Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant ␣ 5 and ␤ 1 proteins and ␣ 5 ␤ 1 integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of ␣ 5 ␤ 1 to fibronectin. Co-immunoprecipitation of GT1b and ␣ 5 ␤ 1 from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified ␣ 5 ␤ 1 from SCC12 cells and insect recombinant ␣ 5 ␤ 1 , particularly the ␣ 5 subunit, further suggest interaction between ganglioside and ␣ 5 ␤ 1 . The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of ␣ 5 ␤ 1 from SCC12 and insect cells or poorly glycosylated recombinant ␣ 5 ␤ 1 from Escherichia coli cells. The GT1b-␣ 5 ␤ 1 interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in ␣ 5 ␤ 1 . The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate ␣ 5 ␤ 1 -mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the ␣ 5 subunit of ␣ 5 ␤ 1 integrin. Human keratinocyte motility on a fibronectin (FN)1 matrix is critical for the re-epithelialization of healing wounds, for the spread of cutaneous malignancy, and in cutaneous embryogenesis. Although the molecular events that influence this migration are poorly understood, the interaction between keratinocyte ␣ 5 ␤ 1 integrin and the arginine-glycine-aspartic acid (RGD) site of FN is known to be key (1). Increased expression of ␣ 5 ␤ 1 has been shown in keratinocytes at the migrating edge of wounds in vivo, in lesional skin of patients with psoriasis, and in cultured keratinocytes (2-4). Both receptor clustering on keratinocytes and ligand occupancy of ␣ 5 ␤ 1 are required to activate intracellular signal transduction components (5), including focal adhesion kinase, phosphatidylinositol 3-kinase, protein kinase C, and integrin-linked kinase, leading to cell adhesion to .Gangliosides are glycosphingolipids characterized by the presence of one or more sialic acid moieties in the oligosaccharide chain (9). The role of gangliosides, which are localized to the outer leaflet of the plasma membrane of eukaryotic cells, is largely unknown, but studies with cultured keratinocytes and keratinocyte-derived cells suggest that gangliosides are involved in regulating cellular proliferation, differentiation, and adhesion (10 -13). The discovery that gangliosides inhibit cell attachment and spreading on a FN matrix (14) led investigators to consider gangliosides to be the cell receptors for FN before integrin ␣ 5 ␤ 1 was identified (15,16). Although these early studies showed that the terminal sialic acid residues of gangliosides were critica...

Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) anti-body against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required. Interestingly, the functional scFv was expressed as a soluble and active form in the cytoplasm of an engineered E. coli SHuffle ® strain. Purified anti-GAA scFv, which yielded 2.56 mg from 1 L of culture medium, was obtained from simple and inexpensive procedures for expression and purification. The anti-GAA scFv-based indirect competitive enzyme-linked immunosorbent assay (icELISA) exhibited high sensitivity (linearity: 0.078-1.25 µg/mL) with precision (CV: ≤6.20%) and reliability (recovery: 100.1-101.8%) for GAA determination. In summary, the approach described here is an inexpensive, simple, and efficient expression system that extends the application of anti-GAA scFv-based immunoassays. In addition, when in vitro refolding steps can be skipped, the cost and complexity of scFv antibody production can be minimized. Key words Ganoderma lingzhi; ganoderic acid A; single-chain variable fragment antibody; Escherichia coli; enzyme-linked immunosorbent assay Antibodies are the most frequently used biological agents for routine diagnostics, therapeutics, and studies in various fields. Their applications to chemical analysis provide various advantages such as binding specificity and methodological simplicity that extend beyond conventional chromatographic techniques. The subjects of target molecules for immunoas-says have been expanded from large molecules (i.e., protein, peptide, and DNA) to small molecules (herbicides, natural products, and phytohormones). As such, many monoclonal antibodies (mAbs) against small molecules (haptens), such as amikacin, 1) carbamazepine, 2) aflatoxins, 3) and daidzin, 4,5) have been produced to develop mAb-based immunoassays for qualitative and quantitative analyses, which were shown to be simple and convenient analytical methods. Because the cost of recombinant antibodies (rAbs) produced using Escherichia coli is much lower than that of antibodies produced using hy-bridoma cells or other animal cell lines, 6) immunoassays using E. coli-derived antibodies represent an economical approach for analytical applications. However, the E. coli-based production of rAbs against haptens is limited by an inability to retain rAb reactivity. Normally, refolding steps are required to recover the binding reactivity of the rAb, and the steps are determined by time-consuming trial-and-error procedures. The single-chain variable fragment (scFv) antibody is a simple and small arrangement of the functional rAb, in which the...

Abstract-The effects of propranolol on lipid metabolism were studied in spon taneously hypertensive rats (SHR).Male SHR and corresponding Wistar Kyoto rats (WKY) were used at 5 weeks of age. The SHR were given 10 mg/kg/day of dl-propranolol•HCI by gavage for 10 weeks. Body weight gain in untreated SHR and propranolol-treated SHR (SHR-P) groups were low, as compared with those of the WKY group.Total cholesterol, phospholipid and total lipid of the serum and liver in the SHR-P group were higher than in the SHR group.In the early weeks of treatment, serum triglyceride and non-esterified fatty acid levels in the SHR-P group were slightly lower than those in the SHR group.Aortic lipid levels in the SHR-P group were lower than those in the SHR group.During the later weeks of treatment, blood glucose level in the SHR-P group was higher than in the SHR group.The serum immunoreactive insulin value in the SHR-P group was slightly lower than in the SHR group.These results may suggest that propranolol inhibits hormone-sensitive lipase activity in the early weeks of treatment and influences cholesterol biosynthesis and/or catabolism.

We previously reported the binding specificities of two anti-ganglioside GD2 murine monoclonal antibodies (MAbs), A1-425 and A1-267, both of which are of IgG3 isotype. A1-425 reacts specifically with ganglioside GD2, whereas A1-267 binds preferentially to GD2 but also reacts with GD3 [Tai, T., Kawashima, I., Tada, N., & Dairiki, K. (1988) J. Biochem. 103, 682-687]. In this paper, they were used for comparative analyses of antibody-mediated cytotoxicity, i.e., antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human melanoma and neuroblastoma cell lines. Melanoma cells were found to contain GD2 and/or GD3, whereas neuroblastoma cells expressed only GD2. Both antibodies induced high levels of ADCC and CDC to GD2/GD3-positive cells with human peripheral large granular lymphocytes (LGL) as effector cells and in the presence of human serum, respectively. A good correlation was obtained between the contents of disialogangliosides and the binding level of the antibodies; both melanoma and neuroblastoma cells with larger amounts of GD2/GD3 showed a higher level of antibody binding than did the cells with a smaller amount of GD2/GD3. Surprisingly, ADCC did not correlate well with the binding level of the antibodies. Thus, A1-425 showed stronger lytic activity than A1-267 in spite of the binding level of A1-425 being similar to or lower than that of A1-267 on the cell surfaces. Antigen-antibody complexes composed of GD2 and A1-425 showed higher binding levels to LGL than complexes of GD2 and A1-267. In contrast, free MAb molecules gave minimum binding to LGL.(ABSTRACT TRUNCATED AT 250 WORDS)

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