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Invasion of host cells is essential for tile pathogenicity of Salmonella. The author's group has recently reported the cloning of the rfaE gone of Salmonella typhimurium, previously implicated in biosynthesis of file lipopolysaccharide (LPS)-inner core [Jin et al. (2001); Kim (2002)]. The product of the rfaE gene is involved in ADP-L-glycero-D-manno-heptose biosynthesis, rfaE mutants synthesize heptose-deficient LPS (Re-LPS) consisting only of lipid A and 3-deoxy-Dmanno-octulosonic acid (KDO). Mutants that make incomplete LPS are rough mutants and "deep-rough" mutants affected in the heptose region of the inner core have reduced growth rate and increased sensitivity to high temperature. Complementation of S. typhimurium rfaE mutant strain SL1102 (rfaE543) with rfaE demonstrated conclusively that this gene restored the smooth phenotype, and the LPS produced by the complemented strain was indistinguishable from that of wild type smooth strains. In vitro infection experiments showed that complementation with rfaE permitted invasion of human Chang epithelial cells, larynx epidermal carcinoma HEp-2 cells and intestinal epithelial Henle-407 cells. These data imply that the structure of the LPS that is synthesized is critical for Salmonella invasiveness. [ABSTRACT FROM AUTHOR]

Bacillus circulans F-2 amylase-pullulanase enzyme (APE) displayed dual activity with respect to glycosidic bond cleavage. The enzyme was active on α-1,6 bonds in pullulan, amylopectin, and glycogen, while it showed α-1,4 activity against malto-oligosaccharides, amylose, amylopectin, and soluble starch, but not pullulan. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as two competing substrates was used to distinguish the dual specificity of the enzyme from the single-substrate specificity known for pullulanases and α-amylases. Enzyme activities were inhibited by some metal ions, and by metal-chelating agents with a different mode. The enzyme-inhibitory results of amylase and pullulanase with Hg2+ and Co2+ ions were different, indicating that the activation mechanisms of both enzyme activities are different. Cyclomaltoheptaose inhibited both a-amylase and pullulanase activities with inhibition constants (Ki) of 0.029 and 0.06 mg/ml, respectively. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide confirmed a carboxy group at the active sites of both enzymes. The N-terminal sequence of the enzyme was: Ala-Asp-Ala-Lys-Lys-Thr-Pro-Gln-Gln-Gln-Phe-Asp-Ala-Leu-Trp-Ala-Ala-Gly-ILe-Val-Thr-Gly-Thr-Pro-Asp-Gly-Phe. The purified enzyme displayed Michaelis constant (Km) values of 0.55 mg/ml for amylose, and 0.71 mg/ml for pullulan. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which the two substrates are hydrolyzed at different active sites. These results suggest that amylopullulanases, which possess both α-1,6 and α-1,4 cleavage activities at the same active site, should be distinguished from APEs, which contain both activities at different active sites on the same polypeptide. Also, it is proposed that the Enzyme Commission use the term ‘amylase-pullulanase enzyme’ to refer to enzymes which act on starch and cleave both α-1,6-bonds in pullulan and α-1,4 bonds in amylose at different active sites. [ABSTRACT FROM AUTHOR]

Previously we showed that the human GM3 synthase gene was expressed during the induction of megakaryocytic differentiation in human leukemia K562 cells by phorbol 12-myristate 13-acetate (PMA). In this study we found that treatment of PMA-induced K562 cells with Gö6976, a specific inhibitor of PKC, and U0126, an inhibitor of the extracellular signal-regulated kinase (ERK) reduced expression of GM3 synthase, whereas wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) did not. Moreover, activation of ERK and cAMP response element binding protein (CREB) was prevented by pretreatment with Gö6976 and U0126. PMA stimulated the promoter activity of the 5'-flanking region from -177 to -83 region of the GM3 synthase gene, and mutation or deletion of a CREB site located around -143 of the promoter reduced PMA-stimulated promoter activity, as did the inhibitors Gö6976 and U0126. Our results demonstrate that induction of GM3 synthase during megakaryocytic differentiation in PMA-stimulated human leukemia K562 cells depends upon the PKC/ERK/CREB pathway. [ABSTRACT FROM AUTHOR]

Sialic acid containing glycosphingolipids (gangliosides) are thought to play important roles in the function of various biological phenomena such as atherosclerosis. We have previously shown that the overexpression of the disialoganglioside (GD3) synthase gene effectively suppresses cell proliferation, cell cycle progression, and MMP-9 expression in vascular smooth muscle cells (VSMC). However, the issue of how the overexpression of GD3 synthase gene results in the inhibition of cellular responses in VSMC remains unclear. The findings herein demonstrate that overexpression of the GD3 synthase gene suppresses VSMC responses through the generation of reactive oxygen species (ROS). Superoxide and hydrogen peroxide were generated at increased levels in GD3 synthase gene transfectants in comparison with empty vector (EV) -transfected VSMC. This phenomenon was blocked by antioxidants such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). Increased ROS generation was associated with a decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Further studies revealed that the blockade of ROS function with antioxidants reversed the effect of GD3 synthase gene overexpression on VSMC proliferation and cell cycle regulation in response to platelet-derived growth factor (PDGF). In addition, we found that treatment with antioxidants reversed the decreased matrix metalloproteinase-9 (MMP-9) expression in response to TNF-α as determined by zymography and immunoblot in GD3 synthase gene transfectants. This recovery effect was characterized by the up-regulation of MMP-9 promoter activity, which was transcriptionally regulated at NF-κB and activation protein-1 (activating protein (AP) -1) sites in the MMP-9 promoter. These findings suggest that ROS may play a role in GD3 synthase gene-mediated VSMC phenotypic changes that may contribute to plaque instability in atherosclerosis. [ABSTRACT FROM AUTHOR]

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism. [ABSTRACT FROM AUTHOR]

The expression of matrix metalloproteinase-9 (MMP-9) has been implicated in progression of atherosclerotic lesions. The role and importance of the signaling pathway in the transcriptional regulation of MMP-9 in human aortic smooth muscle cells (HASMC) was examined. Tumor necrosis factor-α (TNF-α) stimulated the secretion of MMP-9 in HASMC, as shown by zymography and immunoblot analysis. At the transcriptional levels, TNF-α also stimulated the 5'-flanking 710-bp promoter activity of MMP-9. Transcription factors NF-κB binding site (-601) and AP-1 binding site (-82) were identified as the cis-elements for TNF-α activation, as determined by gel shift assay and mutation analysis. Treatment with U0126, an inhibitor of the extracellular signal-regulated kinase (ERK), significantly downregulated TNF-α-induced MMP-9 expression and promoter activity, whereas the inactive analog U0124 had no effect. Furthermore, the transactivation of TNF-α-stimulated NF-κB and AP-1 was inhibited by U0126 treatment. Finally, the transient transfection of HASMC with dominant negative Ras (RasN17) suppressed TNF-α-induced ERK activity, MMP-9 production, and promoter activity. Overexpression of RasN17 also abolished the TNF-α-stimulated NF-κB and AP-1 activity. In conclusion, the findings herein indicate the activation of the Ras/ERK pathway contributes to the induction of MMP-9 expression in HASMC. In addition, the transcription factors NF-κB and AP-1 that are involved in the Ras/ERK-mediated control of MMP-9 regulation on HASMC in response to TNF-α have now been identified. J. Cell. Physiol. 198: 417–427, 2004© 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

In order to prevent hyperacute rejection in pig-to-human xenotransplantation, it would be very useful to be able to down-regulate the Gal α1-3 Galβ 1-4 GlcNAc-R (α-Gal epitope) in mouse and swine tissues. When the β-D-mannoside β-1,4-N-acetyIglucosaminyltransferase III (GnT-III) gene was introduced into mouse aorta endothelial cells (MEC) their susceptibility to complement-mediated cell lysis by normal human serum (NHS) was reduced. Expression of GnT-III also suppressed the antigenicity of MEC to human natural antibodies as shown by binding of Griffonia simplicifolia 1 isolectin (GS1B4 lectin) to the α-Gal epitope. Western blot analysis indicated that the reactivity of the glycoproteins of the transfectants to NHS and GSIB4 lectin was reduced to approximately the same extent. Thus GnT-III, a key enzyme involved in the formation of hranched N-linked sugars, reduces the expression of xenoantigens, suggesting that this approach may he of value in clinical xenotransplantation. [ABSTRACT FROM AUTHOR]

Investigates the role of hepatitis B viral protein HBx in the expression of matrix metalloproteinase-9 (MMP-9) gene. Effect of HBx on mitogen-activated protein kinase; Influence of HBx on the activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signal pathways; Link between HBx and MMP-9 expression.

Background: Enterotoxigenic Escherichia coli (ETEC) cause infectious diarrhea and diarrheal death. However, the genetic properties of pathogenic strains vary spatially and temporally, making prevention and treatment difficult. In this study, the genomic features of the major type of ETEC in Korea from 2003 to 2011 were examined by wholegenome sequencing of strain NCCP15740, and a comparative genomic analysis was performed with O6 reference strains. Results: The assembled genome size of NCCP15740 was 4,795,873 bp with 50.54% G+C content. Using rapid annotation using subsystem technology analysis, we predicted 4492 ORFs and 17 RNA genes. NCCP15740 was investigated for enterotoxin genes, colonization factor (CF) genes, serotype, multilocus sequence typing (MLST) profiles and classical and nonclassical virulence factors. NCCP15740 belonged to the O6:H16 serotype and possessed enterotoxin genes encoding heat-stable toxin (STh) and heat-labile toxin (LT); 87.5% of the O6 serotype strains possessed both toxin types. NCCP15740 carried the colonization factors CS2 and CS3, whereas most O6 strains carried CS2-CS3-CS21 (79.2%). NCCP15740 harbored fewer virulence factors (59.4%) than the average observed in other O6 strains (62.0%). Interestingly, NCCP15740 did not harbor any nonclassical virulence genes. Conclusions: The major type of ETEC in Korea had the same MLST sequence type as that of isolates from the USA obtained in 2011 and 2014, but had different colonization factor types and virulence profiles. These results provide important information for the development of an ETEC vaccine candidate. [ABSTRACT FROM AUTHOR]

Background: The Shiga toxin-producing Escherichia coli (STEC) O91:H21 strains NCCP15736 and NCCP15737 were isolated during a single outbreak in Korea, NCCP15736 from a symptomatic carrier and NCCP15737 from an asymptomatic carrier. To investigate genomic differences between the two strains, we performed whole-genome sequencing of both strains and conducted a comparative genomic analysis. Results: Using the Illumina HiSeq 2000 platform and Rapid Annotation using the Subsystem Technology (RAST) server, whole-genome sequences of NCCP15736 and NCCP15737 were obtained and annotated. Phylogenetic analysis of ten E. coli strains showed that NCCP15736 and NCCP15737 are evolutionarily close. The two strains were found to be most close to E. coli O91:NM str. 2009C-3745. The genomic comparison showed that the fimD gene of NCCP15737 is truncated and that the truncation could underlie the defects in infection and pathogenicity of NCCP15737. The two strains showed the same virulence factor profiles, and we identified 25 virulence factors from NCCP15736 and NCCP15737, respectively. We identified ten and nine phage-associated regions in the NCCP15736 and NCCP15737 genomes, respectively; the two strains share five of these. Conclusions: NCCP15736 and NCCP15737 differ at the genomic level, even though they share features such as virulence-related genes. NCCP15737 has a deletion in fimD, which may underlie its asymptomatic character. We conclude that complete genome sequencing and integration of other types of omics data are needed to fully reveal the mechanism underlying the asymptomatic character of NCCP15737. [ABSTRACT FROM AUTHOR]

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 51-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. [ABSTRACT FROM AUTHOR]

Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: β1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis. [ABSTRACT FROM AUTHOR]

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 51-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 51-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. [ABSTRACT FROM AUTHOR]

Background: The metastasis of hematogenous cancer cells is associated with abnormal glycosylation such as sialyl lewis antigens. Although the hepatitis B virus X protein (HBx) plays important role in liver disease, the precise function of HBx on aberrant glycosylation for metastasis remains unclear. Methods: The human hepatocellular carcinoma tissues, HBx transgenic mice and HBx-transfected cells were used to check the correlation of expressions between HBx and Sialyl lewis antigen for cancer metastasis. To investigate whether expression levels of glycosyltransferases induced in HBx-transfected cells are specifically associated with sialyl lewis A (SLA) synthesis, which enhances metastasis by interaction of liver cancer cells with endothelial cells, ShRNA and siRNAs targeting specific glycosyltransferases were used. Results: HBx expression in liver cancer region of HCC is associated with the specific synthesis of SLA. Furthermore, the SLA was specifically induced both in liver tissues from HBx-transgenic mice and in in vitro HBx-transfected cells. HBx increased transcription levels and activities of α2-3 sialyltransferases (ST3Gal III), α1-3/4 fucosyltransferases III and VII (FUT III and VII) genes, which were specific for SLA synthesis, allowing dramatic cell-cell adhesion for metastatic potential. Interestingly, HBx specifically induced expression of N-acetylglucosamine-β1-3 galactosyltransferase V (β1-3GalT 5) gene associated with the initial synthesis of sialyl lewis A, but not β1-4GalT I. The β1-3GalT 5 shRNA suppressed SLA expression by HBx, blocking the adhesion of HBx-transfected cells to the endothelial cells. Moreover, β1-3GalT 5 silencing suppressed lung metastasis of HBx-transfected cells in in vivo lung metastasis system. Conclusion: HBx targets the specific glycosyltransferases for the SLA synthesis and this process regulates hematogenous cancer cell adhesion to endothelial cells for cancer metastasis. [ABSTRACT FROM AUTHOR]

Background: Bee venom has been used to relieve pain and to treat inflammatory diseases, including rheumatoid arthritis, in humans. To better understand the mechanisms of the anti-inflammatory and anti-atherosclerosis effect of bee venom, gel electrophoresis and mass spectrometry were used to identify proteins whose expression was altered in human Vascular Smooth Muscle Cells (hVSMCs) stimulated by tumor necrosis factor alpha after 12 h in the presence of melittin. Results: To obtain valuable insights into the anti-inflammatory and anti-atherosclerosis mechanisms of melittin, two-dimensional (2-D) gel electrophoresis and MALDI-TOF/TOF were used. The proteome study, we showed 33 significant proteins that were differentially expressed in the cells treated with tumor necrosis factor alpha and melittin. Thirteen proteins were significantly increased in the cells treated with tumor necrosis factor alpha, and those proteins were reduced in the cells treated with melittin. Five of the proteins that showed increased expression in the cells treated with tumor necrosis factor alpha are involved in cell migration, including calreticulin, an essential factor of development that plays a role in transcription regulation. The proteins involved in cell migration were reduced in the melittin treated cells. The observed changes in the expression of GRP75, prohibitin, and a select group of other proteins were validated with reverse transcribed-PCR. It was confirmed that the observed change in the protein levels reflected a change in the genes level. In addition, the phosphorylation of EGFR and ERK was validated by analyzing the protein pathway. Conclusion: Taken together, these data established that the expression of some proteins was significantly changed by melittin treatment in tumor necrosis factor alpha stimulated the cells and provided insights into the mechanism of the melittin function for its potential use as an anti-inflammatory agent. [ABSTRACT FROM AUTHOR]

Triptolide (TPL) has been shown to inhibit cell proliferation and induce apoptosis in various human cancer cells; however, the precise mechanism of apoptosis induced by TPL in human melanoma cells has not yet been elucidated. In this study, we investigated the precise mechanism underlying cytocidal effects of TPL on human melanoma cells. Treatment of human melanoma cells with TPL significantly inhibited cell growth and induced apoptosis, as evidenced by flow cytometry and annexin V-fluorescein isothiocyanate analyses. TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3. Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression. We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death. In conclusion, our data firstly demonstrated that TPL induces apoptosis by both extrinsic and intrinsic apoptosis pathways in human melanoma cells and identified that RIP shuttles between Fas and FAK to mediate apoptosis. [ABSTRACT FROM AUTHOR]

Pluripotent human embryonic stem cells (hESCs) provide appropriate systems for developmental studies and prospective donor cell sources for regenerative medicine. Identification of surface markers specific to hESCs is a prerequisite for studying hESC biology and can be used to generate clinical-level donor cell preparations that are free from tumorigenic undifferentiated hESCs. We previously reported the generation of monoclonal antibodies that specifically recognize hESC surface antigens using a decoy immunization strategy. In this study, we show that monoclonal antibody 57-C11 recognizes a phosphorylated form of adenovirus early region 1B-associated protein 5 (E1B-AP5). E1B-AP5 is a nuclear RNA-binding protein, but we report that 57-C11-reactive E1B-AP5 is expressed on the surface of undifferentiated hESCs. In undifferentiated hESCs, 57-C11-reactive E1B-AP5 is localized to SSEA3-, SSEA4-, TRA-1-60-, TRA-1-81-, OCT4-, SOX2-, and NANOG-positive hESCs. In mixtures of undifferentiated hESCs and hESC-derived neurons, 57-C11 exclusively recognizes undifferentiated hESCs but not hESC-derived neuronal cells. Further, the expression of 57-C11-reactive E1B-AP5 decreases upon differentiation. Our results demonstrate that 57-C11-reactive E1B-AP5 is a novel surface molecule that is involved in the undifferentiated state of hESCs. As far as we know, this is the first report demonstrating that heterogeneous nuclear RNA-binding protein is expressed on the surface of undifferentiated hESCs. [ABSTRACT FROM AUTHOR]

The Euonymus alatus (Thunb.) Sieb. has long been used as a crude drug. In this paper, we investigate the effects of E. alatus on cultured hepatocyte cell system and lipid peroxidation in hydrogen peroxide (H2O2) treatment conditions. The study covers the physiological activity (the antioxidative activity and the nitrite-scavenging effect) of E. alatus. H2O2 that can produce intracellular free radical was used for inducer of the peroxidation of cellular lipids. Treatment of E. alatus attenuated in cell killing enhanced by increasing concentrations of H2O2. The increased malondialdehyde level induced by H2O2 treatment was reduced by pre-treatment of E. alatus. Furthermore, addition of E. alatus in cell culture medium significantly reduced cell killing and content of intracellular antioxidants. Changes in nitrite-scavenging effect of E. alatus at various concentrations (5–25 mg/ml) and various pH levels (pH 1.2, 4.2 and 6.0) were also observed. The present study was also done to investigate the effects of E. alatus on cultured hepatocyte cell system, H2O2-induced cytotoxicity and antioxidative enzyme activities, including catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase in H2O2 treatment conditions. E. alatus treatment had significant protective or elevating activities on these antioxidative enzyme activities compared to a normal group. The results indicate that E. alatus provides a strong antioxidant protection of cells against H2O2-induced oxidative stress. [ABSTRACT FROM AUTHOR]

Triphenylmethane reductase (TMR) catalyzes the NADH-dependent reduction of triphenylmethane dyes. Sequence alignment revealed a region with a conserved G XXG XXG motif near its N-terminus, which corresponds to a conserved structural motif of known dinucleotide-binding proteins. To verify whether some of these glycine residues are important for the enzyme catalysis, these three glycine residues (Gly-7, Gly-10 and Gly-13) were individually replaced by alanine using site-directed mutagenesis. The secondary structures of these mutants, as measured by circular dichroism spectroscopy, did not show remarkable differences as compared with the wild type. The Vmax/ Km values of mutants G7A and G13A for both Basic fuchsin and NADH were increased about three and twofold over that of the wild type, respectively, whereas the Vmax/ Km value of mutant G10A were decreased about sixfold. These results suggest that these three glycine residues are involved in the interaction with both substrate and cofactor for the catalytic activity of TMR. [ABSTRACT FROM AUTHOR]

To investigate the role of matrix metalloproteinase-9 (MMP-9) in the pre-ovulatory follicular fluid and culture media during in vitro fertilisation (IVF) cycle and to develop the zymographic pre-diagnosis marker for successful implantation and pregnancy in human IVF. Controlled clinical study. IVF Laboratory, Women's Hospital Infertility Clinic and Dongguk University, Korea. Women undergoing in vitro fertilisation treatment. Experiments were designed for controlled clinical study with women undergoing IVF treatment. MMP-9 expressions in follicular fluid and culture media samples that had been collected during transvaginal oocyte retrieval were measured using zymography. MMP-9 activities and expressions were strongly correlated to a higher rate of fertilisation and pregnancy. Fertilisation rates and ultrasonic evidence of intrauterine pregnancy by four weeks after embryo transfer. MMP-9 activity was significantly higher in the pregnant group than in the non-pregnant group ( P< 0.01). In contrast, MMP-2 activity was present in the follicular fluid and culture media of all women, and no difference in its expressions was found between the pregnant and non-pregnant groups. No correlation was found between the MMP-9 expression in follicular fluid and culture media and the fertilisation rates. The expression of MMP-9 in the follicular fluid and culture media is a prerequisite for successful pregnancy in IVF cycle. The zymography of MMP-9 activity in follicular fluids of human and culture media was developed as a pre-diagnostic method and zymographic diagnosis marker for successful fertilisation, implantation and pregnancy in human IVF. [ABSTRACT FROM AUTHOR]

Dietary isothiocyanates (ITCs) have shown protective effects against certain chemically induced cancers in animal models. These inhibitory effects are associated with reduced levels of extracellular signal-regulated kinase (ERK) 1/2 activity and the arrest of the G1 cell cycle. Benzyl isothiocyanate (BITC) treatment down-regulates cyclins and CDKs and up-regulates the expression of the CDK inhibitor p21, but up-regulation of p27 or p53 was not detected. Since antiatherogenic effects are not needed for antiproliferation, we determined whether BITC exerted inhibitory effects on matrix metalloproteinase-9 (MMP-9) activity in TNF-a-induced vascular smooth muscle cells (VSMCs). BITC inhibited TNF-a-induced MMP-9 secretion in VSMC in a dose dependent manner. This inhibition was characterized by the down-regulation of MMP-9, which is transcriptionally regulated at the NF-?B site, and the activation protein-1 (AP-1) site in the MMP-9 promoter. These findings indicate that BITC is an effective agent for inhibiting cell proliferation, the G1 to S phase cell cycle progress, and MMP-9 expression through the transcription factors NF-?B and AP-1 in TNF-a-induced VSMC. 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

UDP-N-acetylglucosamine:α-D-mannosideβ-1,4 N-acetylglucosaminyltransferase III (GnT-III) is a key enzyme in N-glycan biosytnesis. Human GnT-III enzyme activity was found to be elevated in the serum of patients with hepatomas and liver cirrhosis and in hepatocellular carcinoma tissues. Therefore, to understand the relationship between the elevation in GnT-III activity and hepatitis B viral (HBV) hepartocarcinogenesis, we investigated GnT-III gene expression in the HBV-infected cells.A cell line, HFH-T1, producing HBV was produced by natural infection of human fetal hepatocytes. A 170-bp band corresponding to the pre-S1 region of HBV was detected in the culture medium by polymerase chain reaction. Virions were also isolated from the culture medium by sucrose density gradient centrifugation. The synthesis of bothα-fetoprotein and albumin as an indicator that these cells were functional hepatocytes and the extent of differentiation was examined. Polymerase chain reaction and Western blot analysis using a monoclonal antibody, GT273, which was prepared using human aglycosyl recombinant GnT-III were used for HBV DNA and GnT-III detection.Two types of HBV-related particles were secreted into the culture medium; one was a Dane particle (40 nm in size) containing HBV DNA and the other was a subviral hepatitis B surface antigen particle (20 nm in size) that did not contain the viral genome. The secretion from the cell line was diminished by the number of passages and, thus, this cell was renamed as HFH-T2. A decreased level of the HBV was secreted from the cells after a rest period. HFH-T2 cells showed a weak staining forα-fetoprotein and a moderate staining for albumin in the cytoplasm around the nucleus. High levels of a 0.7 kb DNA fragment originating from GnT-III DNA were detected in HFH-T2 cells. Western blot analysis using a monoclonal antibody, GT273, whixh was prepared using human aglycosyl recombinant GnT-III showed a single band, corresponding to Mr 63 kDa, whereas aglycosyl GnT-III showed a band at Mr 53 kDa, with a molecular weight difference of about 10 kDa. This indicates that HFH-T2 cells express glycosylated GnT-III. GnT-III activities were 347.2 ± 53.6 pmol/mg of protein/h in HFH-T2, 276 ± 26.3 in Hep3B, 252.5 ± 23.3 in HepG2 and 30.7 ± 3.4 in NIH-3T3. GnT-III activity was higher in HFH-T2 cells than in the hepatoma cell lines, Hep3B and HepG2.A human fetal hepatocyte cell line was transformed by infection with HBV and the cell line expressed high levels of GnT-III as the levels of secretion of HBV decreased. The decrease in HBV secretion from HFH-T2 cells could be due to a high level of expression of GnT-III. Such a cell line could be used to investigate relationships between HBV infection and glycosyltransferase gene expression. Furthermore, this cell line will be useful in future studies on the effect of the expression of GnT-III on other glycosyltransferase. [ABSTRACT FROM AUTHOR]

Our previous studies have dearly shown that the angiogenic enzymes, matrix metalloproteinase (MMP) -2/9, are directly involved in human hepatic tumorigenesis and metastasis and suggest that the MMP-2/9 inhibitors, which have dual inhibitory activities on enzyme activity and transcription, represent the best candidates for achieving tumor regression. Many anti-cancer drugs have strong cellular cytotoxicity and side effects, indicating that strong anti-cancer drugs that have no or minimal cytotoxicity and side effects need to be developed. The specific aim of the present study was to develop powerful anti-cancer drugs with specific tumor regression and anti-metastatic potential having the dual inhibitory activities of specific MMP-2 and -9 enzyme activities and gene transcription at the molecular level. Caffeic acid (CA), a strong and selective MMP-9 activity and transcription inhibitor, was isolated from the plant Euonymus alatus and its derivative, caffeic acid phenethyl ester (CAPE), was synthesized. CA and CAPE selectively inhibited MMP-2 and -9 but not -1, -3, -7, or cathepsin K. Treatment of HepG2 cells with CA (100 µg/mL) and CAPE (5 µg/mL) suppressed phorbol 12-myristate 13-acetate (PMA) -induced MMP-9 expression by inhibiting the function of NF-κB, but not AP-1. We confirmed that CA and CAPE suppressed the growth of HepG2 tumor xenografts in nude mice in vivo. The subcutaneous and oral administrations of CA and CAPE significantly reduced the liver metastasis. These results confirm the therapeutic potential of the compounds and suggest that the antimetastatic and anti-tumor effects of CA and CAPE are mediated through the selective suppression of MMP-9 enzyme activity and transcriptional down-regulation by the dual inhibition of NF-KB as well as MMP-9 catalytic activity. [ABSTRACT FROM AUTHOR]

We previously cloned and characterized the promoter region of the human NeuAcα2,3Galβ1,3GalNAcα2,6- sialyltransferase (hST6GalNAc IV) gene [Kim et al. (2003)]. In the present study, we identified a region of 294 bp upstream of exon 1 of the gene that produced maximal transcriptional activity in human Jurkat T cells. Site-directed mutagenesis and transient transfection assays demonstrated that Sp1 and MZF1 elements in this region were required for the promoter activity. Further analysis by electrophoretic mobility shift assays using specific competitors and antibody revealed that Sp1 and MZF1 nuclear proteins interacted with these elements. These results indicate that Sp1 and MZF1 are involved in the transcriptional regulation of the hST6GalNAc IV gene in Jurkat T cells. [ABSTRACT FROM AUTHOR]

Sialic acid-containing glycosphingolipids (gangliosides) have been implicated in the regulation of various biological phenomena such as atherosclerosis. Recent report suggests that exogenously supplied disialoganglioside (GD3) serves a dual role in vascular smooth muscle cells (VSMC) proliferation and apoptosis. However, the role of the GD3 synthase gene in VSMC responses has not yet been elucidated. To determine whether a ganglioside is able to modulate VSMC growth, the effect of overexpression of the GD3 synthase gene on DNA synthesis was examined. The results show that the overexpression of this gene has a potent inhibitory effect on DNA synthesis and ERK phosphorylation in cultured VSMC in the presence of PDGF. The suppression of the GD3 synthase gene was correlated with the down-regulation of cyclinE/CDK2, the up-regulation of the CDK inhibitor p21 and blocking of the p27 inhibition, whereas up-regulation of p53 as the result of GD3 synthase gene expression was not observed. Consistently, blockade of GD3 function with anti-GD3 antibody reversed VSMC proliferation and cell cycle proteins. The expression of the GD3 synthase gene also led to the inhibition of TNF-α-induced matrix metalloproteinase-9 (MMP-9) expression in VSMC as determined by zymography and immunoblot. Furthermore, GD3 synthase gene expression strongly decreased MMP-9 promoter activity in response to TNF-α. This inhibition was characterized by the downregulation of MMP-9, which was transcriptionally regulated at NF-κB and activation protein-1 (AP-1) sites in the MMP-9 promoter. Finally, the overexpression of MMP-9 in GD3 synthase transfectant cells rescued VSMC proliferation. However, MMP-2 overexpression was not affected by cell proliferation. These findings suggest that the GD3 synthase gene represents a physiological modulator of VSMC responses that may contribute to plaque instability in atherosclerosis. [ABSTRACT FROM AUTHOR]

The X protein of hepatitis B virus (HBx) plays a major role on hepatocellular carcinoma (HCC). Apolipoprotein B (apoB) in the liver is an important glycoprotein for transportation of very low density lipoproteins and low density lipoproteins. Although lipid accumulation in the liver is known as one of the factors for the HCC, the relationship between HBx and apoB during the HCC development is poorly understood. To better understand the biological significance of HBx in HCC, liver Chang cells that specifically express HBx were established and characterized. In this study we demonstrate that overexpression of HBx significantly up-regulates the expression of UDP-N-acetylglucosamine:β-D-mannoside-1,4-N-acetylglucosaminyltransferase-III (GnT-III), an enzyme that functions as a bisecting-N-acetylglucosamine (GIcNAc) transferase in apoB, and increases GnT-III promoter activity in a chloramphenicol acetyl-transferase assay. GnT-III expression levels of HBx-transfected cells appeared to be higher than that of hepatocarcinoma cells as well as GnT-III-transfected cells, indicating that HBx may has a strong GnT-III pro-motor-enhancing activity. Intracellular levels of apoBs, which contained the increased bisecting GlcNAc, were accumulated in HBx-transfected liver cells. These cells as well as GnT-III-transfected liver cells revealed the inhibition of apoB secretion and the increased accumulation of intracellular triglyceride and cholesterol compared with vector-transfected cells. Moreover, overexpression of GnT-III and HBx in liver cells was shown to down-regulate the transcriptional level of microsomal triglyceride transfer protein, which regulates the assembly and secretion of apoB. Therefore, our study strongly suggested that the HBx increase in intracellular accumulation of aberrantly glycosylated apoB resuited in inhibition of secretion of apoB as well as intracellular lipid accumulation by elevating the expression of GnT-III. [ABSTRACT FROM AUTHOR]

Matrix metalloproteases (MMP) and α-fetoproteins (AFP) are involved in hepatitis B virus (HBV)-induced chronic hepatitis. In the present study, we have determined the correlation between the MMP-9/MMP-9 ratio and AFP levels in the serum of patients during chronic viral B hepatitis. Twenty-eight healthy individuals (18 men and 10 women) with a mean age of 36.3 years (range 23–58 years) and 50 patients (42 men, 8 women) with a mean age of 39.7 years (range 22–61 years) participated in the study. Forty-eight participants had HBV and the remaining two were either hepatitis G virus (HGV) or hepatitis C virus (HCV) carriers. Values of patients were compared with those obtained from 12 blood donor controls (5 men, 7 women), mean age 36 years (range 21–46 years). Patient's sera were subjected to examination of hepatitis B surface (HBs) and hepatitis B early (Hbe) antigen, SGOT, SGPT, AFP, MMP-2 and MMP-9. Serum levels of MMP-2 and MMP-9 activities were measured by a zymogram protease assay and densitometric measurement. The ratios of MMP-9 to MMP-2 were calculated by dividing the densitometric results. Compared with the healthy controls, the mean serum concentrations of MMP-2 were slightly increased in the chronic HBV patients. In contrast, compared with the healthy controls, the mean serum concentrations of MMP-9 were significantly increased in the chronic HBV patients. When the ratios of the MMP-9/MMP-2 and amounts of the serum AFP were compared, a specific correlation between these two parameters was observed. Higher amounts of AFP were detected in the patients with a low ratio of MMP-9/MMP-2. Patients with hepatocellular carcinoma (HCC) and cirrhosis showed relatively low MMP-9/MMP-2 ratios in chronic hepatitis B. In addition, AFP levels of HCC and cirrhosis were higher than in chronic HBV patients. These results indicate that the AFP level and ratio of MMP-9 and MMP-2 is highly correlated in chronic HBV-induced hepatitis. Because the serum MMP activities were significantly varied between each stage of AFP production in liver disease, an individual profile of these parameters might serve as an easy accessing serum marker to monitor the progression of liver disease. [ABSTRACT FROM AUTHOR]

Endogenous expression of human membrane type ganglioside sialidase (Neu3) was examined in various cell lines including NB-1, U87MG, SK-MEL-2, SK-N-MC, HepG2, Hep3B, Jurkat, HL-60, K562, ECV304, Hela and MCF-7. Expression was detected in the neuroblastoma cell lines NB-1 and SK-N-MC, and also in erythroleukemia K562 cells, but not in any other cells. We isolated a Neu3 cDNA from K562 cells and expressed a His-tagged derivative in a bacterial expression system. The purified recombinant product of approximately 48 kDa had sialidase activity toward 4-methyl-umbelliferyl-α-D-1-acetylneuraminic acid (4MU-NeuAc). The optimal pH of the purified Neu3 protein for GD3 ganglioside was 4.5. The enzyme also efficiently hydrolyzed GD3, GD1a, GD1b and GM3 whereas sialyllactose, 4MU-NeuAc, GM1 and GM2 were poor substrates, and it had no activity against sialylated glycoproteins such as fetuin, transferrin and orosomucoid. We conclude that the sialidase activity of Neu3 is specific for gangliosides. [ABSTRACT FROM AUTHOR]

The pharmacological effects of Polygonum cuspidatum water extract (PCWE) on lipid biosynthesis were investigated in cultured human hepatocyte HepG2 cells. The addition of PCWE (5 and 20 μg/ml), which had no effect on cell proliferation and cellular protein content, caused a marked decrease in the cellular cholesterol content, particularly, the cholesteryl ester content following 24 h of incubation. The incorporation of 14C-oleate into the cellular cholesteryl ester fraction was also reduced remarkably during incubation for 6 and 24 h. The effect of PCWE on acyl-coenzyme A–cholesterol acyltransferase (ACAT) activity were studied in vitro to explore the mechanism by which PCWE inhibits cholesterol ester formation. The data confirmed that PCWE, in a dose dependent manner, remarkably inhibits ACAT activity. Among the main active chemicals of P. cuspidatum, resveratrol, a kind of flavonoid, decreased ACAT activity in a dose-dependent manner from the level of 10−3 M. Theses results strongly suggest that PCWE reduces the cholesteryl ester formation in human hepatocytes by inhibiting ACAT. [Copyright &y& Elsevier]

β-D-Mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyses the attachment of an N- acetytglucosamine (GlcNAc) residue to mannose in the β(1-4) configuration in N-glycans, and forms a bisecting GlcNAc. We have generated transgenic mice that contain the human GnT-III gene under the control of the mouse albumin enhancer/promoter [Lee et al., (2003)]. Overexpression of this gene in mice reduced the antigenicity of N-glycans to human natural antibodies, especially in the case of the α-Gal epitope, Galα1-3Galβ1-4GlcNAc-R. Study of endothelial cells from the GnT-III transgenic mice revealed a significant reduction in antigenicity, and a dramatic decrease in both complement- and natural killer cell-mediated mouse cell lysis. Changes in the enzymatic activities of other glycosyltransferases, such as α1,3-galactosyltransferase, and α-6-D-mannoside β-1,6 N-acetylglucosaminyltransferase V, did not point to any interaction between GnT-III and these enzymes in the transgenic mice, suggesting that this approach may be useful in clinical xenotransplantation. [ABSTRACT FROM AUTHOR]

Geiji-Bokryung-Hwan (GBH), consisting of herbes of Cinnamomi ramulus (Geiji), Poria cocos (Bokryun), Mountan cortex radicis (Mokdanpi), Paeoniae radix (Jakyak), and Persicae semen (Doin), on antiplatelet activity in human platelet suspensions was studied. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes and GBH (15 and 30 μg/ml) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. GBH did not significantly affect nitrate production in collagen (10 μg/ml)-induced human platelet aggregation. Various concentrations of GBH (0, 5, 10, 15, and 30 μg/ml) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 μg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 min). These results indicated that the antiplatelet activity of GBH may possibly be due to the inhibition of phospholipase C (PLC) activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists. In conclusion, GBH suppressed PLC in a dose-dependent manner, and may have pharmaceutical applications. These data suggest that GBH extracts merit investigation as a potential anti-atherosclerogenic agent in humans. [Copyright &y& Elsevier]

Presents the summary of a research on changes in ganglioside levels during glutamate-induced neuronal cell death. Upregulation of GM3 in glutamate-induced neuronal cell death; Effects of RNA interference-mediated silencing of GM3 synthase on neuronal cell death; Activation of 12-LOX; Relationship between GM3 levels and neuronal cell death.

Angiogenesis is associated with growth, invasion, and metastasis of human solid tumors. Aberrant activation of endothelial cells and induction of microvascular permeability by a vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) signaling pathway is observed in pathological angiogenesis including tumor, wound healing, arthritis, psoriasis, diabetic retinopathy, and others. Here, we show that GM3 regulated the activity of various downstream signaling pathways and biological events through the inhibition of VEGF-stimulated VEGFR-2 activation in vascular endothelial cells in vitro. Furthermore, GM3 strongly blocked VEGF-induced neovascularization in vivo, in models including the chick chorioallantoic membrane and Matrigel plug assay. Interestingly, GM3 suppressed VEGF-induced VEGFR-2 activation by blocking its dimerization and also blocked the binding of VEGF to VEGFR-2 through a GM3-specific interaction with the extracellular domain of VEGFR-2, but not with VEGF. Primary tumor growth in mice was inhibited by subcutaneous injection of GM3. Immunohistochemical analyses showed GM3 inhibition of angiogenesis and tumor cell proliferation. GM3 also resulted in the suppression of VEGF-stimulated microvessel permeability in mouse skin capillaries. These results suggest that GM3 inhibits VEGFR-2-mediated changes in vascular endothelial cell function and angiogenesis, and might be of value in anti-angiogenic therapy. [ABSTRACT FROM AUTHOR]

Ganglioside GM3 inhibits the growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Our previous results have shown that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation. However, the precise molecular mechanism(s) for the transcriptional regulation of a PTEN gene induced by GM3 remains unclear. Here, we show, for the first time, that GM3 induces transcription factor AP-2α-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5′-flanking region of the PTEN gene showed that the region between −1175 and −1077 from the translational initiation site, which contains the AP-2α binding site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis, and chromatin immunoprecipitation assay obviously indicated that the AP-2α is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against AP-2α diminished the enhancement of AP-2α and PTEN expressions in GM3-induced colon cancer cells. The transient expression of AP-2α also results in the induction of PTEN transcription in AP-2α-negative colon cancer cells. Additionally, GM3 induced AP-2α-mediated PTEN expression through the inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2α transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of the PTEN gene. [ABSTRACT FROM AUTHOR]