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Sialic acids of glycoconjugates play crucial roles in various biological processes, such as cell-cell communication and cell-substrate interaction. A sisalyltransferase, ST6GalNAc IV (Neu5Ac-alpha2,3-Gal-beta1,3-GalNAc-alpha2,6-sialyltransferase), catalyzes the formation of alpha2-6-linkages onto GalNAc residues of O-glycosidically linked Ser/Thr of proteins. In this study, we cloned the pig ST6GalNAc IV (pST6GalNAc IV) and investigated its functional characterization. pST6GalNAc IV cDNA has been isolated from pig liver tissues and it contains an entire open reading frame (ORF, 906 bp) coding for 302 amino acid residues. Entire ORF of pST6GalNAc IV containing sialylmotif 'L'-(Large), 'S'-(Small) and '-VS' (Very small) has a high degree of sequence similarity with Homo sapiens (90%), Pan troglodytes (91%) and Mus musculus (87%). Expression of pST6GalNAc IV mRNA in various pig tissues was identified by reverse transcription polymerase chain reaction (RT-PCR) analysis. pST6GalNAc IV mRNA was highly expressed in tongue, muscle and heart, whereas it was not expressed in pancreas. For functional characterization of pST6GalNAc IV gene in pig kidney PK15 cells, we have also established pST6GalNAc IV-transfected PK15 cells, which are stably expressing the pST6GalNAc IV gene. The glycosylation pattern of pST6GalNAc IV-transfected PK15 cells was detected by flow cytometry and immunofluorescence analysis with Maackia amurensis agglutinin (MAA), Maackia amurensis hemagglutinin (MAL II), Sambucus nigra agglutinin (SNA) and peanut agglutinin (PNA) lectins. The specific carbohydrate structures of Neu5Acalpha2-3Galbeta1-3(Neu5Acalpha2-6)GalNAc tetrasaccharide or Neu5Acalpha2-6GalNAc disaccharide recognized by MAL-II and SNA were revealed to be newly synthesized by pST6GalNAc IV. From the results, it was suggested that the pig pST6GalNAc IV gene is capable of synthesizing Neu5Acalpha2-3Galbeta1-3(Neu5Acalpha2-6)GalNAc tetrasaccharide structures on O-glycoproteins.

The Gram-negative bacteria cell wall component lipopolysaccharide (LPS) is considered as the most potent activators for macrophages. LPS-activated macrophages produces an array of proinflammatory cytokines such as tumor necrosis facto-a (TNF-a), interleukin (IL)-1b, IL-6 and other inflammatory mediators, such as nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) as well as matrix metalloproteinases (MMPs). [1][2][3][4][5] MMPs are a family of zinc requiring endopeptidases that play an important role in the turnover of extracellular matrix (ECM). Among them, MMP-2 and MMP-9 (92 kDa gelatinase) are capable of degrading basement membrane proteins. Various inflammatory cells such as macrophages, mast cells, lymphocytes, eosinophils and neutrophils produce and/or releases MMP-9 by LPS and other inflammatory mediators, such as TNF-a, IL-8, and granulocyte colony-stimulating factor. [6][7][8] Over expression or inappropriate expression of MMP-9 may contribute to the pathogenesis of conditions such as atherosclerosis, inflammation, tumor invasion and metastasis, rheumatoid arthritis, and periodontal disease. 9)It has been well established that MMP-9 induction is regulated by the transcription factor, nuclear factor-kB (NFkB).10,11) Inactive NF-kB is located in the cytoplasm by the inhibitor protein inhibitor of kB (IkB). When stimulated with any of a variety of stimuli, such as LPS, TNF-a, IL-1b and cellular stress, IkB is degraded and then NF-kB is translocated from the cytosol to the nucleus, and binds to its cognate DNA binding sites.12) LPS-stimulated macrophages activate several intracellular signaling pathways, including the NF-kB pathway and three of the mitogen-activated protein kinases (MAPKs) pathways such as extracellular signalregulated kinase (ERK) 1 and 2, c-Jun N-terminal kinase (JNK) and p38.12) Several lines of evidence have demonstrated that LPS also regulates MMP-9 expression via the MAPK signaling pathway. 13,14) Saururus chinensis (S. chinensis) has been traditionally used since long for the treatment of edema, jaundice, gonorrhea, pneumonia, and several inflammatory diseases. Previous published studies on the chemical nature of S. chinensis have reported a content of more than 20 lignans.15,16) Isolated tetrahydrofurans protect against cell adhesion, 17) inflammation, 18,19) hypercholesterolemia, 20) and sepsis. 21) Sesquinelignans such as saucerenol D inhibited the LPS-induced iNOS expression by blocking NF-kB activation.22) Previously, we had reported on isolation of compounds that protect against sepsis from this plant along with four new lignans including saucerneol G (SG) and their topoisomerase inhibitory activity.23) During the study of SG on anti-inflammatory effect using RAW264.7 cells, SG showed the MMP-9-inhibitory activity. However, the underlying mechanisms in the inhibitory activity of SG on MMP-9 induction in LPSstimulated macrophage cell line RAW264.7 cells have not been elucidated till date.Thus, in this study, we evaluated the effects of SG on LPSstimulated MMP-9 induction and co...