Nobuko Miyazawa

Immunological detection of glycated proteins in normal and streptozotocin-induced diabetic rats using anti hexitol-lysine IgG

A polyclonal antibody specific for the Amadori compound, a product of an early stage of the Maillard reaction, was raised in rabbits by immunization with hexitol-lysine (1-glucitol-lysine or 1-mannitol-lysine) coupled with various carrier proteins. The affinity purified antibody has a high titre and preferentially recognizes the glucose adduct, in the presence of sodium borohydride, as judged on enzyme-linked immunosorbent assay as well as immunoblot analysis. The glycated proteins (Amadori products) in various tissues of normal and streptozotocin-induced diabetic rats were examined by immunoblot analysis. In diabetic conditions, kidney, liver, lens, brain and lung proteins are more susceptible to glycation than other tissue proteins. Heart, spleen, adrenal gland and muscle proteins exhibit similar extents of glycation in both normal and diabetic conditions. This is the first demonstration of a specific antibody against the Amadori compound being raised with a synthetic compound, and of the tissue distribution of glycated proteins in normal and diabetic conditions. The antibody was very useful for in vitro and in vivo experiments on the Maillard reaction.

Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. It has been shown that 15–20% of patients with familial ALS (FALS) have defects in the Sod1 gene that encodes Cu, Zn‐superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu, Zn‐SODs in FALS, the susceptibility of mutants to glycation was examined. Mutated Cu, Zn‐SODs (G37R, G93A, and I113T) related to FALS and wild type were produced in a baculovirus/insect cell expression system. Glycated and nonglycated proteins were separated on a boronate column, and the nonglycated fraction was then incubated with glucose. The mutated Cu, Zn‐SODs were found to be highly susceptible to glycation compared with the wild‐type enzyme as estimated by Western blot analysis using an anti‐hexitol lysine antibody. The mutated Cu, Zn‐SOD incubated with glucose generated higher levels of hydrogen peroxide than the wild‐type enzyme. Mutated Cu, Zn‐SODs were also shown to be highly susceptible to fructation, and the fructated mutant also produced higher levels of hydrogen peroxide than the wild type. These results suggest that high susceptibility of mutated Cu, Zn‐SODs to glycation could be the origin of the oxidative stress associated with neuronal dysfunction in FALS.

Specific detections of the early process of the glycation reaction by fructose and glucose in diabetic rat lens

The glycation reaction by fructose, as well as that by glucose, in control and diabetic rat lens was analyzed by using antibodies which specifically recognize adducts of lysine with fructose and with glucose. Levels of fructose adducts in diabetic rat lens were 2.5 times that of the control, and correlated with sorbitol levels. This was mainly due to enhanced glycation of β‐ and γ‐crystallins by fructose under diabetic conditions. These data suggest that glycation by fructose may also play a role in cataract formation under conditions of diabetes and aging.

Effects of exercise stress and cold stress on glutathione and γ-glutamyltransferase in rat liver

Abstract Effects of acute and chronic stress (exercise and cold) on glutathione and γ-glutamyltransferase (γGT) in the rat liver were investigated. Such stress, except for in the case of acute exercise, had no definite influence on the glutathione level. On the other hand, γGT activity in both the extramicrosomal and microsomal fractions varied substantially, suggesting that acute exercise increases the release ability of the microsomal membrane of the rat liver, and that swimming training and long-term cold exposure stabilize the membrane. Immunoreactive γGT, however, did not always correlate with the enzyme activity, especially in the extramicrosomal fraction. Cross-adaptation appeared to exist between swimming training and chronic cold exposure.

Preparation and characterization of monoclonal antibodies to an N-linked oligosaccharide.

Two monoclonal antibodies to an N-linked oligosaccharide, MT-5 and MT-9, have been prepared by immunization with a pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar. The reactivity of these antibodies was monitored by their reaction with human asialoglycophorin in a solid-phase enzyme-linked immunosorbent assay. Both antibodies reacted with the sugar chains of various human glycoproteins such as immunoglobulin G, transferrin, gamma-glutamyl transpeptidase, alpha 1-acid glycoprotein, and alpha-fetoprotein. Treatment of asialoglycophorin with beta-N-acetylhexosaminidase or alpha-mannosidase resulted in reduction of the binding to these antibodies. The reactivity of MT-5 to asialoglycophorin was slightly inhibited by D-mannose and N-acetylglucosamine, whereas that of MT-9 was inhibited by D-mannose, N-acetyl-D-glucosamine, chitobiose, and L-fucose. The epitope specificity of MT-5 appears to be a sugar chain containing biantennary N-acetyl-D-glucosamine residues, the bisected N-acetyl-D-glucosamine residue, and a trimannosyl core. The epitope to which MT-9 is directed may be a complex made up of beta-mannose, chitobiose, and L-fucose. These studies indicate that immunization with pyridylaminated sugars can produce antibodies that recognize N-linked oligosaccharides. Monoclonal/polyclonal antibodies to the N-linked sugar chains of glycopeptides would be useful in such studies of proteins.

Physiological Relevance of Aldehyde Reductase and Aldose Reductase Gene Expression

Carbonyl compounds which are produced as intermediate metabolism during ordinary metabom or are present in food or drugs are known to be toxic to living organisms because of their high degree of reactivity. It has also been suggested that elevation in protein carbonyl groups is also a likely cause of aging (Stadtman, 1992). Cells contain defense systems against these compounds (Flynn, 1982) in the form of aldo-keto reductases, which includle aldehyde and aldose reductases, which catalyze the reduction of a variety of aldehydes to alcohols in an NADPH-dependent manner (Jez et al., 1997). Cytotoxic compounds which tain an aldehyde moiety, such as tripeptidyl aldehyde (Inoue et al., 1993),trioses (Van der Jagt et al., 1992) and methotrexate (Callahan and Beverley, 1992) are deified by enzymes in this gene family. The glycation reaction represents another source carbonyl compounds. This reaction occurs during normal aging and at accelerated rates diabetes, and is involved in the pathogenesis of diabetic complications (Fujii et al. 998). Glycation alters the activity of some enzymes such as Cu,Zn-SOD (Arai et al., 1; Ookawara et al., 1992), carbonic anhydrase (Kondo et al., 1987), sorbitol dehy- drogen (Hoshi et al., 1996), and aldehyde reductase (Takahashi et al., 1995b), and is also involved in production of dicarbonyl compounds such as 3-deoxyglucosone and methyloxal (Figure 1). The cross-linking of long-lived proteins such as collagen and lens crystallins are induced by these compounds and correlates with aging and diabetes. These dicarbonyl compounds are highly toxic and induce apoptosis in susceptible cells (Okado al., 1996). The production of aldehydes is enhanced during pathological conditions, includ diabetes and cancer. Concomitantly, aldehyde-reducing activity is also increased in hepatoma cell lines (Canuto et al., 1994). This is mainly due to an elevated expresn of the aldose reductase gene (AKR1B) as has been demonstrated in chemically induceepatoma tissues (Zeindl-Eberhart et al., 1994; Takahashi et al., 1995a).

Differential Expression of Mn- and Cu,Zn-Superoxide Dismutases in Various Tissues of LEC Rats

Oxy-radicals are thought to play important roles in the course of multistep carcinogenesis [1–3]. Accurate detection of oxy-radicals is impossible, however, because of their instability and short half-life. Therefore, evaluation of the levels of enzymes involved in the metabolism of oxy-radicals appears to be the most reliable means of assessing oxy-radical metabolism in vivo.

Reducing Sugars Induce Apoptosis in Pancreatic β-Cells by Provoking Oxidative Stress via a Glycation Reaction

Reducing sugars brought about apoptosis in isolated rat pancreatic islet cells as well as in a pancreatic β-cell-derived cell line, HIT. This apoptosis was characterized biochemically by internucleosomal DNA cleavage and morphologically by nuclear shrinkage, chromatic condensation, and apoptotic body formation. N-acetyl-L-cysteine and aminoguanidine inhibited the apoptosis. Proteins in β-cells were actually glycated by the binding with an antibody that can specifically recognize the protein glycated by fructose. The FACS analysis using dichlorofluorescin diacetate showed that reducing sugars increased intracellular peroxide levels preceding the induction of apoptosis. Levels of carbonyl and malondialdehyde were also increased. These results suggest that reducing sugars trigger oxidative modification and apoptosis in pancreatic β-cells by provoking oxidative stress mainly via a glycation reaction, which may explain the deterioration of β-cells under diabetic conditions.