiation of mp AD-MSCs. Our findings suggest that mp AD-MSCs could be a potential candidate for use as an additive, such as an immunosuppressant, in stem cell transplantation.

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

Background/aim The aim of this study was to reveal the novel roles of calmodulin 2 (CALM2) in hepatocellular carcinoma (HCC) progression. Materials and methods The effects of knockdown of CALM2 expression by siRNA were investigated using various experimental approaches in both cellular and molecular levels. Results Silencing of CALM2 inhibited HCC cell proliferation and colony formation through induction of apoptosis. At the molecular level, CALM2-specific knockdown led to the common dysregulation of 154 genes in HCC cells. Notably, E2F transcription factor 5 (E2F5), which is functionally associated with migration, invasion and proliferation, was generally down-regulated. These functional associations were confirmed in HCC clinical samples. Reflecting the molecular changes, CALM2 knockdown reduced the migration and invasion abilities of HCC cells and abrogated the potency of tumor formation in vivo. Conclusion Targeting CALM2 may be a molecular strategy for both primary HCC treatment and prevention of metastasis or recurrence.

In recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-rasG12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-rasG12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.

Although tacrolimus (FK-506) has been shown to be an effective monotherapy for psoriasis, it does not always work well. Currently, combination therapy is frequently used to manage psoriasis because clinical trials have shown it may provide additive or synergistic benefits and reduce risks of adverse effects. Myeloid-derived suppressor cells (MDSCs) have potent immunomodulatory and anti-inflammatory properties in autoimmune diseases. We previously reported that MDSCs had protective effects in a murine model of imiquimod (IMQ)-induced psoriasis. The present study was undertaken to investigate the systemic immunomodulatory and therapeutic efficacy effects of MDSC plus FK-506 in an IMQ-induced mouse model of psoriasis and to investigate the immunomodulatory mechanisms involved. Systemic MDSC plus FK-506 therapy was found to have a significant anti-psoriatic effect in the murine model, to reduce levels of pro-inflammatory cytokines Th1 cytokines (TNF-α and IFN-γ) and Th17 cytokines (IL-17A and IL-23) in serum and skin. However, treatment with MDSCs or FK-506 alone had little impact. Furthermore, the anti-psoriatic effects of MDSC plus FK-506 were associated with histopathological reductions in inflammatory infiltration, epidermal hyperplasia, and hyperkeratosis. In addition, this combined treatment also attenuated IMQ-induced splenomegaly, and increased the proportion of CD4+CD25+FoxP3+ regulatory T (Treg) cells and decreased the proportions of CD4+IFN-γ+ Th1 cells and CD4+IL-17+ Th17 cells in spleen. Taken together, our results show systemic combination therapy with MDSCs and FK-506 had a better therapeutic effect in our IMQ-induced psoriasis model than either agent alone, and suggest that this combinatorial therapy might be useful for the management of autoimmune skin diseases like psoriasis.

Mesenchymal stem cells, also known as multipotent stromal progenitor cells, can differentiate into cells of mesodermal lineage. Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation and several signaling molecules. These molecules are localized in glycosphingolipid-enriched microdomains on the cell surface and are regulated by glycosphingolipid composition. Transforming growth factor-beta (TGF-&beta, ) signaling plays a critical role in chondrogenic differentiation. However, the role of gangliosides in chondrogenesis is not understood. In this study, the relationship between the ganglioside GM3 and TGF-&beta, activation, during chondrogenic differentiation, was investigated using an aggregate culture of human synovial membrane-derived mesenchymal stem cells. We showed that the gangliosides GM3 and GD3 were expressed after the chondrogenic differentiation of hSMSC aggregates. To test whether GM3 affected the chondrogenic differentiation of hSMSC aggregates, we used GM3 treatment during chondrogenic differentiation. The results showed that the group treated with 5 &mu, M GM3 had higher expression of chondrogenic specific markers, increased toluidine blue, and safranin O staining, and increased accumulation of glycosaminoglycans compared with the untreated group. Furthermore, GM3 treatment enhanced TGF-&beta, signaling via SMAD 2/3 during the chondrogenic differentiation of hSMSC aggregates. Taken together, our results suggested that GM3 may be useful in developing therapeutic agents for cell-based articular cartilage regeneration in articular cartilage disease.

Pig-human xenotransplantation can trigger cell-mediated immune responses. We explored the role of gangliosides in inflammation related to immune rejection in xenotransplantation. Co-culture of xenogeneic cells (pig-MSCs and RAW264.7) was used to emulate xenotransplantation conditions. MTT assay results indicated that cell viability was significantly decreased in pADMSCs co-cultured with RAW264.7 cells. GM1 and GM3 were highly expressed in pADMSCs co-cultured with RAW264.7 cells. pADMSCs co-cultured with RAW264.7 cells strongly expressed pro-inflammatory proteins such as COX-2, iNOS, p50, p65, pIκBα, and TNF-α. GM1-knockdown pADMSCs co-cultured with RAW 264.7 cells did not show significantly altered cell viability, but pro-inflammatory proteins were markedly inhibited. Co-culture of pADMSCs with RAW264.7 cells induced significant phosphorylation (p) of JNK1/2 and pERK1/2. However, pERK1/2 and pJNK1/2 were decreased and MEK1/2 and Raf1 were suppressed in GM1-knockdown pADMSCs co-cultured with RAW264.7 cells. Thus, the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways were significantly upregulated in response to increases of GM1 in co-cultured xenogeneic cells. However, the inflammatory response was suppressed in co-culture of GM1-knockdown pADMSCs with RAW264.7 cells via down-regulation of the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways. Therefore, the ganglioside GM1 appears to play a major role in the inflammatory response in xenotransplantation via the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways.

Glycosphingolipids are important components of the outer layer of the plasma membrane in the majority of eukaryotic cells. Specifically, gangliosides are sialic acid‑containing glycosphingolipids that participate in cell‑cell recognition, adhesion, proliferation, differentiation and signal transduction, and are integral components of cell surface microdomains and lipid rafts. Stem cells are defined functionally as cells that have the capacity to self‑renewal and differentiate to generate various cell types. Due to different synthesis patterns and locations of gangliosides, they have been used as molecular markers of stem cells. The current review describes the presence of gangliosides in various types of mouse stem cells, including pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) and neural stem cells, and the functional roles of gangliosides in various processes, including cell proliferation and neural differentiation. Thus, this review will aid the understanding of gangliosides patterns and functions in mouse stem cells, and outline markers for the identification of stem cells.

Aristolochia manshuriensis Kom (AMK) is an herb used as a traditional medicine; however, it causes side effects such as nephrotoxicity and carcinogenicity. Nevertheless, AMK can be applied in specific ways medicinally, including via ingestion of low doses for short periods of time. Non-alcoholic steatohepatitis (NASH) induced the hepatocyte injury and inflammation. The protective effects of AMK against NASH are unclear; therefore, in this study, the protective effects of AMK ethyl acetate extract were investigated in a high-fat diet (HFD)-induced NASH model. We found decreased hepatic steatosis and inflammation, as well as increased levels of lipoproteins during AMK extract treatment. We also observed decreased hepatic lipid peroxidation and triglycerides, as well as suppressed hepatic expression of lipogenic genes in extract-treated livers. Treatment with extract decreased the activation of c-jun N-terminal kinase 1/2 (JNK1/2) and increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). These results demonstrate that the protective effect of the extract against HFD-induced NASH occurred via reductions in reactive oxygen species production, inflammation suppression, and apoptosis related to the suppression of JNK1/2 activation and increased ERK1/2 phosphorylation. Taken together, these results indicate that that ethyl acetate extract of AMK has potential therapeutic effects in the HFD-induced NASH mouse model.

Plant-derived multiple monoclonal antibody (mAb) CO17-1A × BR55 (mAbP CO17-1A × BR55) produced in transgenic tobacco plants were cross-pollinated with mAb CO17-1A and mAb BR55. Human anti-colorectal cancer multiple mAb CO17-1A × BR55 was cloned using pBI121 vector. Mice were given a single intraperitoneal injection of azoxymethane (AOM) with 10 mg/kg body weight. Starting 1 week after the injection, mice received 2% dextran sulfate sodium (DSS) in the drinking water for 1 week. In addition, the mice were injected intraperitoneal with mAbs dissolved in phosphate buffered saline (100 μg/mouse) twice per week for 4 weeks. Apoptotic cell death, expression of pro-apoptotic proteins, activity of inflammatory cytokines and ERK pathway phosphorylation were assayed by Western blot and TUNEL kit. mAbP CO17-1A × BR55 meaningfully and efficiently suppressed the development of AOM/DSS-induced colorectal inflammation and colorectal tumors, as determined by a reduced activation of inflammatory cytokines, number ...

Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from various tissues in the adult body. MSCs should be characterized by three criteria for regenerative medicine. MSCs must (1) adhere to plastic surfaces, (2) express specific surface antigens, and (3) differentiate into mesodermal lineages, including chondrocytes, osteoblasts, and adipocytes, in vitro. Interestingly, MSCs have immunomodulatory features and secrete trophic factors and immune receptors that regulate the microenvironment in host tissue. These specific and unique therapeutic properties make MSCs ideal as therapeutic agents in vivo. Specifically, pre-clinical and clinical investigators generated inflammatory and fibrotic diseases models, and then transplantation of MSCs into diseases models for therapeutic effects investigation. In this review, we characterize MSCs from various tissues and describe their applications for treating various inflammation and fibrotic diseases.

Overexpression of human epidermal growth factor receptor type 2 (HER2) is considered as a prognostic factor of breast cancer, which is positively associated with recurrence when cancer metastasizes to the lymph nodes. Here, we expressed the single variable domain on a heavy chain (VHH) form of anti-HER2 camelid single domain antibody in tobacco plants and compared its in vitro anticancer activities with the anti-HER2 full size antibody. The gene expression cassette containing anti-HER2 camelid single domain antibody VHH fused to human IgG Fc region with KDEL endoplasmic reticulum (ER) (VHH-FcK) was transferred into the tobacco plant via the Agrobacterium-mediated transformation. The transformants were screened with polymerase chain reaction and Western blot analyses. Enzyme-linked immunosorbent assay (ELISA) confirmed the binding of the purified anti-HER2 VHH-FcK to the HER2-positive breast cancer cell line, SK-BR-3. Migration assay results confirmed anticancer activity of the plant-derived anticancer camelid single chain antibody. Taken together, we confirmed the possibility of using anti-HER2 VHH-FcK as a therapeutic anticancer agent, which can be expressed and assembled and purified from a plant expression system as an alternative antibody production system.

Bisphenol A (BPA) is synthetic organic compound that exhibits estrogen-like properties and it induces mitochondrial superoxide production. Melatonin (Mela) protects against BPA-mediated cell damage and apoptosis. However, the antioxidative effects of Mela against BPA-induced superoxide production in porcine oocytes are still not known. In this study, we investigated the antioxidative effects of Mela against BPA-derived superoxide on oocyte maturation in pigs. To investigate the effects of the superoxide specific scavenger, Mito-TEMPO, on porcine oocyte maturation in response to BPA exposure apoptosis proteins, we treated the oocytes with Mito-TEMPO (0.1 &micro, M) after pre-treating them with BPA (75 &micro, M) for 22 h. As expected, the reduction in meiotic maturation and cumulus cell expansion of cumulus-oocyte-complexes (COCs) in the BPA (75 &micro, M) treated group was recovered (p &lt, 0.01) by treatment with Mito-TEMPO (0.1 &micro, M). An increase in the levels of mitochondrial apoptotic proteins (AIF, cleaved Cas 3 and cleaved Parp1) in response to BPA-induced damage was also reduced by Mito-TEMPO treatment in porcine COCs. Interestingly, we confirmed the positive effects of Mela with respect to superoxide production upon BPA exposure during oocyte maturation and also confirmed the reduction in mitochondrial apoptosis in Mela (0.1 &micro, M)-treated porcine COCs. These results provide evidence for the first time that antioxidative effects of Mela on BPA-derived superoxide improve porcine oocyte maturation.

Cloned pigs produced by somatic cell nuclear transfer (SCNT) are important as a potential alternative source of organs. Although SCNT has created new possibilities for targeted gene modification, the successful cloning of pigs is rare. Here, we successfully conducted serial SCNT for three generations. We determined that the piglet genome was inherited from donor cell nuclei using microsatellite analysis of each generation. The methylation of differentially methylated regions (DMRs) in H19 was gradually reduced over the three generations of serial SCNT. By contrast, methylation of the insulin-like growth factor 2 (Igf2) DMR increased in the F1 generation, compared to the F0, and remained at the higher level in the F2 and F3 generations. The methylation patterns of housekeeping genes such as GAPDH and β-actin were unchanged in the serially cloned pigs. In addition, expression levels of H19 and Igf2 were variable for each generation of serial SCNT piglets, but there was no clear relationship between the meth...

Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants.

Despite the application of numerous supplements to improve in vitro culture (IVC) conditions of mammalian cells, studies regarding the effect of fetal bovine serum (FBS) on mammalian early embryogenesis, particularly in relation to redox homeostasis, are lacking. Herein, we demonstrated that early development of in vitro-produced (IVP) porcine embryos highly depends on the combination of FBS supplementation timing and embryonic reactive oxygen species (ROS) requirements. Interestingly, FBS significantly reduced intracellular ROS levels in parthenogenetically activated (PA) embryos regardless of the developmental stage. However, the beneficial effect of FBS on early embryogenesis was found only during the late phase (IVC 4–6 days) treatment group. In particular, developmental competence parameters, such as blastocyst formation rate, cellular survival, total cell number and trophectoderm proportion, were markedly increased by FBS supplementation during the late IVC phase. In addition, treatment with FBS elevated antioxidant transcript levels during the late IVC phase. In contrast, supplementation with FBS during the entire period (1–6 days) or during the early IVC phase (1–2 days) greatly impaired the developmental parameters. Consistent with the results from PA embryos, the developmental competence of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) embryos were markedly improved by treatment with FBS during the late IVC phase. Moreover, the embryonic stage-specific effects of FBS were reversed by the addition of an oxidant and were mimicked by treatment with an antioxidant. These findings may increase our understanding of redox-dependent early embryogenesis and contribute to the large-scale production of high-quality IVP embryos.

Gangliosides are ubiquitous components of cell membranes that can act as mediators of cell adhesion and signal transduction. However, their interaction with adhesion molecules in vascular grafts following xenotransplantation is not yet clearly understood. In this study, human umbilical vein endothelial cells (HUVECs) were cocultured with porcine aortic endothelial cells (PAECs) in order to investigate the expression patterns of gangliosides, inflammation factors, and the mRNA levels of adhesion molecules by PCR and western blot. The expression of gangliosides GM3 and GM1 was detected in both HUVECs and PAECs, while GD3 was expressed only in PAECs. However, when HUVECs were cocultured with PAECs, GD1a expression was newly detected. In addition, we observed the mRNA expression levels of adhesion molecules including such as E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and platelet endothelial cell adhesion molecule-1 were higher in cocultured HUVECs with PAECs than in eit...

Seafoods and seaweeds represent some of the most important reservoirs of new therapeutic compounds for humans. Seaweed has been shown to have several biological activities, including anticancer activity. This review focuses on colorectal and breast cancers, which are major causes of cancer-related mortality in men and women. It also describes various compounds extracted from a range of seaweeds that have been shown to eradicate or slow the progression of cancer. Fucoidan extracted from the brown algae Fucus spp. has shown activity against both colorectal and breast cancers. Furthermore, we review the mechanisms through which these compounds can induce apoptosis in vitro and in vivo. By considering the ability of compounds present in seaweeds to act against colorectal and breast cancers, this review highlights the potential use of seaweeds as anticancer agents.

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.

Human dental pulp-derived stem cells (hDPSCs) have been considered as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. Gangliosides, which sialic acid-containing glycosphingolipids are abundantly expressed in the plasma membrane and which play an important role in various processes, such as proliferation and differentiation. This study investigated the possible role of gangliosides in dopaminergic neural differentiation. When hDPSCs were cultured under dopaminergic neural differentiation conditions, the expression of dopaminergic neural cell markers genes such as TH, Pitx-3, Nurr-1, and DAT were detected. Immunofluorescence analysis showed that ganglioside biosynthesis was associated with the dopaminergic neural differentiation of hDPSCs. Specifically, GD3 and GT1b were expressed during dopaminergic neural differentiation. These results suggest that gangliosides may play a role in the dopaminergic neural differentiation process of hDPSCs.

Gangliosides are sialic acid-containing glycosphingolipids, which are the most abundant family of glycolipids in eukaryotes. Gangliosides have been suggested to be important lipid molecules required for the control of cellular procedures, such as cell differentiation, proliferation, and signaling. GD1a is expressed in interstitial cells during ovarian maturation in mice and exogenous GD1a is important to oocyte maturation, monospermic fertilization, and embryonic development. In this context, GM1 is known to influence signaling pathways in cells and is important in sperm−oocyte interactions and sperm maturation processes, such as capacitation. GM3 is expressed in the vertebrate oocyte cytoplasm, and exogenously added GM3 induces apoptosis and DNA injury during in vitro oocyte maturation and embryogenesis. As a consequence of this, ganglioside GT1b and GM1 decrease DNA fragmentation and act as H2O2 inhibitors on germ cells and preimplantation embryos. This review describes the functional roles of gangliosides in spermatozoa, oocytes, and early embryonic development.

Gangliosides are complex glycosphingolipids that are the major component of cytoplasmic cell membranes, and play a role in the control of biological processes. Human mesenchymal stem cells (hMSCs) have received considerable attention as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. In this study, we focus on various functional roles of gangliosides in the differentiation of hMSCs into osteoblasts or neuronal cells. A relationship between gangliosides and epidermal growth factor receptor (EGFR) activation during osteoblastic differentiation of hMSCs was observed, and the gangliosides may play a major role in the regulation of the differentiation. The roles of gangliosides in osteoblast differentiation are dependent on the origin of hMSCs. The reduction of ganglioside biosynthesis inhibited the neuronal differentiation of hMSCs during an early stage of the differentiation process, and the ganglioside expression can be used as a marker for the identification of neuronal differentiation from hMSCs. [BMB Reports 2013; 46(11): 527-532]

Gangliosides, which are glycosphingolipids containing sialic acid, play important regulatory roles in cell proliferation and adhesion, survival and immunosuppressive activity. In this study, we investigated whether gangliosides can affect cell viability in the porcine kidney (PK) cell line, PK15, when stimulated with lipopolysaccharide (LPS). As the amount of LPS that PK15 cells were treated with was increased, the cell proliferation decreased, whereas nitric oxide (NO) production increased. High-performance thin-layer chromatography (HPTLC) and immunofluorescence analyses showed that GM3 and GM2 ganglioside expression significantly decreased in LPS-stimulated PK15 compared to unstimulated PK15. UDP-glucose ceramide glucosyltransferase (Ugcg), which catalyzes the initial step in the glycosphingolipid biosynthesis pathway, was knocked-down in PK15 by using short hairpin RNA (shRNA). Western blot and HPTLC analyses showed that the Ugcg protein expression decreased and the ganglioside expression decreased in...

A male silkworm pupae extract with herbal mixtures such as Rubus coreanus Miquel, Chinese matrimony vine Acanthopanax senticosus and tocopherol can be effectively used to recover or boost stamina. In this study, the effect of the male silkworm pupae extract on the reproductive system of Sprague–Dawley male rats was investigated. No clinical symptoms, and no dying or dead animals were found among experimental male silkworm larvae extract, male silkworm pupae extract and control rat groups during the study. No significant differences were found in body weights, feed or water consumption, or macroscopic examination among the three groups. No lesion was found upon necropsy. However, sperm in the group treated with male silkworm pupae extract were significantly more active than sperm in the control group. Sperm counts in both male groups treated with male silkworm larvae extract and male silkworm pupae extract were significantly higher than sperm counts in the control group. Analysis of phospholipid–hydroperoxide glutathione peroxidase (PHGPx) gene expression showed that the male silkworm pupae extract increased expression in testes by 26.7%. This study shows that the male silkworm pupae extract has potential to be used to enhance the function of the human reproductive system.

Agonistic anti-4-1BB antibodies (Abs) play a central role in immunomodulatory conditions that control the pathogenesis of immune-mediated autoimmune and allergic diseases. However, the effects of agonistic anti-4-1BB Abs have not been examined in an experimental mouse model of psoriasis. Therefore, we investigated the protective effects of agonistic anti-4-1BB Abs, using imiquimod (IMQ)-induced psoriasis-like dermatitis in mice, a condition histologically and clinically similar to human psoriasis. We found that administration of agonistic anti-4-1BB Abs (10 mg/kg) significantly alleviated the severity of IMQ-induced psoriasis-like skin inflammation in mice, with reduced histologic symptoms, including inflammatory infiltration, parakeratosis, and hyperkeratosis. Subsequent analyses revealed that the production of Th17 cytokines (IL-17A and IL-23) in the serum and skin of IMQ-induced mice was significantly inhibited by agonistic anti-4-1BB Abs (10 mg/kg), although Th1 cytokines (TNF-α and IFN-γ) were not. Moreover, administration of agonistic anti-4-1BB Abs (10 mg/kg) induced a relative increase of CD4 + FoxP3 + regulatory T (Treg) cells in the spleen and draining lymph node (DLN). Taken together, our data provide evidence that agonistic anti-4-1BB Abs possesses immunosuppressive properties in IMQ-induced psoriasis-like skin inflammation, providing insight into the immunomodulatory effect of agonistic anti-4-1BB Abs for psoriasis immunotherapy.

Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs.

Flavonoids have a range of biological activities, including anti-allergic, anti-inflammatory, anti-microbial, and anti-cancer activities, as shown by in vitro studies. In this study, we investigated whether luteolin can be applied to the suppression of lipopolysaccharide (LPS)-stimulated inflammatory responses in murine macrophages. Luteolin was found to reduce nitric oxide (NO) production from LPS-stimulated Raw 264.7 cells. moreover, expression of inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) and pro-inflammatory cytokine tumor necrotic factor-α (TNF-α) at the mRNA and protein levels were decreased. These inhibitory effects were found to be caused by the blockage of nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB) activation and the phosphorylation of mitogen-activated protein (MAP) kinase famaily, extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAP kinase. Furthermore, pre-treatment with luteolin reduced ganglioside expression levels and inhibited GT1b expression in Raw 264.7 cells. On the basis of these observations, we suggest that luteolin has potential as an anti-inflammatory drug candidate, and ganglioside GT1b may play a role in the inflammatory.

Transgenic plants offer a source for the sustainable, safe, and large-scale production of therapeutic recombinant proteins. In this study, both murine anti-colorectal cancer mAb (mAbMC) and human anti-rabies mAb 57 (mAbHR), expressed in a single plant were investigated for their cancer cell binding activity and rabies virus neutralization activity, respectively. Transgenic plants, expressing murine anti-colorectal cancer mAb CO17-1A (mAbMC) and human anti-rabies mAb 57 (mAbHR), respectively, were crossed to reproduce F1 transgenic plant, expressing both mAbs. PCR and immunoblot analyses demonstrated that heavy (HC) and light chain (LC) genes of mAbMC and mAbHR were present, and that both mAbs were expressed in F1 transgenic lines, respectively. Quantitative immunoblot for purified mAb also showed the presence of both mAbs in F1 transgenic lines. However, Cell ELISA analysis showed that in mAbPC and mAbPR purified from the F1 transgenic lines (mAbPC×R), the binding activity to SW948 human colorectal carcinoma cells was lower than mAbMC, and in vitro mAbMC, mixed with mAbHR (mAbMHC+R). The in vitro rabies virus neutralization assay demonstrated that the mAbPC×R, from the F1 transgenic plants, had lower bioactivity against rabies virus than mAbH57, and mAbMHC+R. N-glycan structure analysis revealed that mAbMC and mAbHR had Golgi type (94 and 14%) and ER type (6 and 86%), respectively, and the purified mAbs from the F1 transgenic plants had Golgi type (75%) and ER type (25%). These results indicate that the F1 transgenic plant produced both mAbPC and mAbPR; however, the HC and LC proteins of each anti-rabies virus and anti-colorectal cancer mAbs were assembled randomly, resulting in chimerism in HC and LC assembly for mAb.

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17-1A recognizes the tumor-associated antigen GA733-2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti-colorectal cancer large single chain (lsc) mAb based on mAb CO17-1A in the baculovirus-insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17-1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C-terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus-infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti-colorectal lsc mAb recognizing human colorectal cancer cells.

Anti-colorectal cancer mAb CO17-1A (IgG2a) recognizes the antigen GA733, which is highly expressed on the surface membrane of human colorectal carcinoma cells. In this study, a transgenic tobacco system for the production of mAb CO17-1A was developed. The mAb construct included a KDEL sequence, an endoplasmic reticulum (ER) retention signal attached to the C-terminus of the heavy chain, to target accumulation of mAb into ER. An immunoblot showed significantly enhanced levels of expression of the plant-derived mAbK (mAbPK) CO17-1A compared to mAbP CO17-1A mAb without the KDEL sequence. An ELISA assay using human colorectal carcinoma cells confirmed that expression of mAbPK was also significantly higher than that of mAbP. Glycosylation analysis revealed that mAbP had plant-specific glycans; whereas, mAbPK primarily had oligomannose glycans. FACS showed that the Fc domains of both mAbPK and mammalian-derived mAb (mAbM) had similar binding activity to the FcγRI receptor (CD64). However, the Fc domains of the mAbP had slightly lower binding activity to the FcγRI receptor than both mAbPK and mAbM. The antibody-dependent cell cytotoxicity of mAbPK, against human colorectal cancer cells, was as efficient as mAbM; whereas mAbP was very low. These results suggest that KDEL localized and accumulated mAbP in the ER and eventually enhanced the expression of mAbP with oligomannose glycan and similar anti-cancer biological activity to the parental mAbM.

An enzyme with polySia degrading activity was purified from a culture filtrate of Pseudomonas fluorescens JK-0412 to apparent homogeneity using DEAE-Sepharose CL-6B column chomatography and fast performance liquid chomatography separation on a Mono-Q column. The molecular mass of the purified enzyme (tentatively named Endo-PS) was approximately 20 kDa on SDS-PAGE and 120 kDa on native-PAGE gels, suggesting that the active form is a hexamer. Although 12 residues of the Endo-PS N-terminal amino acid sequence showed 75% homology to the 21 kDa chitin binding protein (CBP21) of Serratia marcescens 2170, no significant similarity to other known proteins was observed. Apparent K m and V max values of Endo-PS toward the artificial substrate 4-methylumbelliferyl-sialic acid (4-MU-Neu5Ac) were 0.08 mM and 16 nmol/mg/min, respectively. The enzyme was maximally active at 37°C and pH 8.0. Interestingly, the enzyme was shown to hydrolyze the natural substrate, α2,8-linked polySia (colominic acid), in an endo-acting manner. However, no activity toward α2,3- or α2,6-sialyllactose was observed. Under optimal conditions, oligoSia ranging from 2 to 30 residues long were liberated by the cleavage of polySia, as identified by HPAEC-PED. Therefore, the purified enzyme Endo-PS was found to be a polySia-specific sialidase. This is the first report to describe the properties of a bacterial polySia-specific sialidase. Therefore, this enzyme may be a useful tool for both industrial oligoSia production and research on the structure and biological functions of polySia in nature.

Aims To investigate neuroprotective effects of three major anthocyanins (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, and petunidin-3-O-glucoside) isolated from the black soybean (Glycine max L.) cv. Cheongja 3 seed coat against H2O2-induced cell death of human brain neuroblastoma SK-N-SH cells. Main methods Cell viability, reactive oxygen species (ROS) generation, production and expression of heme oxygenase (HO)-1 and inactivation of mitogen-activated protein (MAP) kinase cascades were determined by MTT assay, 2,7-dichlorofluorescein diacetate (DCF-DA) assay, reverse transcriptase polymerase chain reaction (RT-PCR), and western blotting, respectively. Key findings Pretreatment with anthocyanins reduced the cytotoxicity of H2O2 on SK-N-SH cells, dose-dependently reduced the intracellular ROS level and inactivated apoptosis signal-regulating kinase (ASK1, Thr845), p38, and c-Jun N-terminal kinase (JNK) proteins. The HO-1 and Neu1 mRNA levels were increased by H2O2 (25 μM) and further elevated by the pretreatment with anthocyanins. Sialic acids added to the culture plates not only attenuated the cytotoxicity of H2O2 (25 μM) but also reduced intracellular ROS level. These results suggest that Cheongja 3 black soybean seed coat anthocyanins have brain neuroprotective effects against oxidative stress (H2O2) by inhibiting the activation of ASK1–JNK/p38 pathways, scavenging ROS, stimulating the expression of HO-1 and, more interestingly, recruiting cellular free sialic acids through up-regulation of Neu1 sialidase gene expression. Significance This is the first report indicating potent health benefits of black soybean seed coat anthocyanins in neuroprotection by triggering mobilization of cellular free sialic acid and utilizing it as an additional biological antioxidant in brain neural cells.

Gangliosides play important roles in the control of several biological processes, including proliferation and transmembrane signaling. In this study, we demonstrate the effect of ganglioside GM1 on the proliferation of mouse induced pluripotent stem cells (miPSCs). The proliferation rate of miPSCs was lower than in mouse embryonic stem cells (mESCs). Fluorescence activated cell sorting analysis showed that the percentage of cells in the G2/M phase in miPSCs was lower than that in mESCs. GM1 was expressed in mESCs, but not miPSCs. To confirm the role of GM1 in miPSC proliferation, miPSCs were treated with GM1. GM1-treated miPSCs exhibited increased cell proliferation and a larger number of cells in the G2/M phase. Furthermore, phosphorylation of mitogen-activated protein kinases was increased in GM1-treated miPSCs. [BMB Reports 2012; 45(12): 713-718]

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 0.5 mg/mL of silkworm extract and alcohol than with commercial Dawn808™ and alcohol, indicating that silkworm pupae extracts have alcohol detoxification activity.

Gangliosides play an important role in neuronal differentiation processes. The regulation of ganglioside levels is related to the induction of neuronal cell differentiation. In this study, the ST8Sia5 gene was transfected into mESCs and then differentiated into neuronal cells. Interestingly, ST8Sia5 gene transfected mESCs expressed GQ1b by HPTLC and immunofluorescence analysis. To investigate the effects of GQ1b over-expression in neurogenesis, neuronal cells were differentiated from GQ1b expressing mESCs in the presence of retinoic acid. In GQ1b expressing mESCs, increased EBs formation was observed. After 4 days, EBs were co-localized with GQ1b and nestin, and GFAP. Moreover, GQ1b co-localized with MAP-2 expressing cells in GQ1b expressing mESCs in 7-day-old EBs. Furthermore, GQ1b expressing mESCs increased the ERK1/2 MAP kinase pathway. These results suggest that the ST8Sia5 gene increases ganglioside GQ1b and improves neuronal differentiation via the ERK1/2 MAP kinase pathway.

BACKGROUND: Plant expression system for mass production of recombinant proteins has several advantages over other existing expression systems with economical and safety issues. Breast cancer is a cancer originating from breast tissue and comprises almost 25% of all cancers in women world widely. Lewis-Y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cancer cell surface. In this study, the anti-breast cancer mAb BR55, which recognizes the epitope Lewis-Y, was expressed in the plant expression system. METHODS AND RESULTS: We have developed plant system for production of mAb BR55 with or without KDEL (the ER retention signal). This ER retention signal was attached to C-terminus of protein to help retain the recombinant glycoprotein carrying oligomannose glycans and enhance glycoprotein accumulation. PCR analysis was performed and confirmed the presence of recombinant genes. Western blot validated that the recombinant proteins mAb BR55 with or without KDEL were expressed in transgenic plants, moreover, the expression level of the mAb BR55 with KDEL was higher compared to the mAb BR55 without KDEL. CONCLUSION: These results indicate that KDEL fusion is a good way to produce proteins and plant can be an ideal expression system to obtain proteins and enhance accumulation of proteins.

The accelerated cooling rate associated with vitrification reduces injuries attributed to cryopreservation and improves the post-freezing developmental competence of vitrified embryos. In this study, embryos were vitrified and warmed and morphologically evaluated for their development to blastocysts. Survival rates between the fresh (96.7%±3.8%) and vitrified embryos (90.7%±5.1%) did not differ significantly (P >0.05). The mitochondrial membrane potential of fresh control cells measured by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanide iodide staining was similar to that of cryoprotected and vitrified embryos. Mitochondrial staining with rhodamine 123 did not differ among the fresh, cryoprotected, and vitrified embryos. Moreover, the distribution of H2O2, assessed by 2′,7′-dichlorodihydrofluorescein diacetate staining, did not differ among the groups. The results showed that the developmental rate did not differ significantly among the fresh (87.8%±11.3%), cryoprotected (83...