Heajin Park

Differentiating xanthogranulomatous cholecystitis from wall-thickening type of gallbladder cancer: Added value of diffusion-weighted MRI

AIM To evaluate the benefit of diffusion-weighted imaging (DWI) in differentiating xanthogranulomatous cholecystitis from the wall-thickening type of gallbladder cancer. MATERIALS AND METHODS This retrospective study was approved by the institutional review board. Fourteen patients with xanthogranulomatous cholecystitis and 19 patients with the wall-thickening type of gallbladder cancer were included. Qualitative (visual diffusion restriction compared to liver parenchyma) and quantitative [apparent diffusion coefficient (ADC)] analyses were performed. Conventional MRI findings including dynamic enhancement pattern between the two groups were also analysed. Two observers independently reviewed conventional magnetic resonance imaging (MRI) images and subsequently reviewed combined conventional MRI and DWI images. Pairwise comparison of the receiver operating characteristic (ROC) curves was used to compare diagnostic performances. RESULTS In conventional MRI findings, xanthogranulomatous cholecystitis showed significant continuity of enhancing mucosal line [79% (11/14) versus 26% (5/19), p = 0.003] and intramural T2-high signal intensity [64% (9/14) versus 21% (4/19), p = 0.012] compared to the wall-thickening type of gallbladder cancer. The enhancement pattern of gallbladder cancer compared to liver parenchyma showed earlier onset than that of xanthogranulomatous cholecystitis (p = 0.001). Diffusion restriction was more frequently seen in the wall-thickening type of gallbladder cancer (68%, 13/19) than in xanthogranulomatous cholecystitis (7%, 1/14; p < 0.001). The mean ADC value of xanthogranulomatous cholecystitis was higher than that of the wall-thickening type of gallbladder cancer with statistical significance (1.637 × 10(-3) mm(2)/s versus 1.076 × 10(-3) mm(2)/s, p = 0.005). Diagnostic performance [area under ROC curve (Az)] of both observers improved significantly after additional review of DWI; Az improved from 0.737 to 0.930 (p = 0.027) for observer 1 and from 0.675 to 0.938 (p = 0.008) for observer 2. CONCLUSION Addition of DWI to conventional MRI improves discrimination between xanthogranulomatous cholecystitis and the wall-thickening type of gallbladder cancer.

Effects of Watercress Containing Rutin and Rutin Alone on the Proliferation and Osteogenic Differentiation of Human Osteoblast-like MG-63 Cells

Most known osteoporosis medicines are effective for bone resorption, and so there is an increasing demand for medicines that stimulate bone formation. Watercress (N. officinale R. Br.) is widely used as a salad green and herbal remedy. This study analyzed a watercress extract using ultra-performance liquid chromatography/mass spectrometry, and identified a rutin as one of its major constituents. Osteogenic-related assays were used to compare the effects of watercress containing rutin (WCR) and rutin alone on the proliferation and differentiation of human osteoblast-like MG-63 cells. The reported data are expressed as percentages relative to the control value (medium alone; assigned as 100%). WCR increased cell proliferation to 125.0±4.0% (mean±SD), as assessed using a cell viability assay, and increased the activity of alkaline phosphatase, an early differentiation marker, to 222.3±33.8%. In addition, WCR increased the expression of collagen type I, another early differentiation marker, to 149.2±2.8%, and increased the degree of mineralization, a marker of the late process of differentiation, to 122.9±3.9%. Rutin alone also increased the activity of ALP (to 154.4±12.2%), the expression of collagen type I (to 126.6±6.2%), and the degree of mineralization (to 112.3±5.0%). Daidzein, which is reported to stimulate bone formation, was used as a positive control; the effects of WCR on proliferation and differentiation were significantly greater than those of daidzein. These results indicate that WCR and rutin can both induce bone formation via the differentiation of MG-63 cells. This is the first study demonstrating the effectiveness of either WCR or rutin as an osteoblast stimulant.

Cleavage of the terminal N-acetylglucosamine of egg-white ovalbumin N-glycans significantly reduces IgE production and Th2 cytokine secretion.

Ovalbumin (OA) is one of the most abundant of the glycoprotein allergens, and induces a T-helper type 2 immune response that results in an IgE-mediated hypersensitivity. In this study, the terminal carbohydrates of N-glycans from intact OA were cleaved with the exoglycosidases galactosidase, mannosidase, and N-acetylglucosaminidase to generate degalactosylated-OA, demannosylated-OA, and de-N-acetylglucosaminylated-OA, respectively, in order to evaluate their role in allergenicity. The exoglycosidase digestion procedure did not result in either degradation or contamination of the three deglycosylated sample, and the digestion efficiency was confirmed by comparing the results of glycan analysis of the three exoglycosidase-treated OAs with that of glycans of intact OA. Mice were immunized with either intact or exoglycosidase-treated OAs, and their respective allergic reactions were compared. IgE production in the de-N-acetylglucosaminylated-OA group was reduced to 58.8% of that in the intact OA group. In addition, the production levels of the cytokines interleukin-4 and interleukin-5 were significantly reduced in the de-N-acetylglucosaminylated-OA group to 53.4% and 45.8% of the levels in the intact OA group, respectively. However, there were almost no changes (or only slight reductions) in the degalactosylated-OA and demannosylated-OA groups, respectively. These results indicate that cleavage of the terminal carbohydrate, and particularly N-acetylglucosamine, reduces the allergenicity of OA. This is the first report of the effect of cleavage of the terminal carbohydrate on glycoprotein allergenicity.

Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human β1,4-galactosyltransferase and human CTLA4Ig

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4IgP) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4IgM). However, limitations of hCTLA4IgP include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing (β1-2)-xylose and α1,3-fucose, which cause immunogenic reactions in humans. In the present study, human β1,4-galactose-extended hCTLA4IgP (hCTLA4IgP-Gal) was expressed through the coexpression of human β1,4-galactosyltransferase (hGalT) and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4IgP-Gal indicated that not only were the two glycans containing (β1-4)-galactose newly extended, but also glycans containing both β1,2-xylose and α1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4IgP, respectively. Unlike hCTLA4IgP, hCTLA4IgP-Gal was effective as an acceptor via (β1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4IgP-Gal in Sprague–Dawley rats was 1.9 times longer than that of hCTLA4IgP, and the clearance pattern of hCTLA4IgP-Gal was close to that for hCTLA4IgM. These results indicate that the coexpression with hGalT and hCTLA4IgP is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.

Effects of selective cleavage of high-mannose-type glycans of Maackia amurensis leukoagglutinin on sialic acid-binding activity

BACKGROUND Maackia amurensis leukoagglutinin (MAL) is a glycoprotein and sialic acid-binding lectin that is used widely in the detection and characterization of sialoglycoconjugates and human cancer cells. However, its N-linked glycan structure and role have yet to be determined. METHODS The N-linked glycans were analyzed using high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the secondary structure was investigated using circular dichroism analysis. A hemagglutination assay was performed. Furthermore, surface plasmon resonance analysis, and fluorescence microscopy and fluorescence-activated cell-sorting analysis were conducted to assess the sialoglycoprotein-binding ability and its usefulness in the detection of human breast cancer MCF-7 cells, respectively. RESULTS Analysis of the N-linked glycan structure of MAL confirmed the presence of eight glycans, comprising two α1,3-fucosylated paucimannosidic-type and six high-mannose-type glycans. Glycan analysis of MAL that had been treated with peptide N-glycosidase F (de-M-MAL) revealed that while the two α1,3-fucosylated paucimannosidic glycans remained attached following the treatment, the six high-mannose-type glycans had been completely cleaved from the original MAL. There were almost no secondary structural changes between MAL and de-M-MAL; however, the lectin activities exhibited by MAL, such as hemagglutination and binding to a sialoglycoprotein, were completely absent in de-M-MAL, and the ability to detect human breast cancer MCF-7 cells was 77% lower in de-M-MAL than in MAL. CONCLUSION The high-mannose-type glycans in intact MAL are closely associated with its lectin activities. GENERAL SIGNIFICANCE This is the first report of the N-linked glycan structure of MAL and the effect of high-mannose-type glycans on lectin activities.

Effects of Glucosinolates from Turnip (Brassica rapa L.) Root on Bone Formation by Human Osteoblast-Like MG-63 Cells and in Normal Young Rats

Turnip (Brassica rapa L.) root ethanol extract (TRE) was prepared, and its chemical constituents were characterized by ultra‐performance liquid chromatography and mass spectrometry. Thirteen glucosinolates (GSLs) were identified, comprising eight aliphatic, four indolic, and one aromatic compounds. The effects of these GSLs on bone formation were investigated in vitro by incubating human osteoblast‐like MG‐63 cells with TRE and then analyzing their viability, alkaline phosphatase (ALP) activity, collagen content, and mineralization and in vivo by administering TRE orally to normal young rats (500 mg/kg/day) and assessing subsequent changes in serum osteocalcin and bone microstructure in these animals. No TRE‐related toxicity was found, and the levels of cell viability, ALP activity, collagen synthesis, and mineralization were significantly increased relative to the negative control. In particular, stimulatory effects on the differentiation of MG‐63 cells were strongly enhanced as compared with a positive control (daidzein). Serum osteocalcin was also significantly increased, and some important bone microstructural parameters were improved in TRE‐administered rats compared with their saline‐administered counterparts. GSLs therefore appear to have a stimulatory effect on bone formation in both MG‐63 cells and normal young rats. This is the first report on the usefulness of turnip root and its GSL compounds for bone formation. Copyright

Significant reduction in allergenicity of ovalbumin from chicken egg white following treatment with ascidian viscera N-acetylglucosaminidase.

Ovalbumin (OA) is the most abundant ingredient of chicken egg-white allergenic proteins. In the present study we investigated the possibility of reducing OA allergenicity by treatment with a natural protein exhibiting N-acetylglucosaminidase (NA) activity. Ascidian is cultivated as a food resource in northeast Asia. The ascidian viscera NA (AVNA) with almost no other exoglycosidases or proteolytic enzymes was isolated by applying size-exclusion chromatography to a protein precipitate of ascidian viscera. Intact OA was mixed with AVNA containing 0.2, 1.0, and 5.0 Units of NA. Anion-exchange chromatography was then used to isolate OA from AVNA-treated OA. The electrophoretic patterns and N-glycans of each isolated OA from AVNA-treated OA (iOA) were analyzed, and the terminal N-acetylglucosamines of iOA were selectively cleaved with no other degradation occurring. A competitive indirect enzyme-linked immunosorbent assay using rabbit anti-OA sera was performed to investigate the allergenicity of iOA, which was found to be significantly reduced depending on the increased NA activity compared to that of intact OA. These results indicate that OA allergenicity was reduced using a simple and mild treatment process with AVNA, and suggest that ascidian NA is an efficient natural protein for reducing the allergenicity of OA without requiring the use of harsh physical treatments or chemical conjugation.

Binding Specificity of Philyra pisum Lectin to Pathogen-Associated Molecular Patterns, and Its Secondary Structure

We recently reported a Philyra pisum lectin (PPL) that exerts mitogenic effects on human lymphocytes, and its molecular characterization. The present study provides a more detailed characterization of PPL based on the results from a monosaccharide analysis indicating that PPL is a glycoprotein, and circular dichroism spectra revealing its estimated α-helix, β-sheet, β-turn, and random coil contents to be 14.0%, 39.6%, 15.8%, and 30.6%, respectively. These contents are quite similar to those of deglycosylated PPL, indicating that glycans do not affect its intact structure. The binding properties to different pathogen-associated molecular patterns were investigated with hemagglutination inhibition assays using lipoteichoic acid from Gram-positive bacteria, lipopolysaccharide from Gram-negative bacteria, and both mannan and β-1,3-glucan from fungi. PPL binds to lipoteichoic acids and mannan, but not to lipopolysaccharides or β-1,3-glucan. PPL exerted no significant antiproliferative effects against human breast or bladder cancer cells. These results indicate that PPL is a glycoprotein with a lipoteichoic acid or mannan-binding specificity and which contains low and high proportions of α-helix and β-structures, respectively. These properties are inherent to the innate immune system of P. pisum and indicate that PPL could be involved in signal transmission into Gram-positive bacteria or fungi.

N-glycan Remodeling Using Mannosidase Inhibitors to Increase High-mannose Glycans on Acid α-Glucosidase in Transgenic Rice Cell Cultures

Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.

Four unreported types of glycans containing mannose-6-phosphate are heterogeneously attached at three sites (including newly found Asn 233) to recombinant human acid alpha-glucosidase that is the only approved treatment for Pompe disease

Myozyme is a recombinant human acid alpha-glucosidase (rhGAA) that is currently the only drug approved for treating Pompe disease, and its low efficacy means that a high dose is required. Mannose-6-phosphate (M6P) glycosylation on rhGAA is a key factor influencing lysosomal enzyme targeting and the efficacy of enzyme replacement therapy (ERT); however, its complex structure and relatively small quantity still remain to be characterized. This study investigated M6P glycosylation on rhGAA using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The glycans released from rhGAA were labeled with procainamide to improve mass ionization efficiency and the sensitivity of MS/MS. The relative quantities (%) of 78 glycans were obtained, and 1.0% of them were glycans containing M6P (M6P glycans). These were categorized according to their structure into 4 types: 3 newly found ones, comprising high-mannose-type M6P glycans capped with N-acetylglucosamine (GlcNAc) (2 variants, 17.5%), hybrid-type M6P glycans (2 variants, 11.2%), and hybrid-type M6P glycans capped with GlcNAc (3 variants, 6.9%), as well as high-mannose-type M6P glycans (3 variants, 64.4%). HCD-MS/MS spectra identified six distinctive M6P-derived oxonium ions. The glycopeptides obtained from protease-digested rhGAA were analyzed using nano-LC-ESI-HCD-MS/MS, and the extracted-ion chromatograms of M6P-derived oxonium ions confirmed three M6P glycosylation sites comprising Asn 140, Asn 233 (newly found), and Asn 470 attached heterogeneously to nine M6P glycans (two types), eight M6P glycans (four types), and seven M6P glycans (two types), respectively. This is the first study of rhGAA to differentiate M6P glycans and identify their attachment sites, despite rhGAA already being an approved drug for Pompe disease.