Seiko Horiuchi

Lysophosphatidylcholine plays an essential role in the mitogenic effect of oxidized low density lipoprotein on murine macrophages

We previously demonstrated that the growth of starch-induced murine macrophages was stimulated by modified low density lipoproteins, such as oxidized low density lipoprotein (Ox-LDL) and acetylated low density lipoprotein (acetyl-LDL), and that the mitogenic effect of Ox-LDL was much greater than that of acetyl-LDL (Yui, S., Sasaki, T., Miyazaki, A., Horiuchi, S., and Yamazaki, M. (1993) Arterioscler. Thromb. 13, 331-337). The present study was undertaken to elucidate the factor(s) that are involved in this growth-stimulating effect of Ox-LDL. The growth-stimulating effect of acetyl-LDL on murine resident macrophages was negligibly weak compared with that of Ox-LDL. However, the treatment of acetyl-LDL with phospholipase A2 led to an increase in lysophosphatidylcholine (lyso-PC) (75% of total phospholipids) and a concomitant increase in the mitogenic activity of acetyl-LDL. In contrast, cell-free incubation of Ox-LDL with high density lipoprotein resulted in a decrease in lyso-PC content and a concomitant loss of growth-stimulating activity. These results suggest that lyso-PC may play an essential role in the mitogenic activity of Ox-LDL.

Advanced glycation endproduct-modified superoxide dismutase-1 (SOD1)-positive inclusions are common to familial amyotrophic lateral sclerosis patients with SOD1 gene mutations and transgenic mice expressing human SOD1 with a G85R mutation.

Abstract To clarify the biological significance of the neuronal Lewy body-like hyaline inclusions and astrocytic hyaline inclusions characteristically found in patients with familial amyotrophic lateral sclerosis with superoxide dismutase-1 (SOD1) gene mutations and in transgenic mice expressing human SOD1 with G85R mutation, the detailed protein composition in both types of inclusions was immunohistochemically analyzed using 45 different antibodies. Both types of inclusions had very strong immunoreactivity for SOD1. The SOD1-positive inclusions in both cell types were also immunoreactive for the insoluble advanced glycation endproducts (AGEs) such as Nɛ-(carboxymethyl)lysine (CML), pyrraline and pentosidine: both inclusions in both conditions were ultrastructurally composed of the granule-coated fibrils that had immunoreactivities to CML and pyrraline. Both types of inclusions were negative for stress-response proteins (SRPs), 4-hydroxy-2-nonenal (HNE), acrolein, nitric oxide synthases (NOSs) and nitrotyrosine as representative markers of oxidative stress. The neurons and astrocytes of the normal individuals and non-transgenic mice showed no significant immunoreactivity for SOD1, AGEs, SRPs, HNE, acrolein, NOSs or nitrotyrosine. Our results suggest that a portion of the SOD1 composing both type of inclusions, probably toxic mutant SOD1, is modified by the AGEs, and that the formation of the AGE-modified SOD1 is one of the mechanisms responsible for the aggregation involving no significant oxidative mechanisms.

Differential effect of subspecies of lipoprotein containing apolipoprotein A-I on cholesterol efflux from cholesterol-loaded macrophages : functional correlation with lecithin : cholesterol acyltransferase

Two species of lipoprotein containing apoA-I, one containing only apoA-I (LpA-I), and the other containing apoA-I and apoA-II (LpA-I/A-II), were tested for their effects on macrophage foam cells. Rat macrophages were converted to foam cells by incubation with radiolabeled acetylated LDL. Incubation with LpA-I or LpA-I/A-II decreased the cellular cholesteryl esters (CE) mass. However, the free cholesterol (FC) mass was only reduced by LpA-I. All the radioactivity excreted into the medium was associated with LpA-I or LpA-I/A-II; 39% of the excreted radioactivity was esterified in LpA-I and 10% in LpA-I/A-II. Upon complete inactivation of lecithin: cholesterol acyltransferase (LCAT) activity with dithiobisnitrobenzoic acid, the cholesterol reducing capacity of LpA-I was weakened significantly. However, the CE mass reducing capacity of LpA-I/A-II was not affected. When LpA-I and LpA-I/A-II were combined, the cholesterol reducing capacity of the mixture was similar to that of LpA-I alone. However, LpA-I re-isolated from the medium showed a lower esterification rate than did the re-isolated LpA-I/A-II, thereby indicating that the cholesterol esterified in LpA-I was transferred to LpA-I/A-II. These results suggest that (i) the function of LpA-I is closely linked to the LCAT activity while that of LpA-I/A-II is not, and (ii) LpA-I in concert with LpA-I/A-II induces a series of extracellular events; LCAT-mediated esterification of excreted FC by LpA-I and a subsequent CE transfer to LpA-I/A-II. These mechanisms might be important for net cholesterol efflux from macrophage foam cells in physiological states.

Advanced glycation end products in human optic nerve head

AIMS To localise advanced glycation end products (AGEs) in human optic nerve head. METHODS Optic nerve samples from 13 elderly individuals (seven diabetics and six non-diabetics) were obtained at necropsy. Pyrraline, an advanced glycation end product, was immunohistochemically localised in the optic nerve heads. RESULTS In the diabetic subjects, moderate to intense immunoreactivity for pyrraline was detected in sclera, pia mater, cribriform plates, connective tissues in the optic nerve, and around vessels in the optic nerve and pia mater. Immunoreactivity for pyrraline was also detected around retinal vessels. In the non-diabetic subjects, slight or no immunoreactivity for pyrraline was found in cribriform plates and around the optic nerve vessels. CONCLUSION Accumulation of AGEs in cribriform plates and around vessels in the optic nerve may contribute to the development of optic neuropathy in diabetic patients.

Advanced glycation end products in Descemet's membrane and their effect on corneal endothelial cell.

Purpose. The purpose of this study was to evaluate the effect of advanced glycation end products (AGEs) in Descemet’s membrane on the attachment and spreading of the corneal endothelial cells. Methods. An anti-AGEs monoclonal antibody (6D12), which recognizes a N e -carboxymethyl lysine (CML)-protein adduct as an epitope, was used for immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Fresh bovine Descemet’s membrane was incubated for 4 weeks in the buffered solution with 500 mM of glucose-6-phosphate (G-6-P). In the incubated Descemet’s membrane, the immunohistochemical localization of CML was examined. Type I collagen-, type IV collagen-, fibronectin-, or laminin-coated 96-well plates were glycated by G-6-P. The amount of CML was determined by ELISA using 6D12. Cultured bovine corneal endothelial cells were seeded onto glycated or non-glycated extracellular matrix (ECM) in 96-well plates and allowed to attach for 3 hours. The number and the surface area of the attached cells were examined. Results. Immunoreactivity to CML was detected in Descemet’s membrane incubated in the buffered solution containing G-6-P. Glycation of fibronectin and laminin decreased the number and the surface area of the attached corneal endothelial cells. Aminoguanidine in the incubation mixture inhibited CML formation of ECM components and increased the number and the surface area of the attached corneal endothelial cells in a dose-dependent manner. Conclusions. AGE formation on fibronectin and laminin attenuated the attachment and spreading of the corneal endothelial cells. AGEs’ formation in Descemet’s membrane may be responsible for the corneal endothelial cell loss with aging and corneal endothelial abnormalities in diabetic patients

Characterization of subspecies of apolipoprotein A-I-containing lipoprotein in homozygotes for familial lecithin:cholesterol acyltransferase deficiency.

We characterized the two species of lipoproteins containing apolipoprotein A-I (apoA-I), one containing only apoA-I (LpA-I) and the other containing apoA-I and apoA-II (LpA-I/A-II), in four homozygotes for familial lecithin: cholesterol acyltransferase (LCAT) deficiency. Two homozygotes lacked both LCAT mass and activity, whereas the other two had some residual LCAT mass and activity. In these patients, the amount of all apoA-I-containing lipoproteins was one fourth that of normal control subjects, and > 60% was LpA-I. The chemical composition of both LpA-I and LpA-I/A-II is characterized by markedly decreased ratios of neutral to polar lipids compared with those of normals and the sizes of LpA-I and LpA-I/A-II particles are shifted to smaller and larger diameter ranges when compared with those of normal particles. Changes in particle diameter are also reflected in slower electrophoretic mobilities of both LpA-I and LpA-I/A-II particles. All of these abnormalities were more evident in the two homozygotes who lacked LCAT activity. Incubation of LCAT-deficient plasma with LCAT markedly corrected the chemical and physical abnormalities in both LpA-I and LpA-I/A-II particles. These data, taken together, emphasize the importance of LCAT in modifying the chemical composition, size, and shape of LpA-I and LpA-I/A-II particles.

The molecular forms of γ-glutamyl transferase in bile and serum of icteric rats

Abstract Alterations in the molecular form of γ-glutamyl transferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) were studied in the bile and serum of rats under surgical ligation of the bile duct. Polyacrylamide gel electrophoresis of the bile, followed by the enzyme stain, revealed a major, slowly migrating broad band and a minor, faster migrating band. The former was converted to the latter upon limited proteolysis of the bile with a very small amount of papain. This conversion was accompanied by a decrease in molecular size of the enzyme. Both enzyme forms were specifically adsorbed to a concanavalin A-Sepharose column. Most of the papain-treated enzyme preparation could be eluted from the column by α-methyl- d -glucoside, a haptenic sugar of this lectin. On the other hand, the predominant form of the enzyme in the untreated bile was eluted only in the presence both of the sugar and Triton X-100. Based on the chromatographic behavior of the two enzyme forms (detergent-solubilized and protease-solubilized form) purified from rat renal brush border membrane on concanavalin A-Sepharose column, it was concluded that the predominant form of the enzyme in the bile was the detergent-solubilized form and that the minor component represents the protease-solubilized enzyme. The serum from icteric rats was also found to contain both types of the enzyme. However, the relative amount of the protease-solubilized form to the detergent-solubilized form in the serum was much greater than that in the bile. These findings suggested that γ-glutamyl transferase in the hepatobiliary membrane systems was solubilized into the bile mainly as the detergent solubilized form, and that, during the process of translocation into the blood circulation, the enzyme was partly converted to the protease-solubilized form by some protease-like action.