Katsumi Murata

Cigarette smoking induces formation of 8-hydroxydeoxyguanosine, one of the oxidative DNA damages in human peripheral leukocytes

Active oxygen species (AOS) such as O2- and H2O2 have been shown to be generated from both gas and tar phases of cigarette smoke and it has been suggested that they are involved in carcinogenesis due to cigarette smoking. Therefore, we investigated the effect of cigarette smoking on oxidative DNA damages in human peripheral blood cells using 8-hydroxydeoxy-guanosine (8-OH-dG) as a marker. From ten healthy male volunteers aged 20-22 years, 5 ml of blood was taken before and 10 minutes after smoking 2 cigarettes in 10 minutes. After lysis of blood cell membranes leukocyte DNA was isolated using a DNA extractor and 8-OH-dG levels were determined using high performance liquid chromatography (HPLC) with electrochemical detection. The mean levels of 8-OH-dG increased significantly (P less than 0.05) from 3.3 +/- 0.8/10(6) dG (mean +/- SD) to 5.1 +/- 2.5 after smoking. These results indicate that cigarette smoking induces oxidative DNA damage in peripheral blood cells in a relatively short time.

Polydispersity of acidic glycosaminoglycan components in human liver and the changes at different stages in liver cirrhosis.

The aim of this study was to characterize acidic glycosaminoglycan components in normal human liver and in alcoholic cirrhosis, and to determine whether the proportions of individual glycosaminoglycans change with advancing cirrhosis. Acidic glycosaminoglycans are components of extracellular matrices and consist of repeating disaccharides of hexosamine and hexuronic acid with molecular weights ranging from 5 X 10(3) to 5 X 10(4), except for hyaluronic acid, whose molecular weight ranges from 3 X 10(4) to 1.6 X 10(6). The acidic glycosaminoglycan components in normal liver and at different stages of liver cirrhosis were found to be polydisperse as to the molecular weight and the degree of sulfation. The increased content of glycosaminoglycans with advancing liver cirrhosis was related to those of heparan sulfate and dermatan sulfate components. Heparan sulfates were the most prominent components of smaller molecular weight fractions. In the normal state, moderate amounts of dermatan sulfate and the oversulfated isomer were present in intermediate molecular weight fractions, but increasing amounts of the components shifted to higher molecular weight fractions with advancing cirrhosis. A small amount of hyaluronic acid was found in higher molecular weight fractions and the amount increased at the initial stage as a reversible phenomenon. The possible roles of hepatic glycosaminoglycan components in the process of fibrosis are discussed.

Distribution of acidic glycosaminoglycans in the intima, media and adventitia of bovine aorta and their anticoagulant properties

SUMMARY 1. The constituents of acidic glycosaminoglycans in the tunica intima, media and adventitia were studied by means of an enzymatic assay with chondroitinases and by electrophoretic characterization. 2. The content of the acidic glycosaminoglycans was higher in the intima than in the adventitia based on defatted dry tissue weight. The gel filtration pattern showed that, with chondroitinase-AC, the proportion of the intact glycosaminoglycans is greater in the adventitia than in the intima. 3. Electrophoresis before and after digestion with chondroitinases indicated that the three layers contain chondroitin-4- and -6-sulfates, dermatan sulfate, heparan sulfates and hyaluronic acid. In the case of the adventitia, bands migrating like the heparin standard were also detected. 4. Paper chromatographic separation of the unsaturated disaccharides after digestion with chondroitinases revealed that the proportion of chondroitin-6-sulfate is predominant in the intima, whereas the proportion of heparan sulfates, hyaluronic acid and dermatan sulfate is one and one-half-2 times higher in the adventitia than in the intima. 5. Anticoagulant activity, measured by thrombelastography, of the acidic glycosaminoglycans in the three layers clearly indicated that the glycosaminoglycans in the adventitia possess the highest potency among the three layers. It was considered that the higher activity of the glycosaminoglycans in the adventitia is due to the relatively greater proportion of heparan sulfates and dermatan sulfate.

Urinary excretion of macromolecular acidic glycosaminoglycans in Werner's syndrome.

The urinary acidic glycosaminoglycans (AGAG) of Werners syndrome were isolated, purified and characterized by gel-chromatography, cellulose acetate electrophoresis, chemical analysis, streptomyces hyaluronidase susceptibility and viscometry. The AGAG appeared at the first peaks of the 0.6 M and 0.8 M fractions obtained through Sephadex G-100 were mainly a a hyaluronic acid (HA). HA was composed of 16% of the urinary AGAG. The AGAG at the first peak had the maximum molecular weight of 360 000 in the 0.8 M fraction followed by lesser molecular weights in the other fractions.

Inhibitory effects of human aortic and venous acid glycosaminoglycans on thrombus formation

Abstract 1. (1) The effects of acid glycosaminoglycans extracted from human vascular tissues on thrombus formation were studied in relation to the thrombogenic theory of atherosclerosis, by means of a modified circular loop technique. 2. (2) Vascular acid glycosaminoglycans showed prolonged thrombus formation time and reduced thrombus weight, indicating that they possess anti-thrombogenic properties. These activities of vascular acid glycosaminoglycans were found to be much greater in venous tissue than in arterial tissue. 3. (3) Comparative effects of chondroitin sulfate isomers on thrombus formation were studied in view of the fact that human vascular tissues contain mainly a chondroitin sulfate family. The most potent anti-thrombogenic activity was found to be in chondroitin sulfate B among the chondroitin sulfate isomers, whereas the activities of chondroitin sulfates A and C were less than that of the B isomer. Above finding accords with electrophoretic evidence that a greater amount of chondroitin sulfate B is present among venous acid glycosaminoglycans than among aortic ones.

Werner's Syndrome: Twenty-four Cases with a Review of the Japanese Medical Literature

Twenty‐four cases of Werners syndrome were studied to define the clinical manifestations in comparison with those found in a clinical review of 153 cases in the Japanese medical literature. The conspicuous characteristics of the 24 patients were short stature, stocky trunk with thin limbs, low body weight, bird‐like or “masked” face, early graying or loss of hair, high‐pitched or hoarse voice, bilateral cataracts, and various scleroderma‐like signs. Three signs not stressed previously were hyperreflexia, flat feet, and irregular dental development. The onsets and incidences of these manifestations during the seven‐year serial study are described.

The Acidic Glycosaminoglycans in Leukocytes: An Application of Enzymatic Methods

Acidic glycosaminoglycans (AGAG) in bovine leukocytes were studied by an enzymatic approach with the chondroitinases and streptomyces hyaluronidase. The appearance of a single spot corresponding to the unsaturated 4-sulfated disaccharide after digestion of leukocyte AGAG with chondroitinases indicated that chondroitin sulfate in leukocyte AGAG consists of chondroitin 4-sulfate only. Dowex 1-X2 column fractionation, followed by electrophoretic identification and thin layer chromatography, as well as enzymatic digestion of intact AGAG followed by gel filtration indicated that small amounts of hyaluronic acid and possibly heparan sulfates are also present in leukocyte AGAG. The present studies showed that the content of chondroitin 4-sulfate, hyaluronic acid, and heparan sulfates in leukocyte AGAG is 75-80%, 10-15%, and approximately lo%, respectively.

Acidic glycosaminoglycans in human heart valves

Abstract Acidic glycosaminoglycans (AGAG) in normal human heart valves were analyzed with an enzymatic assay method using AGAG-lyases. The AGAG content was high in the mitral and aortic valves and to a lesser extent in the tricuspid valve. The amount in the three valves was high at maturation and then decreased with advancing age. No definite difference in the AGAG composition was found among the three valves. The main AGAG was hyaluronic acid (HA) constituting approximately half of the total AGAG, and dermatan sulfate (DS) and chondroitin sulfate (CS) isomers each accounted for 1 4 to 1 5 of the total AGAG. Heparan sulfate (HS) was detected to constitute several % of the total AGAG. A minor amount of oversulfated DS was also present. With advancing age, the proportions of HA and chondroitin 4-sulfate tended to decrease whereas those of chondrotin 6-sulfate, DS and HS tended to increase. A possible function of the valvular AGAG is discussed with respect to the constitutional change.

Chondroitin sulfate isomers in normal human urine.

Abstract 1. 1. Enzymic analysis of urinary chondroitin sulfate isomers in normal subjects was carried out on the basis of unsaturated disaccharide subunits produced by the digestion of urinary chondroitin sulfates with chondroitinases and chondrosulfatases. 2. 2. By paper chromatographic and electrophoretic separations, four unsaturated disaccharides were detected as the products of degradation of urinary chondroitin sulfate isomers with chondroitinases. 3. 3. Minor constituents of unsaturated nonsulfated disaccharide and unsaturated di-sulfated disaccharide were demonstrated in addition to the major components of unsaturated 4-sulfated and 6-sulfated disaccharides in normal human urine. This finding indicates that nosulfated and/or undersulfated chondroitin sulfate, as well as oversulfated chondroitin sulfate, are present in normal urine. The distribution of the unsaturated disaccharides fractioned by NaCl elution from Dowex columns reflected the diverse spectrum of urinary chondroitin sulfate isomers.

Acidic glycosaminoglycan, lipid and water contents in human coronary arterial branches.

The acidic glycosaminoglycans (AGAG) of the human coronary arterial tree (the main left and right branches and their distal portions) were analyzed by enzymatic methods employing chondroitinases, hyaluronidase and heparitinase. The AGAG content of human coronary arteries was highest in the left branch, intermediate in the right branch and lowest in the distal portions. Some compositional differences in AGAG were found in these three parts. The amount of AGAG in the coronary arterial tree decreased with increasing severity of atherosclerosis. The main AGAG were heparan sulfate (HS) and chondroitin 6-sulfate (C-6S), constituting 33-38% and 24-36% of the total AGAG, respectively. Dermatan sulfate (DS) and chondroitin 4-sulfate (C-4S) each comprised 1/5-1/10 of the total AGAG. Hyaluronic acid (HA) and oversulfated DS comprised smaller proportions of the total AGAG. A small amount of heparin was occasionally detected in the coronary arterial tree, particularly in the distal portions. The lipid content of the main branches was increased in mildly atherosclerotic parts but diminished in severely affected parts. The water content was relatively higher in the main branches and decreased with severity of atherosclerosis. A possible function of these AGAG in atherosclerosis is discussed with respect to the compositional changes.