T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells.

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-ß receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Regulatory mechanism for the human glioblastoma cell-specific expression of the human GD1c/GT1a/GQ1b synthase (hST8Sia V) gene.

In this study we observed that human GD1c/GT1a/GQ1b synthase (hST8Sia V) is particularly expressed in human glioblastoma cells. To address the mechanism regulating human glioblastoma-specific gene expression of the hST8Sia V, after the transcription start site (TSS) was identified by the 5'-rapid amplification of cDNA end with total RNA from human glioblastoma U87MG cells, the 5'-flanking region (2.5 kb) of the hST8Sia V gene was isolated and its promoter activity was examined. By luciferase reporter assay, this 5'-flanking region revealed strong promoter activity in only U-87MG cells, but not in other tissue-derived cancer cells. 5'-deletion mutant analysis showed that the region from -1140 to -494 is crucial for transcription of the hST8Sia V gene in U87MG cells. This region contains the activator protein-1 (AP-1) binding site, the main target of the c-Jun N-terminal kinase (JNK) downstream. The AP-1 binding site at -1043/-1037 was proved to be indispensable for the hST8Sia V gene-specific expression in U87MG cells by site-directed mutagenesis. Moreover, the transcriptional activation of hST8Sia V gene in U87MG cells was strongly inhibited by a specific JNK inhibitor, SP600125. These results suggest that the hST8Sia V gene-specific expression in U87MG cells is controlled by JNK/AP-1 signaling pathway.

Context-aware heatstroke relief station placement and route optimization for large outdoor events.

BACKGROUND: Heatstroke is becoming an increasingly serious threat to outdoor activities, especially, at the time of large events organized during summer, including the Olympic Games or various types of happenings in amusement parks like Disneyland or other popular venues. The risk of heatstroke is naturally affected by a high temperature, but it is also dependent on various other contextual factors such as the presence of shaded areas along traveling routes or the distribution of relief stations. The purpose of the study is to develop a method to reduce the heatstroke risk of pedestrians for large outdoor events by optimizing relief station placement, volume scheduling and route. RESULTS: Our experiments conducted on the planned site of the Tokyo Olympics and simulated during the two weeks of the Olympics schedule indicate that planning routes and setting relief stations with our proposed optimization model could effectively reduce heatstroke risk. Besides, the results show that supply volume scheduling optimization can further reduce the risk of heatstroke. The route with the shortest length may not be the route with the least risk, relief station and physical environment need to be considered and the proposed method can balance these factors. CONCLUSIONS: This study proposed a novel emergency service problem that can be applied in large outdoor event scenarios with multiple walking flows. To solve the problem, an effective method is developed and evaluates the heatstroke risk in outdoor space by utilizing context-aware indicators which are determined by large and heterogeneous data including facilities, road networks and street view images. We propose a Mixed Integer Nonlinear Programming model for optimizing routes of pedestrians, determining the location of relief stations and the supply volume in each relief station. The proposed method can help organizers better prepare for the event and pedestrians participate in the event more safely.

Regulation of human ß-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human ß-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189 bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

Transcriptional Activation of Human GD3 Synthase (hST8Sia I) Gene in Curcumin-Induced Autophagy in A549 Human Lung Carcinoma Cells.

Curcumin, a natural polyphenolic compound isolated from the plant Curcuma longa, is known to induce autophagy in various cancer cells, including lung cancer. In the present study, we also confirmed by LC3 immunofluorescence and immunoblotting analyses that curcumin triggers autophagy in the human lung adenocarcinoma A549 cell line. In parallel with autophagy induction, the gene expression of human GD3 synthase (hST8Sia I) responsible for ganglioside GD3 synthesis was markedly elevated in response to curcumin in the A549 cells. To investigate the transcriptional activation of hST8Sia I associated with the autophagy formation in curcumin-treated A549 cells, functional characterization of the 5'-flanking region of the hST8Sia I gene was carried out using the luciferase reporter assay system. Deletion analysis demonstrated that the -1146 to -646 region, which includes the putative c-Ets-1, CREB, AP-1, and NF-κB binding sites, functions as the curcumin-responsive promoter of hST8Sia I in A549 cells. The site-directed mutagenesis and chromatin immunoprecipitation assay demonstrated that the NF-κB binding site at -731 to -722 was indispensable for the curcumin-induced hST8Sia I gene expression in A549 cells. Moreover, the transcriptional activation of hST8Sia I by the curcumin A549 cells was strongly inhibited by compound C, an inhibitor of AMP-activated protein kinase (AMPK). These results suggest that curcumin controls hST8Sia I gene expression via AMPK signal pathway in A549 cells.

Upregulation of Human ST8Sia VI (α2,8-Sialyltransferase) Gene Expression by Physcion in SK-N-BE(2)-C Human Neuroblastoma Cells.

In this research, we firstly demonstrated that physcion, an anthraquinone derivative, specifically increased the expression of the human α2,8-sialyltransferase (hST8Sia VI) gene in SK-N-BE(2)-C human neuroblastoma cells. To establish the mechanism responsible for the up-regulation of hST8Sia VI gene expression in physcion-treated SK-N-BE(2)-C cells, the putative promoter region of the hST8Sia VI gene was functionally characterized. Promoter analysis with serially truncated fragments of the 5'-flanking region showed that the region between -320 and -240 is crucial for physcion-induced transcription of hST8Sia VI in SK-N-BE(2)-C cells. Putative binding sites for transcription factors Pax-5 and NF-Y are located at this region. The Pax-5 binding site at -262 to -256 was essential for the expression of the hST8Sia VI gene by physcion in SK-N-BE(2)-C cells. Moreover, the transcription of hST8Sia VI induced by physcion in SK-N-BE(2)-C cells was inhibited by extracellular signal-regulated protein kinase (ERK) inhibitor U0126 and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, but not c-Jun N-terminal kinase (JNK) inhibitor SP600125. These results suggest that physcion upregulates hST8Sia VI gene expression via ERK and p38 MAPK pathways in SK-N-BE(2)-C cells.

Serum Deprivation-Induced Human GM3 Synthase (hST3Gal V) Gene Expression Is Mediated by Runx2 in Human Osteoblastic MG-63 Cells.

Serum deprivation (SD) is well known to induce G0/G1 cell cycle arrest and apoptosis in various cells. In the present study, we firstly found that SD could induce G1 arrest and the differentiation of human osteoblastic MG-63 cells, as evidenced by the increase of osteoblastic differentiation markers, such as bone morphogenetic protein-2 (BMP-2), osteocalcin and runt-related transcription factor 2 (Runx2). In parallel, gene expression of human GM3 synthase (hST3Gal V) catalyzing ganglioside GM3 biosynthesis was upregulated by SD in MG-63 cells. The 5'-flanking region of the hST3Gal V gene was functionally characterized to elucidate transcriptional regulation of hST3Gal V in SD-induced MG-63 cells. Promoter analysis using 5'-deletion constructs of the hST3Gal V gene demonstrated that the -432 to -177 region functions as the SD-inducible promoter. Site-directed mutagenesis revealed that the Runx2 binding sites located side-by-side at positions -232 and -222 are essential for the SD-induced expression of hST3Gal V in MG-63 cells. In addition, the chromatin immunoprecipitation assay also showed that Runx2 specifically binds to the hST3Gal V promoter region containing Runx2 binding sites. These results suggest that SD triggers upregulation of hST3Gal V gene expression through Runx2 activation by BMP signaling in MG-63 cells.

Ganglioside GM3 participates in the TGF-ß1-induced epithelial-mesenchymal transition of human lens epithelial cells.

TGF-ß (transforming growth factor-ß)-induced EMT (epithelial-mesenchymal transition) induces the proliferation and migration of the HLE (human lens epithelial) cells. Ganglioside GM3, simple sialic-acid-containing glycosphingolipids on mammalian cell membranes, regulates various pathological phenomena such as insulin resistance and tumour progression. However, the relationship between ganglioside GM3 and TGF-ß-induced EMT in the HLE B-3 cells is poorly understood. In the present study we demonstrated that ganglioside GM3 was involved in TGF-ß1-induced EMT in HLE B-3 cells. Our results indicated that the expression of ganglioside GM3 and GM3 synthase mRNA were significantly increased in TGF-ß1-induced HLE B-3 cells. Reporter gene analysis also demonstrated that transcriptional activation of the GM3 synthase gene was regulated by Sp1 (specificity protein 1) in HLE B-3 cells upon TGF-ß1 stimulation. Interestingly, the inhibition of ganglioside GM3 expression by d-PDMP [d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol] and GM3 synthase shRNA (short hairpin RNA) resulted significantly in the suppression of cell migration and EMT-related signalling in HLE B-3 cells stimulated by TGF-ß. Furthermore, exogenous treatment of ganglioside GM3 rescued the expression of EMT molecules and cell migration suppressed by the depletion of ganglioside GM3 in TGF-ß1-induced HLE B-3 cells. We also found that ganglioside GM3 interacted with TGFßRs (TGF-ß receptors) in TGF-ß1-induced HLE B-3 cells. Taken together, these results suggest that ganglioside GM3 induced by TGF-ß1 regulates EMT by potential interaction with TGFßRs.

Cordycepin-mediated transcriptional regulation of human GD3 synthase (hST8Sia I) in human neuroblastoma SK-N-BE(2)-C cells.

In the present study, we firstly found that cordycepin elevated the gene expression of the human GD3 synthase (hST8Sia I) in human neuroblastoma SK-N-BE(2)-C cells. To elucidate the mechanism underlying the upregulation of hST8Sia I gene expression in cordycepin-treated SK-N-BE(2)-C cells, functional characterization of the promoter region of the hST8Sia I gene was performed. Analysis of promoter activity using varying lengths of 5'-flanking region showed a dramatic increase by cordycepin in the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1, and NF-κB. Site-directed mutagenesis for these binding sites and chromatin immunoprecipitation assay revealed that the NF-κB binding site at -731 to -722 is essential for the cordycepin-induced expression of the hST8Sia I in SK-N-BE(2)-C cells. Moreover, the hST8Sia I expression induced by cordycepin was significantly repressed by pyrrolidinedithiocarbamate, an inhibitor of NF-κB. These results suggested that cordycepin induces upregulation of hST8Sia I gene expression through NF-κB activation in SK-N-BE(2)-C cells.

Promotion of neurite outgrowth by 3,5,7,3',4'-pentamethoxyflavone is mediated through ERK signaling pathway in Neuro2a cells.

In this study, the effects of 3,5,7,3',4'-pentamethoxyflavone (KP1), a major bioactive ingredient isolated from the Kaempferia parviflora rhizomes, on a neurite outgrowth in Neuro2a cells and its mechanism have been investigated. KP1 increased concentration-dependently the percentage of neurite-bearing cells. KP1 showed a remarkable capability to elicit neurite outgrowth in Neuro2a cells, as evidenced by morphological alterations and immunostaining using anti-class III ß-tubulin and anti-NeuN antibodies. KP1 also displayed a higher neurogenic activity than retinoic acid (RA), a promoter of neurite outgrowth in Neuro2a cells. KP1 treatment caused significant elevation in phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and glycogen synthase kinase-3ß (GSK-3ß). However, KP1-triggered neurite outgrowth was markedly inhibited by treatment with the ERK inhibitor U0126, whereas p38 MAPK inhibitor SB203580 and GSK-3ß inhibitor SB216763 did not influence KP1-induced neurite outgrowth. These results demonstrate that KP1 elicits neurite outgrowth and triggers cell differentiation of Neuro2a cells through ERK signal pathway.

Triptolide induces apoptosis through extrinsic and intrinsic pathways in human osteosarcoma U2OS cells.

Triptolide, a diterpene derived from Tripterygium wilfordii Hook f., a Chinese medicinal herb, has been reported to inhibit cell proliferation and induce apoptosis in various human cancer cells, but its anticancer effects on human osteosarcoma cells have not yet been elucidated. In this study, we investigated whether triptolide induces apoptosis in human osteosarcoma cells and the underlying molecular mechanisms. We firstly demonstrated that triptolide inhibited cell growth and induced apoptosis in U2OS cells. Western blot analysis showed that the levels of procaspase-8, -9, Bcl-2, Bid and mitochondrial cytochrome c were downregulated in triptolide-treated U2OS cells, whereas the levels of Fas, FasL, Bax, cytosolic cytochrome c, cleaved caspase-3 and cleaved PARP were upregulated. These results suggest that triptolide induces apoptosis in U2OS cells by activating both death receptor and mitochondrial apoptotic pathways.

Transcription of human ß-galactoside α2,6-sialyltransferase (hST6Gal I) is downregulated by curcumin through AMPK signaling in human colon carcinoma HCT116 cells.

BACKGROUND: In this study, we observed that in human colon carcinoma HCT116 cells mRNA level of the human ß-galactoside α2,6-sialyltransferase (hST6Gal I) was decreased by curcumin. FACS analysis using the α2,6-sialyl-specific lectin (SNA) also showed a noticeable decrease in binding to SNA by curcumin. OBJECTIVE: To investigate the mechanism for curcumin-triggered downregulation of hST6Gal I transcription. METHODS: The mRNA levels of nine kinds of hST genes were assessed by RT-PCR after curcumin was treated in HCT116 cells. The level of hST6Gal I product on cell surface was examined by flow cytometry analysis. Luciferase reporter plasmids with 5'-deleted constructs and mutants of the hST6Gal I promoter were transiently transfected into HCT116 cells, and the luciferase activity was measured after treatment with curcumin. RESULTS: Curcumin led to significant transcriptional repression of the hST6Gal I promoter. Promoter analysis using deletion mutants proved that the - 303 to - 189 region of the hST6Gal I promoter is required for transcriptional repression in response to curcumin. Among putative binding sites for transcription factors IK2, GATA1, TCF12, TAL1/E2A, SPT, and SL1 in this region, by site-directed mutagenesis analysis the TAL/E2A binding site (nucleotides - 266/- 246) was proved to be crucial for curcumin-triggered downregulation of hST6Gal I transcription in HCT116 cells. The transcription activity of hST6Gal I gene in HCT116 cells was markedly suppressed by compound C, an AMP-activated protein kinase (AMPK) inhibitor. CONCLUSION: These indicate that gene expression of hST6Gal I in HCT116 cells is controlled through AMPK/TAL/E2A signal pathway.

Upregulation of human GD3 synthase (hST8Sia I) gene expression during serum starvation-induced osteoblastic differentiation of MG-63 cells.

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase (hST8Sia I) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway.

JNP3, a new compound, suppresses PMA-induced tumor cell invasion via NF-κB down regulation in MCF-7 breast cancer cells.

The expression of matrix metalloproteinase (MMPs)-9 is critical for cell migration and can lead to invasion and metastasis of cancer cells. In the present study, we examined the inhibitory effects of JNP3, a new compound which was isolated from traditional Chinese medicine, on cell invasion and MMP-9 activation in phorbol myristate acetate (PMA)-induced MCF-7 cells. Treatment with JNP3 significantly and selectively inhibited PMA-induced MMP-9 secretion, mRNA expression and protein levels, and these results led to reduction of cell invasion and migration in PMA-induced MCF-7 cells. The results of MMP-9 promoter assay and EMSA showed that JNP3 specifically inhibited PMA-induced MMP-9 gene expression by blocking NF-κB-dependent transcriptional activity. In addition, PMA-induced phosphorylation of ERK1/2 and JNK were suppressed by JNP3 treatment, whereas the phosphorylation of p38 MAPK was not affected by JNP3. These results suggest that JNP3 can be potential anti-cancer agents through specific inhibition of NF-κB-dependent MMP-9 gene expression.

Oleifolioside A, a New Active Compound, Attenuates LPS-Stimulated iNOS and COX-2 Expression through the Downregulation of NF-κB and MAPK Activities in RAW 264.7 Macrophages.

Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

Cordycepin induces apoptosis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells.

In this study, the effect of cordycepin (3'-deoxyadenosine), a major component of Cordyceps militaris, an ingredient of traditional Chinese medicine was investigated for the first time on apoptotsis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells. Cordycepin significantly inhibited the proliferation of human neuroblastoma SK-N-BE(2)-C and human melanoma SK-MEL-2 cells with IC50 values of 120 microM and 80 microM, respectively. Cordycepin treatment at 120 microM and 80 microM, respectively, induced apoptosis in both cells and caused the increase of cell accumulation in a time-dependent manner at the apoptotic sub-G1 phase, as evidenced by the flow cytometry (FCM) and annexin V-fluorescein isothiocyanate (FITC) analyses. Western blot analysis revealed the induction of active caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage by cordycepin treatment. These results suggest that cordycepin is a potential candidate for cancer therapy of neuroblastoma and melanoma.

Curcumin-mediated transcriptional regulation of human N-acetylgalactosamine-α2,6-sialyltransferase which synthesizes sialyl-Tn antigen in HCT116 human colon cancer cells.

Human N-acetylgalactosamine-α2,6-sialyltransferase (hST6GalNAc I) is the major enzyme involved in the biosynthesis of sialyl-Tn antigen (sTn), which is known to be expressed in more than 80% of human carcinomas and correlated with poor prognosis in cancer patients. Athough high expression of hST6GalNAc I is associated with augmented proliferation, migration and invasion in various cancer cells, transcriptional mechanism regulating hST6GalNAc I gene expression remains largely unknown. In this study, we found that hST6GalNAc I gene expression was markedly augmented by curcumin in HCT116 human colon carcinoma cells. To understand the molecular mechanism for the upregulation of hST6GalNAc I gene expression by curcumin in HCT116 cells, we first determined the transcriptional start site of hST6GalNAc I gene by 5'-RACE and cloned the proximal hST6GalNAc I 5'-flanking region spanning about 2 kb by PCR. Functional analysis of the hST6GalNAc I 5' flanking region of hST6GalNAc I by sequential 5'-deletion, transient transfection of reporter gene constructs and luciferase reporter assays showed that -378/-136 region is essential for maximal activation of transcription in response to curcumin in HCT 116 cells. This region includes putative binding sites for transcription factors c-Ets-1, NF-1, GATA-1, ER-α, YY1, and GR-α. ChIP analysis and site-directed mutagenesis demonstrated that estrogen receptor α (ER-α) binding site (nucleotides -248/-238) in this region is crucial for hST6GalNAc I gene transcription in response to curcumin stimulation in HCT116 cells. The transcription activity of hST6GalNAc I gene induced by curcumin in HCT116 cells was strongly inhibited by PKC inhibitor (Gö6983) and ERK inhibitor (U0126). These results suggest that curcumin-induced hST6GalNAc I gene expression in HCT116 cells is modulated through PKC/ERKs signal pathway.

Transcriptional activation of pig Galß1,3GalNAc α2,3-sialyltransferase (pST3Gal I) gene by TGF-ß1 in porcine kidney PK-15 cells.

In this study we investigated for the first time the transcriptional regulation of pig Galß1,3GalNAc α2,3-sialyltransferase (pST3Gal I) in response to TGF-ß1 in porcine kidney PK-15 cells. The pST3Gal I gene was found to span about 90kb and to be composed of 8 exons including 2 exons in the 5'-untranslated region. RT-PCR analysis indicated that the induction of pST3Gal I by TGF-ß1 is regulated at the transcriptional level. Functional analysis of the 5'-flanking region of the pST3Gal I gene revealed the -1257 to -976 region functions as the TGF-ß1-inducible promoter and that the Smad-binding site at -1020 is crucial for TGF-ß1-induced expression of pST3Gal I in PK-15 cells. In addition, the transcriptional activity of pST3Gal I induced by TGF-ß1 in PK-15 cells was strongly inhibited by SIS3, which is a specific Smad-3 inhibitor. In summary, our results identified the core promoter region in the pST3Gal I promoter and demonstrated that Smad-3 binding to the Samd-3 binding site at -1020 is essential for transcriptional activation of pST3Gal I in TGF-ß1-induced PK-15 cells.

Fermented Viola mandshurica inhibits melanogenesis in B16 melanoma cells.

We assessed the effects of chloroform extract of fermented Viola mandshurica (CEFV) on melanogenesis B16 melanoma cells. CEFV treatment significantly decreased melanin content and tyrosinase activity in dose-dependent manners. To elucidate the mechanism of the inhibitory effects of CEFV on melanogenesis, we performed RT-PCR and Western blotting for melanogenesis-related genes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF). CEFV strongly inhibited mRNA as well as the protein expression of tyrosinase and MITF, but had no significant effect on TRP-1 or TRP-2 expressions. It markedly decreased the phosphorylation of cAMP responsive element binding protein (CREB), and induced the duration of extracellular signal-regulated kinase (ERK) activation, leading to reduction of MITF expression and subsequently that of tyrosinase. Therefore, we suggest that CEFV induces downregulation of melanogenesis through decreased CREB phosphorylation and ERK activation.

Isolated compounds from black bean inhibit the classical pathway of the complement.

The present study evaluated the anticomplement effects from isolated compounds of black bean in classical pathway complement system. Using column chromatograph, four compounds kaempferol 3-O-ß-D-glucopyranoside (1), kaempferol 5-O-ß-D-glucopyranoside (2), 3-O-[α-L-rhamnopyranosyl(1-2)-ß-D-glucopyranosyl(1-2)-ß-D-glucuronopyranosyl]olean-12-en-3ß,22ß,24-triol (3) and 3-O-[ß-D-glucopyranosyl(1-2)-ß-D-galactopyranosyl(1-2)-ß-D-glucuronopyranosyl]olean-12-en-3ß,22ß,24-triol (4) were isolated and evaluated for in vitro anticomplement activity. 3 showed inhibitory activity against complement system with 50% inhibitory concentrations (IC(50)) values of 98.2 µg/ml. This is the first report of anticomplement activity of isolated compounds from black bean.