The glycosylation sites and structural characteristics of oligosaccharides on recombinant human thrombomodulin.

Thrombomodulin (TM) is an anticoagulant glycoprotein on the surface of endothelial cell that directly inhibits the procoagulant activities of thrombin, and the TM-thrombin complex accelerates thrombin-catalyzed activation of protein C. Soluble TM in urine has no glycosaminoglycan (GAG) chain which accelerates the anticoagulant activities. Therefore, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C127 cells. The glycosylation sites were determined by amino acid sequence analysis of peptides digested with trypsin after S-carboxymethylation. The structures of N-linked oligosaccharides were estimated by two-dimensional sugar mapping of pyridylaminated oligosaccharides that were treated with exoglycosidase. The disaccharide composition analysis of the GAG chain was performed by HPLC using digestion with chondroitinase ABC, ACII and B. Consequently, it was revealed that the N-linked oligosaccharides were assigned to Asn29, Asn98, Asn364, Asn391; those structures were estimated biantennary, 2-6 branched triantennary and 2-4 branched triantennary complex type oligosaccharides that were linked by fucose at the ratio of 1.0:0.5:0.1, respectively. Moreover, the attachment site of the GAG chain was assigned to Ser472. It was then estimated that the GAG chain contained chondroitin-4-sulfate and dermatan sulfate, which were repeated approximately 30 times. In this paper, the GAG attachment site and structural characteristics of GAG-UTM, were confirmed. Moreover, structures of the N-linked oligosaccharides of GAG-UTM are described for the first time.

Importance of sialic acid in recombinant thrombomodulin in terms of pharmacokinetics and separation of desialyzed glycoprotein.

Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM) expressed in mouse C-127 cells has potent antithrombotic activity available as an anticoagulant. GAG-UTM, a glycoprotein with sialic acid, was investigated regarding the influence of the terminal sialic acid on its pharmacokinetics upon rapid intravenous injection in rat. Asialo GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with alpha-2, 6-sialyltransferase, containing sialic acid similarly to native sialo GAG-UTM, had only a short half-life in plasma, suggesting that the binding site of sialic acid on galactose was not only sialyzed with alpha-2, 6-sialyltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however, had a long half-life. These results suggest that terminal sialic acid may be important to the pharmacokinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became significant for quality control. In order to analyze sialo- and asialo-types in the early stage of purification, we investigated separation and analysis methods for both types and found a suitable sample of each: RCA-120-Agarose column for separation and ELISA using anti-thrombomodulin antibody and RCA lectin for analysis.

Solar urticaria inhibited by visible light.

A 25-year-old man with solar urticaria is described. The action spectrum ranged from 400 to 500 nm. An inhibition spectrum was found to be in the visible light range above 660 nm. Simultaneous or alternate exposure to 'blue-violet light' and 'red light' mostly inhibited weal formation. The urticarial reaction was not blocked by local injection of antihistamines and not prevented by histamine depletion with polymyxin B sulfate. These data suggest that histamine may not play a major role in weal production in this case.

Effect of beta-carotene on ultraweak chemiluminescence of UVB-irradiated squalene.

An emission spectral analysis was carried out on chemiluminescence emitted from UVB-irradiated squalene. The main emission species produced by the transition of (1 delta g) (1 delta g) and (1 delta g) (1 sigma g +) to (3 sigma g -) (3 sigma g -) were found by spectroscopic analysis of the chemiluminescence. When beta-carotene was added to the irradiated squalene, its spectral pattern changed drastically and many peaks disappeared.

Chemiluminescence of UV-irradiated linolenic acid and squalene.

An emission spectral analysis was carried out on ultraweak chemiluminescence emitted from UVB-irradiated linolenic acid and squalene. The main emission species produced by the transition of (1 delta g) (1 delta g) dimer and an additional weak band near 477.5 nm (0, 0) by the transition of (1 delta g (1 epsilon g+) to (3 epsilon g-) (3 epsilon g-) were found by spectroscopic analysis of chemiluminescence in both cases of irradiated linolenic acid and of squalene. A distinct peak around 410-420 nm was observed in irradiated squalene and the emitter seems to be due to the excited carbonyl compound.

Histopathological studies on sun-exposed hexachlorobenzene-induced porphyric rat skin.

Hexachlorobenzene (HCB) is a porphyrogenic agent. The inducement of skin changes was attempted through repeated exposure of the skin of HCB-induced porphyric rats to sunlight. The following skin changes were produced in the porphyric rats; erythema, erosion, crust, skin thickening and scarring. Histopathological examination revealed the presence of acanthosis, vacuolization of the malpighian cells, subepidermal vesicle, fibrosis, dilatation and increase of the blood vessels and perivascular cell infiltration composed of lymphocytes, histiocytes and mast cells. The PAS stainability of blood vessel walls was slightly intensified. The assumption is that photosensitive flares were elicited within the short 2-month period though destruction of endothelial cells was not prominent. There were no distinct skin changes, clinically or histopathologically, in any of the three control groups.