Hisayo Kitamura

The specificity of monoclonal antibody A2B5 to c‐series gangliosides

To examine the specificity of monoclonal antibody A2B5, four A2B5‐reactive gangliosides (designated as G‐1, G‐2, G‐3 and G‐4) were purified from bonito fish brain. Ganglioside‐1, ‐2, and ‐3 migrated above GD1b, below GQ1b, and far below GQ1b on thin‐layer chromatography. Ganglioside‐4 had the slowest chromatographic mobility and migrated below G‐3. The structures of these gangliosides were characterized by overlay analysis with glycolipid‐specific ligands, product analysis after sialidase or mild acid treatment, and electrospray ionization‐mass spectrometry (ESI‐MS). Accordingly, G‐1, G‐2 and G‐3 were identified to be GT3, GQ1c and GP1c, respectively. The ganglioside G‐4 was shown to have the following structure: NeuAc‐NeuAc‐NeuAc‐Galβ1‐3Gal NAcβ1‐4(NeuAc‐NeuAc‐NeuAcα2‐3)Galβ1‐4Glcβ1‐1′Cer. The antibody A2B5 reacted with these c‐series gangliosides, but not with GD3 and other gangliosides and neutral glycosphingolipids. The antigenic epitope for A2B5 was assumed to include the trisialosyl residue connected to the inner galactose of the hemato‐ or ganglio‐type oligosaccharide structure of gangliosides. Phylogenetic analysis of brain gangliosides using the A2B5 preparation demonstrated that c‐series gangliosides are enriched in lower animals, especially bony fish of different species. The monoclonal antibody A2B5 would be a useful tool for examining the distribution and function of c‐series gangliosides.

Occurrence of gangliosides in the common squid and pacific octopus among protostomia

Acidic lipids from tissues of the common squid Todarodes pacificus and the pacific octopus Octopus vulgaris were characterized. Hepatopancreatic tissues of both animals had complex compositions of resorcinol-positive acidic lipids, many of which became reactive with cholera toxin B subunit and anti-G(M1) antibody after in situ treatment with sialidase on TLC. One of the major acidic lipids in squid tissue was isolated and examined for its structure. This acidic lipid was identified to be the ganglioside G(D1a) based upon the susceptibility to sialidases of different substrate specificity, characterization of reaction products, and electrospray ionization-mass spectrometry of the lipid. Hepatopancreatic tissues of squid and octopus also contained acidic lipids that reacted with A2B5, a monoclonal antibody specific to c-series gangliosides. Cerebral ganglia of both animals expressed resorcinol-positive acidic lipids, though their compositional patterns differed from the hepatopancreatic tissues. N-Acetylneuraminic acid was identified as the main species in lipid-bound sialic acid in both tissues. The contents of lipid-bound sialic acid in cerebral ganglia were significantly lower than those of hepatopancreatic tissues in both animals. The present study presents the first evidence for the occurrence of gangliosides in protostomia.

Liver gangliosides of various animals ranging from fish to mammalian species

Liver gangliosides of different animal species were analyzed. Bony fish liver contained a major ganglioside that migrated faster than GM3 on thin-layer chromatography (TLC). This ganglioside was identified to be GM4 (NeuAc) by methods including product analysis after sialidase treatment and negative-ion electrospray ionization (ESI)-mass spectrometry (MS). The presence of GM4 (NeuGc) in fish liver was also demonstrated. The main ganglioside band of bovine liver consisted of two different molecular species, i.e. GD1a (NeuAc/NeuAc) and GD1a (NeuAc/NeuGc). Major gangliosides of liver tissue exhibited a distinct phylogenetic profile; GM4 was expressed mainly in lower animals such as bony fish and frog liver, whereas mammalian liver showed ganglioside patterns with smaller proportions of monosialo ganglioside species. While c-series gangliosides were consistently expressed in lower animals, they were found only in mammalian liver of particular species. No apparent trend was observed between the concentration of liver gangliosides and the phylogenetic stage of animals. The present study demonstrates the species-specific expression of liver gangliosides.

Sairei-to Ameliorates Rat Peritoneal Fibrosis Partly through Suppression of Oxidative Stress

Background: Sairei-to is a herbal prescription originating from traditional Chinese medicine. We conducted an experimental study on rat peritoneal fibrosis to clarify the suppressive mechanisms of sairei-to. Methods: Wistar rats were intraperitoneally injected with chlorhexidine gluconate (CG) every day. Peritoneal specimens were collected after 28 days of CG injection and oral administration of sairei-to. Macrophage infiltration, extracellular matrix accumulation, and angiogenesis were evaluated by immunostaining for ED-1, fibronectin, and CD-31, respectively. To observe oxidative stress in the tissue, 4-hydroxy-2-noneal (HNE) accumulation and plasma levels of superoxide dismutase (SOD) activity were detected. As a candidate of antioxidative components in sairei-to, plasma levels of baicalin were determined by high-performance liquid chromatography. Results: Compared with the disease control group, serum total protein levels were significantly recovered in the sairei-to treatment group. Thickness of the submesothelial compact zone, trichrome-stained area, ED-1-positive cells, fibronectin-staining area, and HNE accumulation were suppressed in the treatment group. Concurrently, decreased plasma levels of SOD activity were recovered by sairei-to treatment. Increased CD-31-positive vessel number and area were also suppressed in the sairei-to group. Baicalin was detected in the plasma samples of the sairei-to group at 0.29 ± 0.11 µg/ml (mean±SEM). Conclusion: These results suggest that sairei-to ameliorates peritoneal fibrosis, partly through suppressing oxidative stress and macrophage infiltration.

Serofendic acid protects from iodinated contrast medium and high glucose probably against superoxide production in LLC-PK1 cells.

BackgroundIt is well known that patients with chronic kidney disease, including diabetic nephropathy, often develop cardiovascular diseases. In case of radiographic procedures, reduced renal function may be deteriorated by the use of iodinated contrast medium (CM). This is known as CM-induced nephropathy. In this study, we have focused on the mechanisms of this type of injury in diabetic nephropathy and the preventive effects of serofendic acid.MethodsWe evaluated the cytotoxicity of CM and high glucose on tubular epithelial cells using an LLC-PK1 cell line, and measured cell viability with an alamarBlue assay. We further evaluated superoxide production levels measured by dihydroethidium. We also examined the protective effects of serofendic acid on cytotoxicity with superoxide production of CM and high glucose.ResultsCM reduced cell numbers in a dose-dependent and time-dependent manner in LLC-PK1 cells. Furthermore, cytotoxicity of CM in diluted concentration was additively influenced by high glucose. CM and high glucose increased superoxide production, which was evaluated by the response to dihydroethidium, and was suppressed by serofendic acid. Cytotoxicity of CM, high glucose, and H2O2 was suppressed by serofendic acid, as well as the suppression by N-acetylcysteine on CM toxicity. Interestingly, the recovery by serofendic acid in H2O2- and high glucose-induced cellular injury was to the basal level, in contrast with the partial recovery from CM-induced injury. Finally, serofendic acid suppressed CM-induced injury and high glucose-induced apoptosis.ConclusionsThese results suggest that CM and high glucose induce cytotoxicity and oxidative stress in LLC-PK1 cells and that serofendic acid protects the injury probably from superoxide generation.

Lipopolysaccharide-triggered acute aggravation of mesangioproliferative glomerulonephritis through activation of coagulation in a high IgA strain of ddY mice.

Background: The high IgA (HIGA) strain of ddY mice represents an inbred model of IgA nephropathy that shows mesangioproliferative glomerulonephritis with mesangial IgA deposition. In this study, aggravation of glomerulonephritis in HIGA mice through lipopolysaccharide (LPS)-triggered activation of coagulation was investigated. Methods: Twelve-week-old HIGA and BALB/c mice were intraperitoneally injected with LPS twice at an interval of 3 days, and kidney specimens were collected 7 days after the second LPS injection. In an intervention experiment, the factor Xa inhibitor danaparoid was injected intraperitoneally every day for 7 days after the first LPS injection. Results: LPS injection induced macrophage infiltration and cellular proliferation in the mesangium together with fibrin deposition and monocyte chemoattractant protein 1 mRNA expression, as well as antigen deposition of tissue factor, factor V, factor X, and protease-activated receptor 2. These phenomena were obvious in HIGA mice when compared to BALB/c mice. Interestingly, toll-like receptor 4 was intensely expressed in HIGA mice before LPS injection and subsequently decreased. Danaparoid treatment significantly ameliorated proteinuria, cellular proliferation, and fibrin deposition. Conclusions: The present data suggest that tissue factor and factor V induction by LPS may in part accelerate mesangioproliferative glomerulonephritis through activation of factor X and downstream proinflammatory and procoagulant mechanisms.

Occurrence and tissue distribution of c-series gangliosides in the common squid Todarodes pacificus

We have recently demonstrated that the common squid Todarodes pacificus express acidic lipids that were reactive with a monoclonal antibody A2B5. In the present study, two A2B5-reactive acidic lipids were isolated from squid hepatopancreatic tissue and characterized for their structures by methods including glycolipid overlay analysis, product analysis after sialidase treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, the two acidic lipid were identified as GT3 and GQ1c, respectively. Another A2B5-reactive acidic lipid in the tissue was tentatively assigned to GT2 based upon its reactivity to A2B5 and chromatographic mobility on thin-layer chromatography. The composition and concentration of c-series gangliosides significantly differed among squid tissues (i.e. hepatopancreas, cerebral ganglion, eye lens, and mantle tissue). Interestingly, the percentages of c-series gangliosides within total gangliosides of hepatopancreas and cerebral ganglion were even higher than that of cod fish brain, which is known to be highly enriched with this ganglioside species. These findings strongly support the hypothesis that c-series gangliosides in squid tissues are not derived from ganglioside-containing food intake, but biosynthesized in a tissue-specific manner.

Ion exchange chromatography of aluminum using 3-carboxy-2-naphthylamine-N,N-diacetic acid as a fluorescent post-column chelating reagent.

Ion exchange chromatography of aluminum ion using 3-carboxy-2-naphthylamine-N,N-diacetic acid (CNDA) as a fluorescent post-column chelating reagent was studied. The solution containing ammonium chloride and hydrochloric acid was used for the eluent, and acetate buffer solution containing CNDA was used for the post column chelating reagent. The peak of aluminum was separated from that of calcium, magnesium and zinc, and the chromatogram was not affected by copper(II) and iron(III). The calibration curve gave linear plots with a range of 0.0027-0.54 ppm aluminum, the regression coefficient of correlation (r2) was 1.000, and the detection limit (S/N = 3) was 0.3 ppb, indicating that the method could determine aluminum with high sensitivity. It was demonstrated that CNDA is a useful metallofluorescent reagent for aluminum. This method has been successfully applied to the determination of aluminum in some tea drinks.

Observation of the melting process for ethyl p-aminobenzoate doped with eutectic mixture

Abstract In order to improve the reliability of purity measurements by differential scanning calorimetry (DSC), the melting process of a test material was studied using a system of ethyl p -aminobenzoate and n -butyl p -hydroxybenzoate. The eutectic mixture began melting accompanying the increase in temperature. After the minor component, p -hydroxybenzoate, had entirely melted in the eutectic mixture, it was confirmed by DSC measurement and solid phase-liquid phase titration that the major component, p -aminobenzoate, which remained as a solid, continued to dissolve into the eutectic mixture. In order to completely grasp the melting image of the test material, it was necessary to measure it from a sufficiently low temperature, and work up to the eutectic peak with the minor component. It was found that, by drawing up a solubility curve, the phenomenon in which p -aminobenzoate dissolves into the melted eutectic mixture is, in this system, close to the ideal solution based on Raoults equation, and that the heat of mixing in an infinite dilution was indicated to be a small value of −1.9 J g −1 . For that reason, the heat of fusion and the heat of dissolution coincided within the allowable error. Test materials that have been refined to a certain degree frequently include similar compounds as impurities, and this establishes an approximation of the heat of dissolution with the heat of fusion.

Rapid and simple determination of mercury in traditional Chinese medicines by amalgamgravimetry.

水銀の分析法は環境汚染物質を対象とするものに重点がおかれ,医薬品中の水銀を精度よく定量することは困難である.試料を熱分解し,生じた水銀蒸気をアマルガムとして捕集する重量法は,操作が簡単で水銀を含む廃棄物も少なく,信頼性の高い方法である.この報告では,中薬約10mgを試料とし酸化剤と妨害元素除去剤を充てんした燃焼管により燃焼し,銀を充てんした吸収管に水銀をアマルガムとして捕集する重量法について検討を行った.本法は1回の燃焼時間は25分であり,±0.2%以下の誤差(2σ)で定量することができた.又,水銀を吸収した銀粒は金属の水銀と同様の廃棄物処理法により処理することができた.