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Climatic droplet keratopathy (CDK), known as spheroid degeneration of the cornea, is one of the most frequent degenerative corneal disorders affecting visual function. However, the histochemical nature of the deposits seen in CDK is still unclear.To investigate the pathogenesis of CDK, we investigated the immunohistochemical localisation of advanced glycation end products (AGEs) in surgical specimens of CDK.Immunohistochemical localisation of N(epsilon)-(carboxymethyl)-l-lysine (CML), N(epsilon)-(carboxyethyl)-l-lysine (CEL), pyrraline, pentosidine and imidazolone was examined in three corneas with CDK, six corneas with bullous keratopathy and three corneas without any corneal diseases.In all the specimens with CDK, immunoreactivity was strong in CML, moderate in pyrraline and pentosidine, and weak in imidazolone. Immunoreactivity was absent in CEL. In contrast, no immunoreactivity to CML, pyrraline, pentosidine, imidazolone or CEL was detected in corneas with bullous keratopathy, or in corneas without any corneal diseases.CDK is caused by an aggregation of AGE-modified proteins. The result is consistent with etiological findings that ultraviolet irradiation and ageing, both of which are accelerators of AGE formation, are closely related to the development of CDK.

Cellular interactions with advanced glycation end products (AGE)-modified proteins are known to induce several biological responses, not only endocytic uptake and degradation, but also the induction of cytokines and growth factors, combined responses that may be linked to the development of diabetic vascular complications. In this study we demonstrate that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for AGE-modified bovine serum albumin (AGE-BSA) (K(d) = 27.8 nM), and additionally for EN-RAGE (extracellular newly identified RAGE binding protein) (K(d) = 118 nM). Western blot and RT-PCR analysis showed that RAGE (receptor for AGE) is highly expressed on A549 cells, while the expression of other known AGE-receptors such as galectin-3 and SR-A (class A scavenger receptor), are below the level of detection. The binding of (125)I-AGE-BSA to these cells is inhibited by unlabeled AGE-BSA, but not by EN-RAGE. In contrast, the binding of (125)I-EN-RAGE is significantly inhibited by unlabeled EN-RAGE and soluble RAGE, but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one specific for EN-RAGE and the other specific for AGE-BSA. The latter receptor on A549 cells is distinct from the scavenger receptor family and RAGE.

Interaction of advanced glycation end products (AGE) with AGE-receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE-receptors identified so far are RAGE (receptor for AGE), 80 K-H, OST-48, galectin-3, and SR-A (macrophage scavenger receptor type I and II). Since SR-A belongs to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to the class B scavenger receptor family (SR-B) can recognize AGE-proteins as a ligand. This was tested in the present study at the cellular level using CHO (Chinese hamster ovary) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-BSA (bovine serum albumin) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36 but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized LDL (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE-proteins. Because CD36 is one of the major Ox-LDL receptors and is upregulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE-proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

Background. Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetic glomerulopathy. The role of AGEs in non-diabetic renal damage is not well characterized. First, we studied whether renal AGE accumulation occurs in non-diabetic proteinuria-induced renal damage and whether this is ameliorated by renoprotective treatment. Secondly, we investigated whether renal AGE accumulation was due to intrarenal effects of local protein trafficking.Methods. Pentosidine was measured (by high-performance liquid chromatography) in rats with chronic bilateral adriamycin nephropathy (AN), untreated and treated with lisinopril. Age-matched healthy rats served as negative controls. Secondly, we compared renal pentosidine in mild proteinuric and non-proteinuric kidneys of unilateral AN and in age-matched controls at 12 and 30 weeks. Intrarenal localization of pentosidine was studied by immunohistochemistry.Results. Renal pentosidine was elevated in untreated AN (0.14 +/- 0.04 mu mol/mol valine) vs healthy controls (0.04 +/- 0.01 mu mol/mol valine, P Conclusion. Pentosidine accumulates in non-diabetic proteinuric kidneys in damaged tubules, and renoprotective treatment by angiotensin-converting enzyme (ACE) inhibitors inhibits AGE accumulation, supporting a relationship between abnormal renal protein trafficking, proteinuria-induced tubular damage and tubular pentosidine accumulation. Future studies, applying specific AGE inhibitors, should be conducted to provide insight into the pathophysiological significance of renal AGEs in non-diabetic renal disease.

Mesangial accumulation of GA-pyridine, a novel glycolaldehyde-derived AGE, in human renal disease. Background Advanced glycation end products (AGEs) contribute to diabetic and atherosclerotic end-organ damage, but the mechanisms of AGE-formation and AGE-induced damage are unclear. Glycolaldehyde (GA) is a Maillard-reaction intermediate and can be formed by reaction of L-serine with the myeloperoxidase-system. GA reacts with proteins to form AGEs, such as GA-pyridine, which is specific for protein modification by GA. GA-pyridine accumulates in human atherosclerotic lesions. As atherosclerosis and progressive glomerulosclerosis share many similarities, we hypothesized that GA-pyridine accumulates in renal diseases, especially those with prominent mesangial involvement. Methods Paraffin-embedded renal biopsies from 55 patients with various renal diseases, as well as control tissue, obtained from the unaffected part of kidneys from 10 patients with renal cell carcinoma were immunohistochemically stained with a monoclonal antibody directed against GA-pyridine and were scored semiquantitatively. Additional sections were scored for mesangial matrix expansion (MME) and focal glomerular sclerosis (FGS). Results In normal human kidneys, GA-pyridine was mainly localized in tubular epithelial cells, but not in the glomerular mesangium. Significant mesangial GA-pyridine accumulation was found in disorders with mesangial involvement as a common denominator. In contrast, mesangial GA-pyridine accumulation was less prominent in renal diseases without prominent mesangial involvement. Moreover, mesangial GA-pyridine accumulation was more pronounced in kidneys with higher MME and FGS scores across the different diagnoses. Conclusion GA-pyridine accumulates in the mesangium in human renal disease, in particular in disorders with mesangial involvement. Further studies should elucidate whether mesangial GA-pyridine plays a role in the progression of glomerular damage.

Previous observations by us have clarified that proteins modified by advanced glycation end products (AGEs) are recognized as effective ligands by CD36-overexpressed CHO cells and undergo receptor-mediated endocytosis. CD36, a member of the class B scavenger receptor family, also acts as a fatty acid transporter in adipocytes. Oxidized low-density lipoprotein (Ox-LDL), a ligand for CD36, is known to upregulate CD36 by activating peroxisome proliferator-activated receptor gamma (PPAR-gamma) in macrophages, whereas PPAR-gamma ligands such as troglitazone and 15-deoxy-delta12,14-prostaglandin J2 decrease leptin secretion from adipocytes. The purpose of this study was to examine effects of AGE ligands on leptin expression in adipocytes. Glycolaldehyde-modified bovine serum albumin (GA-BSA) decreased leptin expression at both the protein and mRNA levels in 3T3-L1 adipocytes and mouse epididymal adipocytes. The binding to and subsequent endocytic degradation of GA-BSA by 3T3-L1 adipocytes were effectively inhibited by a neutralizing anti-CD36 antibody. These results indicate that the ligand interaction of GA-BSA with CD36 leads to downregulation of leptin expression in 3T3-L1 adipocytes, suggesting that AGE-induced leptin downregulation is linked to reduction of the insulin sensitivity in metabolic syndrome.

Advanced glycation end products (AGE)-modified proteins as well as oxidized-LDL (Ox-LDL) undergo receptor-mediated endocytosis by CHO cells overexpressing CD36, a member of class B scavenger receptor family. The purpose of the present study was to examine the effects of glycolaldehyde-modified BSA (GA-BSA) as an AGE-ligand and Ox-LDL on leptin expression in adipocytes. GA-BSA decreased leptin expression at both protein and mRNA levels in 3T3-L1 adipocytes and mouse epididymal adipocytes. Ox-LDL showed a similar inhibitory effect on leptin expression in 3T3-L1 adipocytes, which effect was protected by N-acetylcysteine, a reactive oxygen species (ROS) inhibitor. Binding of (125)I-GA-BSA or (125)I-Ox-LDL to 3T3-L1 adipocytes and subsequent endocytic degradation were inhibited by a neutralizing anti-CD36 antibody. Furthermore, this antibody also suppressed Ox-LDL-induced leptin down-regulation. These results clarify that the interaction of GA-BSA and Ox-LDL with CD36 leads to down-regulation of leptin expression via ROS system(s) in 3T3-L1 adipocytes, suggesting that a potential link of AGE- and/or Ox-LDL-induced leptin down-regulation might be linked to insulin-sensitivity in metabolic syndrome.

The levels of plasma 3-deoxyglucosone (3-DG) increase under hyperglycemic conditions and are associated with the pathogenesis of diabetic complications because of the high reactivity of 3-DG with proteins to form advanced glycation end products (AGE). To investigate potential markers for 3-DG-mediated protein modification in vitro and in vivo, we compared the yield of several 3-DG-derived AGE structures by immunochemical analysis and HPLC and measured their localization in human atherosclerotic lesions. When BSA was incubated with 3-DG at 37 degrees C for up to 4 wk, the amounts of N(epsilon)-(carboxymethyl)lysine (CML) and 3-DG-imidazolone steeply increased with incubation time, whereas the levels of pyrraline and pentosidine increased slightly by day 28. In contrast, significant amounts of pyrraline and pentosidine were also observed when BSA was incubated with 3-DG at 60 degrees C to enhance AGE-formation. In atherosclerotic lesions, CML and 3-DG-imidazolone were found intracellularly in the cytoplasm of most foam cells and extracellularly in the atheromatous core. A weak-positive immunoreaction with pyrraline was found in the extracellular matrix and a few foam cells in aortic intima with atherosclerotic lesions. Our results provide the first evidence that CML and 3-DG-imidazolone are major AGE structures in 3-DG-modified proteins, and that 3-DG-imidazolone provides a better marker for protein modification by 3-DG than pyrraline.

We propose a full-DC injector for an energy-recovery linac. The injector is based on a 2 MeV DC accelerator that is commercially available (Dynamitron type), combined with a photo cathode. A bunched beam from the DC accelerator is merged with a recirculating beam and injected into a superconducting buncher-booster, which accelerates the beam up to ∼20 MeV . The buncher-booster is operated in partial energy-recovery mode, in which we can reduce the capacity of the RF generators to 10 kW even for a high-average current operation, 100 mA . We present results of beam dynamics simulation and RF system optimization.

Levels of glycation (fructose-lysine, FL) and advanced glycoxidation and lipoxidation end-products (AGE/ALEs) were measured in total skeletal (gastrocnemius) muscle and myofibril protein and compared to levels of the same compounds in insoluble skin collagen of control and diabetic rats. Levels of FL in total muscle and myofibril protein were 3-5% the level of FL in skin collagen. The AGE/ALEs, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine, were also significantly lower in total muscle and myofibril protein, approximately 25% of levels in skin collagen. The newly described sulfhydryl AGE/ALE, S-(carboxymethyl)cysteine (CMC), was also measured in muscle; levels of CMC were comparable to those of CML and increased similarly in response to diabetes. Although FL and AGE/ALEs increased in muscle protein in diabetes, the relative increase was less than that seen in skin collagen. These data indicate that muscle protein is partially protected against the increase in both glycation and AGE/ALE formation in diabetes.

Urea hydrogen peroxide (UHP) is a stable form of H 2 O 2 and cytotoxic agent. This study describes examination of UHP formation from collagen glycation and relevant glycoxidative damage in chronic renal failure (CRF). Renal fibers were incubated with 50 mM ribose in either serum ultrafiltrate or phosphate-buffered saline in the presence of various concentrations of urea. UHP was determined by a modified ferrous oxidation in xylenol orange (FOX) assay. The presence of urea resulted in an increase in the generation of UHP in a dose-dependent manner of urea in these incubation systems. Pentosidine levels analyzed by HPLC also increased in a dose-dependent manner of urea. Blocking experiments showed that pentosidine and carboxymethyllysine formation was significantly enhanced by hydroxyl radical generated from UHP via Fenton reaction. The renal and cardiac levels of UHP, pentosidine, and carboxymethyllysine in patients with CRF, including seven predialysis and eight hemodialysis subjects, were significantly higher than that in controls ( n = 16). The renal and cardiac levels of UHP closely correlated with the levels of renal and cardiac pentosidine and carboxymethyllysine and inversely correlated with left ventricle ejection fraction in CRF patients. This study provides evidence, for the first time, that UHP can be produced from Maillard reaction. Increased UHP in chronic renal failure enhances the formation of pentosidine and carboxymethyllysine via Fenton reaction (UHP-Fenton pathway).

An energy-recovery linac (ERL) for a high-power free-electron laser (FEL) has been designed and constructed at Japan Atomic Energy Research Institute (JAERI). The construction of the ERL was completed and first energy-recovery operation and first FEL lasing have been demonstrated. We present the design overview and the performance of the JAERI-ERL. Future plans towards a 10-kW FEL are also described.

The sustained saturation in an free-electron laser (FEL) oscillator at perfect synchronism of an optical cavity (dL ¼ 0) was experimentally verified despite the well-known lethargy effect (Phys. Rev. Lett. 86 (2001) 5707). Formation of an FEL field with uniform and constant phase from an incoherent state is one of the key issues to understand the mechanism how the lasing occurs at dL ¼ 0: In this report, we show that phase of an FEL field becomes uniform and constant during passage through undulator periods due to the slippage effect in a model independent manner.

Advanced glycation end products (AGE) are filtered by glomeruli and reabsorbed and metabolized by proximal tubule cells (PTC). In renal failure, decreased renal AGE metabolism likely accounts for the accumulation in serum that is related to uremic complications. In diabetes, AGE generation is increased, and the handling mechanisms in PTC are likely associated with the pathogenesis of tubulointerstitial injury. It is therefore important to clarify the mechanisms of the AGE metabolism to develop a strategy for removing AGE in uremia and to elucidate the pathogenesis of diabetic nephropathy. To this end, this study focused on the molecular analysis of megalin, a multi-ligand endocytic receptor, in PTC. AGE uptake analysis was performed using the rat yolk sac-derived L2 cell line system established for the analysis of megalin's endocytic functions. The cells mediated specific internalization and degradation of AGE, which were significantly blocked by anti-megalin IgG, indicating that megalin is involved in the cellular processes. However, cell surface AGE-binding assays and ligand blot analysis revealed no evidence that megalin is a direct AGE receptor. Affinity chromatography and ligand blot analysis originally revealed that 200-kD and 400-kD proteins in the cells bind to AGE and the 200-kD protein to megalin in a Ca(2+)-dependent manner. The binding of megalin with the 200-kD protein was suppressed by receptor-associated protein (RAP), a ligand for megalin. In conclusion, megalin functions for endocytosis of AGE via an indirect mechanism. L2 cells express novel AGE-binding proteins, one of which may interact with megalin.

We have developed a separation system for N e -(carboxyethyl)lysine (CEL) and N e -(carboxymethyl)lysine (CML) by HPLC equipped with a styrene–divinylbenzene copolymer resin coupled with sulfonic group cation-exchange column and examined whether CEL is formed from proteins modified by glucose via the Maillard reaction. CEL was generated by incubating bovine serum albumin (BSA) with glucose, a reaction inhibited by aminoguanidine, but enhanced by phosphate. Although several aldehydes were detected during incubation of N α -acetyllysine with glucose, incubation of BSA with methylglyoxal alone generated CEL. These results indicate that methylglyoxal is responsible for CEL formation on protein in vitro.

Recent immunohistochemical demonstration of advanced glycation end products (AGE) in several tissues suggests potential involvement of AGEs in aging and age-related disorders. However, little is known about the generation of AGEs during development, and systematic experimental studies of the production of AGEs in aging are few. The aim of the present immunohistochemical study was to examine localization of AGE in fetal, young, and adult rats by using four monoclonal antibodies against AGE-structures—6D12 for Ne-(carboxymethyl)lysine (CML), KNH-30 for Ne-(carboxyethyl)lysine (CEL), 1F6 for fluorolink, and 2A2 for which the epitope is as yet unknown. CML was also determined by RP-HPLC. The results revealed that in rats at birth, all four AGE structures were found in most tissues and cells. The AGE accumulation and distribution increased gradually with aging. In the fetuses at 10 fetal days, all AGE-structures except for fluorolink were found in all ecto-, meso-, and endodermal tissues. AGE-deposition tended to increase with progression of fetal development. HPLC analysis revealed that 724.8±87.3 mmol CML/mol lysine was detected in fetuses at 13 fetal days. These results strongly suggest that AGE accumulation in tissues and cells occurs not only during aging but also from an early stage of ontogeny.

In the Maillard reaction, 3-deoxyglucosone (3-DG) was known to generate from Amadori product and contribute to further AGE formation such as I a -(carboxymethyl)lysine (CML), 3-DG-derived imidazolone (3-DG-imidazolone) and pyrraline. However, main AGE structures generated from 3-DG modification have not yet been elucidated. To solve this issue, we set up the detection system for AGE by immunochemical analysis as well as high-performance liquid chromatography (HPLC) and determined them in 3-DG-modified bovine serum albumin (BSA). When BSA was incubated at 37 °C for up to 4 weeks with 3-DG, the reactivity of antibodies against CML and 3-DG-imidazolone increased steeply with incubation time, whereas the reactivity of antibodies against pyrraline was increased slightly. In contrast, in addition to CML and 3-DG-imidazolone, significant amount of pyrraline was observed when AGE formation was enhanced by high temperature at 60 °C. In HPLC analysis, CML (1.10 mol/mol of BSA), 3-DG-imidazolone (0.84 mol/mol of BSA) and pyrraline (0.33 mol/mol of BSA) were detected in BSA incubated with 3-DG at 37 °C for 4 weeks. Taken together, our analyses clarified that formation of CML and 3-DG-imidazolone is higher than that of pyrraline during the incubation of protein with 3-DG.

Previous studies using peritoneal macrophages obtained from macrophage scavenger receptor-A (MSR-A)-knockout mice showed that the endocytic uptake of advanced glycation endproducts (AGE) by macrophages was mainly mediated by MSR-A. However, it is controversial whether the endocytic uptake of intravenously injected AGE-proteins by liver endothelial cells (LECs) is similarly explained by receptor-mediated endocytosis via MSR-A. The present study was conducted to compare the capacity to endocytose AGE-proteins in LECs and peritoneal macrophages obtained from MSR-A-knockout and litter mate wild type mice. The endocytic degradation of MSR-A-knockout LECs for AGE-BSA was indistinguishable from that of wild type LECs, whereas that of MSR-A-knockout peritoneal macrophages for AGE-BSA was reduced to 30% of wild type cells. Furthermore, formaldehyde-treated serum albumin (f-Alb), a ligand known to undergo scavenger receptor-mediated endocytosis by LECs, was effectively taken up by MSR-A-knockout LECs at a capacity that did not differ from that of wild type LECs. Furthermore, the endocytic uptake of AGE-BSA by LECs was effectively competed for by unlabelled f-Alb. These data indicate that the scavenger receptor-ligands AGE-proteins are endocytosed by LECs through a non-MSR-A pathway.

We demonstrated that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for advanced glycation endproducts (AGE)-modified bovine serum albumin (AGE-BSA) ( K d =27.8 nM) and EN-RAGE (extracellular newly identified RAGE binding protein) ( K d =118 nM). Western blot and RT-PCR analysis showed that receptor for AGE (RAGE) was highly expressed on A549 cells, while the expression of other known AGE-receptors, e.g., galectin-3 and class A scavenger receptor (SR-A), were below the detection level. The binding of 125 I-AGE-BSA to these cells was inhibited by unlabeled AGE-BSA but not by EN-RAGE. In contrast, the binding of 125 I-EN-RAGE was significantly inhibited by unlabeled EN-RAGE and soluble RAGE but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one is specific for EN-RAGE and the other is specific for AGE-BSA. The latter receptor on A549 cells is novel as it is distinct from other known AGE-receptors.

Actions of the Ne-(carboxymethyl)lysine (CML) were investigated on the neovascularization of cultured retinal explants in streptozotocin (STZ)-diabetic rats at the early and the advanced stages. The onset and growth of sprouting and buds in the retinal explant (retinal neovascularization) in the early diabetes were similar to those in the age-matched young control. However, the retinal neovascularization in the advanced diabetes was significantly augmented, compared with those in the early diabetes and the age-matched aged control. Anti-CML antibody (6D12) did not affect the retinal neovascularization in the advanced diabetes. The retinal neovascularization was suppressed by the co-cultured choroidal explant in the early diabetes and facilitated by the choroidal explant in the advanced diabetic and the aged control, respectively. Anti-CML antibody inhibited this retinal neovascularization induced by the co-cultured choroidal explants. Anti-vascular endothelial growth factor (anti-VEGF) antibody also had a similar action as anti-CML antibody in the advanced diabetes. In conclusion, the CML-induced choroidal activity suppressed the retinal neovascularization in the early diabetes. During progression of diabetes, the retinal neovascularization was facilitated in a CML-independent manner. The CML-induced choroidal activity augmented the retinal neovascularization in the advanced diabetes. Both the CML-independent and CML-activated retinal neovascularization in the advanced diabetes may have a pathological role for the induction of proliferative diabetic retinopathy.

In our previous paper, hydroxyl radical was found to be a major reactive oxygen species for N e -(carboxymethyl)lysine (CML) formation through Amadori products. Peroxynitrite (PN) is also known as one of the reactive oxygen species. However, the contribution of PN to the formation of advanced glycation end products (AGE) such as CML has been poorly understood so far. In this work, the effect of PN on CML formation was discussed. First, glycated human serum albumin (glycated HSA) was incubated with 70 μM PN at 37 °C for 1 h. Second, d -glucose was incubated with 1 mM PN at 37 °C for 30 min in advance, followed by further incubation in the presence of HSA at 37 °C for 24 h. The amount of CML in these samples was determined by ELISA. After incubation of glycated HSA with PN for 5 min, CML formation was observed to a significant extent. Furthermore, the reaction of PN-treated glucose with HSA resulted in the production of CML. In contrast, CML was not formed in the parallel incubation without PN. To determine aldehydes produced during PN-induced CML formation, aliquots of the reaction mixtures were mixed with 2,3-diaminonaphthalene (DAN) and then quantified by HPLC. This experiment demonstrated that glucosone is an important intermediate on the formation of CML.

Pre-incubation of cultured scavenger endothelial cells (SEC) with the scavenger receptor (SR) ligand advanced glycation end product (AGE)-modified bovine serum albumin (AGE-BSA) for 3 h reduced subsequent endocytosis of radioiodinated SR ligands AGE-BSA, formaldehyde-treated BSA (FSA) and 125I-oxidatively modified low-density lipoprotein (OxLDL) by 51%, 58% and 32%, respectively. Endocytosis of non-SR ligands, radioiodinated collagen, mannan or heat-aggregated γ-globulin was not affected by preexposure of SEC to AGE-BSA. Pre-incubation with FSA and other ligands different than AGE-BSA did not affect subsequent endocytosis of ligands for any receptor. Subcellular fractionation showed that pre-incubation with AGE-BSA delayed the transport of SR ligands along the endocytic pathway. Quantitative immuno-EM suggested that endocytosis of AGE-BSA in SEC lowered the amount of a newly described SR for AGE-BSA. Endocytic activity reduced by pre-incubation with AGE-BSA could be restored by incubating the cells for 6 h in AGE-free medium. Receptor restoration was inhibited by the protein synthesis inhibitor cycloheximide. We conclude from these findings that endocytosis of AGE-BSA leads to a loss of SR, and delayed intracellular transport in SEC. De novo synthesis of the receptor is required to restore normal endocytic activity.

Aims/hypothesis. We have previously reported that advanced glycation end products are eliminated from the circulation mainly by scavenger receptor-mediated uptake in hepatic sinusoidal endothelial cells. Our experiments showed that the degradation of AGE-modified protein after endocytosis in hepatic sinusoidal endothelial cells occurs slowly compared with that of other scavenger receptor ligands. The aim of this study was to investigate further the mechanism whereby AGE-modified protein affects the important scavenger function of hepatic sinusoidal endothelial cells. Methods. Primary cultures of hepatic sinusoidal endothelial cells were pre-incubated with unlabelled ligand, unbound ligand was washed off, and the endocytic capacity was measured by addition of radiolabelled ligand, and immune electron microscopy. Results. Pre-incubation with unlabelled AGE-modified bovine serum albumin reduced subsequent endocytosis of radiolabelled scavenger receptor ligands AGE-modified bovine serum albumin, formaldehyde-treated serum albumin, oxidized low density lipoprotein and acetylated low density lipoprotein by 50, 56, 32 and 20%, respectively. Non-scavenger receptor-mediated endocytosis was not affected by pre-exposure to AGE-modified protein. Pre-incubation with a number of non-AGE-ligands did not affect subsequent endocytosis via any of the major endocytosis receptors in hepatic sinusoidal endothelial cells. Incubation in fresh medium for 6 h after pre-exposure to AGE-modified protein almost completely restored normal scavenger receptor-mediated endocytic activity. Quantitative immune electron microscopy showed that the amount of a newly described scavenger receptor for AGE-modified protein is reduced after pre-incubation with AGE-modified protein. Subcellular fractionation showed that pre-incubation with AGE-modified protein delays intracellular transport of scavenger receptor ligands. Conclusion/interpretation. Endocytosis of AGE-modified protein leads to loss of scavenger receptors and delayed intracellular transport in hepatic sinusoidal endothelial cells.

Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO(-)) on the formation of N( epsilon )-(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO(-), CML formation was detected by both enzyme-linked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO(-) concentrations. CML was also formed when glucose, preincubated with ONOO(-), was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for alpha-oxoaldehydes. For identifying the aldehydes that contributed to ONOO(-)-induced CML formation, glucose was incubated with ONOO(-) in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO(-) can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive alpha-oxoaldehydes from glucose.

Previous studies from our laboratory demonstrated that N(epsilon)-(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGE), was accumulated in human pyramidal neurons in the hippocampus in an age-dependent manner. This suggests a potential link between AGE-accumulation and the aging process in neurons. The purpose of the present study was to examine whether this notion could be extended to other AGE structures, such as imidazolone and pentosidine. This was done using 19 human brains that were not affected by dementia. The immunohistochemical survey on distribution in brain tissues of imidazolone and pentosidine was carried out with monoclonal antibodies specific for imidazolone and pentosidine. A parallel control experiment was carried out with anti-CML antibody. The results showed that pentosidine and imidazolone were localized in neurons in different areas of human brain tissue, especially in neurons of CA4 in the hippocampus. The characteristic distribution of pentosidine and imidazolone is very similar to that of CML. Furthermore, when the accumulation of these AGE structures was compared with the age of individual brains it was found that accumulation of imidazolone, pentosidine and CML in the CA4 region increased with age. These findings taken together support the notion that the accumulation of AGE structures in the CA4 region might be closely related to the aging process in neurons.

It has been verified experimentally in JAERI-FEL that a high-gain FEL oscillator has the maximum extraction efficiency at the perfect synchronism of optical-cavity length. The simultaneous measurement of FEL efficiency and absolute cavity length has clearly shown that a sharp peak of detuning curve at the perfect synchronism appears in high-gain and low-loss regime and the FEL extraction efficiency exceeds the scaling law of short-bunch FEL oscillators. A numerical analysis indicates that lasing at the perfect synchronism is quasi-stationary superradiance with random fluctuations, which is analogous to a SASE FEL. Second-order autocorrelation measurements show that FEL pulses shorter than four optical cycles are generated successively for a number of round trips at the perfect synchronism, which is consistent with numerical results.

We made a systematic measurement of efficiency detuning curves at several gain and loss parameters. The absolute detuning length (δL) of an optical cavity was measured within an accuracy of 0.1 μm around the maximum efficiency by a pulse-stacking method using an external laser. The FEL gain was controlled by the undulator gap instead of bunch charge, because we can change the gain rapidly while maintaining constant electron bunch conditions. For the high-gain and low-loss regions, the maximum efficiency is obtained at δ L=0 μm and is larger than the value derived from the theoretical scaling law in the superradiant regime, while for the low-gain region the maximum efficiency is obtained for δL shorter than 0 μm and is similar to the scaling law.

We have been developing a fully automated Japanese directory assistance system using touch-tone telephones as input devices. This system is characterized by a newly developed input interface, called the degenerated keyword input method, which reduces the user's input workload. This input method associates one push on a button with one character of kana (Japanese syllabary). Since each touch-tone button on a telephone can represent several different kana characters, using a touch-tone numeral string as the search keyword inevitably leaves some ambiguity. This paper proposes a partial matching method, which is especially suitable for the fully automated Japanese directory assistance system. This method eliminates ambiguity in the degenerated keyword inputs and allows fast and highly accurate database searching. The method features the following. (1) It focuses on the PAT-tree in the field of full-text searching, and derives modified semi-infinite strings (sistrings) at a word level, rather than at a character level, in the PAT-tree for each record. (2) Word boundaries are marked in the index records, and only those candidates whose word boundary position coincides with the last character of the search keyword are selected as retrieval results. This use of word boundaries reduces retrieval noise. This paper also describes the results of evaluation experiments conducted to validate the proposed method. © 2002 Wiley Periodicals, Inc. Syst Comp Jpn, 33(6): 19–31, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/scj.1129

Recent studies have documented carbonyl stress involvement in the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS). The aim of the present study was to assess a role for carbonyl stress in motor neuron degeneration associated with superoxide dismutase-1 (SOD1) mutant familial ALS and its transgenic mouse model, using an immunohistochemical investigation of advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). In the spinal cords from six familial ALS patients with SOD1 A4V mutation and six transgenic mice expressing G93A mutant human SOD1, immunoreactivities for N(epsilon)-(carboxyethyl)lysine, argpyrimidine, pyrraline and N(epsilon)-(carboxymethyl)lysine as AGEs were distinct in almost all of the reactive astrocytes and obscure in the residual neurons, whereas no immunoreactivity for pentosidine as an AGE, or 4-hydroxy-2-nonenal-histidine, malondialdehyde-lysine or acrolein-lysine as ALEs was detectable. Spinal cords from age-matched control humans and mice exhibited no significant immunoreactivities for the examined products. Our results indicate that protein glycation, but not lipid peroxidation, is enhanced in ALS patients with an SOD1 mutation and mutant SOD1 transgenic mice, in which certain AGEs are selectively formed in the spinal cord astrocytes.

Interaction of advanced glycation endproducts (AGE) with AGE receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE receptors identified so far are receptor for AGE (RAGE), 80 K-H, OST-48, galectin-3, and macrophage scavenger receptor, types I and II (SR-A) [Eur. J. Biochem. 230 (1995) 408; Nature 386 (1997) 292.]. Since SR-A is known to belong to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to class B scavenger receptor family (SR-B) can recognize AGE proteins as a ligand. This was tested in the present study at the cellular level by using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-bovine serum albumin (BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36, but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized low-density lipoprotein (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major Ox-LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

We demonstrated the generation of a 500-keV electron beam from a high dc voltage photoemission gun for an energy recovery linac light source [N. Nishimori et al., Appl. Phys. Lett. 102, 234103 (2013)]. This demonstration was achieved by addressing two discharge problems that lead to vacuum breakdown in the dc gun. One is field emission generated from a central stem electrode. We employed a segmented insulator to protect the ceramic insulator surface from the field emission. The other is microdischarge at an anode electrode or a vacuum chamber, which is triggered by microparticle transfer or field emission from a cathode electrode. An experimental investigation revealed that a larger acceleration gap, optimized mainly to reduce the surface electric field of the anode electrode, suppresses the microdischarge events that accompany gas desorption. It was also found that nonevaporable getter pumps placed around the acceleration gap greatly help to suppress those microdischarge events. The applied voltage as a function of the total gas desorption is shown to be a good measure for finding the optimum dc gun configuration.

Background No predictor of postoperative ischemic events has been identified in patients undergoing carotid artery stenting (CAS). We aimed to determine whether Ne-(carboxymethyl)lysine (CML) in debris trapped by an embolic protection filter device is a predictor of postoperative ischemic events. Methods We enrolled 27 patients (73.4 ± 7.2 years; 22 male, 5 female) who underwent CAS for carotid artery stenosis. Diffusion-weighted magnetic resonance imaging was performed before and after the procedure. Protein samples were extracted from the debris. CML and myeloperoxidase were examined by solid phase enzyme-linked immunosorbent assay and Western blot analysis. Results Seventeen patients had 0 or 1 new lesion (nonmultiple lesions) postoperatively, whereas 10 patients had 2 or more new lesions postoperatively (multiple lesions). The CML concentration of the protein sample was significantly higher in patients with multiple lesions than in those with nonmultiple lesions (6.26 ± 2.77 ng/mg protein and 3.36 ± 1.57 ng/mg protein, respectively; P = .010). Statin therapy for dyslipidemia was associated with a lower incidence of multiple lesions and a lower concentration of CML in the protein sample (P = .004 and P = .02, respectively). Receiver operating characteristic analysis showed that the area under the curve for CML was significantly greater than .5 (.877; 95% confidence interval, .742-1.00). Conclusions CML derived from debris may distinguish between patients with postoperative multiple ischemic lesions and those with postoperative nonmultiple lesions who undergo CAS.

The Maillard reaction (also referred to as "glycation") takes place between reducing sugars and compounds with free amino groups during thermal processing of foods. In the final stage of the complex reaction cascade, the so-called advanced glycation end products (AGEs) are formed, including proteins with various glycation structures. It has been suggested that some AGEs could have immunostimulatory effects. Here, we aimed to identify specific glycation structure(s) that could influence the T-cell immunogenicity and potential allergenicity of food allergens, using ovalbumin (OVA, an egg white allergen) as a model allergen. OVA was specifically modified with representative glycation structures: N(ε)-carboxymethyl lysine (CM-OVA), N(ε)-carboxyethyl lysine (CE-OVA), pyrraline (Pyr-OVA), or methylglyoxal-derived arginine derivatives (MGO-OVA). As well as AGE-OVA, a crude glycation product in thermal incubation of OVA with glucose, only Pyr-OVA, and not other modified OVAs, was efficiently taken up by bone marrow-derived murine dendritic cells (BMDCs). The uptake of Pyr-OVA was reduced in scavenger receptor class A (SR-A)-deficient BMDCs, but not in cells treated with inhibitors of scavenger receptor class B, galectin-3, or blocking antibodies against CD36, suggesting that pyrraline binds to SR-A. Compared with other modified OVAs, Pyr-OVA induced higher activation of OVA-specific CD4(+) T-cells in co-culture with BMDCs. Furthermore, compared with native OVA, AGE-OVA and Pyr-OVA induced higher IgE production in mice. Pyrraline could induce better allergen uptake by DCs via association with SR-A and subsequently enhance CD4(+) T-cell activation and IgE production. Our findings help us to understand how Maillard reaction enhances the potential allergenicity of food allergens.

We have demonstrated FEL lasing at the third harmonic in JAERI-FEL driven by a superconducting linac. The lasing was achieved with a super enhanced gold-coated mirror tuned to 7 μm, which is a gold-coated copper mirror with Zn–Se multi-layer deposited on the surface. Lasing at the third harmonic is in the single-supermode regime with an average power of 15 W. The small-signal gain is estimated from the cavity-length detuning curve found to be 7.5%.

To elucidate on the mechanism of the formation of advanced glycation end product (AGE) structure during the incubation of protein with glucose, we analyzed N ⋅ -(carboxymethyl)lysine (CML) in AGE structure using the immunochemical procedure and amino acid analysis system detection methods. We found that CML is a major immunological epitope in proteins modified with AGE, and that the formation of CML from Amadori product was mediated by hydroxy radical generated by the reaction of Fe 2+ with H 2 O 2 . Further, the conversion of CML from Amadori product was enhanced in an alkaline condition.

Japan Atomic Energy Research Institute (JAERI)-FEL has achieved quasi-CW lasing with an average power of 1.7 kW, the initial goal of the R&D program. The FEL extraction efficiency obtained completely exceeds the wellknown limit for non-bunched beam, which is determined by the number of undulator periods. We have conducted numerical studies to characterize lasing dynamics observed at JAERI-FEL. Cavity-length detuning curves numerically obtained show good agreement with experimental results. Lasing behavior numerically obtained exhibits chaotic spiking-mode and superradiance as the cavity-length detuning approaches zero. Broadening of lasing spectrum observed in the experiments is explained by these lasing dynamics. The extraction efficiency becomes maximal at the perfect synchronization of the cavity length, where the lasing is quasi-stationary superradiance. We also compare these results with analytical theory previously reported. r 2001 Elsevier Science B.V. All rights reserved.

Long-term incubation of proteins with reducing sugar proceeds to advanced glycation end products (AGEs). We previously demonstrated the presence of AGE structures in human and animal tissues, and suggested the implication of AGEs in aging and diabetic complications. In this study, we tested whether AGEs present in vivo, such as N e -(carboxymethyl) lysine (CML) adduct, could serve as immunogens for generating autoantibodies. Plasma samples from STZ-induced diabetic rats reacted positively with AGE-proteins as well as CML-BSA, suggesting the presence of autoantibodies against AGE-structures, particularly CML in diabetic rats; the activity of the autoantibody increased with the duration of diabetic states, reflecting the accumulation of AGE proteins in these diabetic rats. An autoantibody fraction purified from the plasma of diabetic patients showed a positive reaction to AGE-proteins as well as to CML proteins, and also to human lens proteins which are known to undergo CML modification in vivo. Patients with renal failure caused by diabetes or nondiabetic pathologies had a higher autoantibody activity against AGE structures than that in normal subjects or diabetic patients without renal failure. These results suggest that AGEs accumulated in vivo serve as immunological epitopes for developing autoantibodies against AGEs, particularly against a CML structure, which might be used as an indicator of diabetic nephropathy or chronic renal failure.

A high power Free-Electron Laser (FEL) has lased at a wavelength of 22 μm at the Japan Atomic Energy Research Institute (JAERI). The maximum power on a macro-pulse average is 1.7 kW , and it corresponds to an FEL energy of 160 μJ / micro -pulse. Extraction efficiency from the electron beam to the FEL radiation was measured to be 5.3% by an energy analyzer, when the maximum FEL power was coupled out. The rms wavelength spread was measured to be 4.6% at the same time. The extraction efficiency, in general, has a maximum value near the zero detuning length of an optical cavity, where (in contrast) the single-pass gain becomes smallest. A high peak current and a long macro-pulse duration are therefore indispensable for realizing high efficiency. The electron beam energy is 16.5 MeV , and the average current is 5.3 mA at a micro-pulse repetition rate of 10.4 MHz . The macro-pulse duration is 500 μs (5000 micro-pulses), long enough to reach saturation near the zero detuning length. The width and the peak current of the electron bunch are 5 ps FWHM and 100 A , respectively, at the undulator.

For determining whether both the spinal cord motor neurons and glial cells are exposed to increased oxidative stress in amyotrophic lateral sclerosis (ALS), we performed an immunohistochemical investigation of end products of lipid peroxidation and protein glycoxidation in spinal cords from seven sporadic ALS patients and seven age-matched control individuals. In the ALS spinal cords, immunoreactivities for adducts of 4-hydroxy-2-nonenal-histidine and crotonaldehyde-lysine as markers of lipid peroxidation, N(epsilon)-(carboxymethyl)lysine as a marker of lipid peroxidation or protein glycoxidation, and pentosidine as a marker of protein glycoxidation were localized in the gray matter neuropil and almost all of the motor neurons, reactive astrocytes and microglia/macrophages, whereas none of the immunoreactivities for N(epsilon)-(carboxyethyl)lysine or argpyrimidine as markers of protein glycoxidation or enzymatic glycolysis, or pyrraline or imidazolone as markers of nonoxidative protein glycation were detectable. The control spinal cords displayed no significant immunoreactivities for any of these examined products. Our results indicate that in sporadic ALS, both lipid peroxidation and protein glycoxidation are enhanced in the spinal cord motor neurons and glial cells, and suggest that the formation of certain products in these abnormal reactions is implicated in motor neuron degeneration.

We used immunohistochemical methods and four monoclonal antibodies for specific molecular structures of advanced glycation end products (AGE)-6D12, KNH-30, 1F6, and 2A2-to examine localization of AGE in fetal, young, and adult rats, and rats with streptozotocin-induced diabetes. 6D12 recognized N(epsilon)-(carboxymethyl)lysine (CML); KNH-30, N(epsilon)-(carboxyethyl)lysine (CEL); and 1F6, fluorolink. The epitope of 2A2 is as yet unknown. Immunoreactivities for these monoclonal antibodies were found in various organs and tissues in postnatal and adult rats, and accumulation increased with aging. In the fetuses, AGE structures were detected at 10 fetal days, and their accumulation increased during ontogeny. Reversed-phase high-performance liquid chromatography revealed CML in fetuses at 13 fetal days and in lungs of 28-week-old rats. In various organs and tissues of fetal, young, and adult rats, CML, CEL, 2A2-positive AGE, and fluorolink accumulated, in that order, which suggests that the accumulation of CML, a nonfluorescent/noncross-linked AGE, occurs earlier than accumulation of fluorolink, a fluorescent/cross-linked AGE. In diabetic rats, hepatocytes, splenic macrophages, renal glomerular endothelial and mesangial cells, testicular Leydig cells, and erythrocytes showed excessive accumulation of AGE, leading to the pathologic changes characteristic of diabetes mellitus. For the induction of these changes, persistent hyperglycemia and hyperketonemia might be important for acceleration of intracellular AGE accumulation in diabetic rats. Thus, AGE accumulation in tissues and cells occurs not only during aging and in diabetes mellitus but also from an early stage of ontogeny.

Cellular interactions of advanced glycation end products (AGE) are mediated by AGE receptors. We demonstrated previously that class A scavenger receptor types I and II (SR-A) and CD36, a member of class B scavenger receptor family, serve as the AGE receptors. In this study, we investigated whether scavenger receptor class B type I (SR-BI), another receptor belonging to class B scavenger receptor family, was also an AGE receptor. We used Chinese hamster ovary (CHO) cells overexpressed hamster SR-BI (CHO-SR-BI cells). (125)I-AGE-bovine serum albumin (AGE-BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-SR-BI cells. (125)I-AGE-BSA exhibited saturable binding to CHO-SR-BI cells (K(d) = 8.3 microg/ml). Endocytic uptake of (125)I-AGE-BSA by CHO-SR-BI cells was completely inhibited by oxidized low density lipoprotein (LDL) and acetylated LDL, whereas LDL exerted only a weak inhibitory effect (20%). Cross-competition experiments showed that AGE-BSA had no effect on HDL binding to these cells and vice versa. Interestingly, however, SR-BI-mediated selective uptake of HDL-CE was completely inhibited by AGE-BSA in a dose-dependent manner (IC(50)10 microg/ml). Furthermore, AGE-BSA partially inhibited (by30%) the selective uptake of HDL-CE in human hepatocarcinoma HepG2 cells (IC(50)30 microg/ml). In addition, [(3)H]cholesterol efflux from CHO-SR-BI cells to HDL was significantly inhibited by AGE-BSA in a dose-dependent manner (IC(50)30 microg/ml). Our results indicate that AGE proteins, as ligands for SR-BI, effectively inhibit both SR-BI-mediated selective uptake of HDL-CE and cholesterol efflux from peripheral cells to HDL, suggesting that AGE proteins might modulate SR-BI-mediated cholesterol metabolism in vivo.

Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) are morphological hallmarks of certain familial amyotrophic lateral sclerosis (FALS) patients with superoxide dismutase-1 (SOD1) gene mutations, and transgenic mice expressing the human SOD1 gene mutation. The ultrastructure of inclusions in both diseases is identical: the essential common constituents are granule-coated fibrils approximately 15-25nm in diameter and granular materials. Detailed immunohistochemical analyses have shown that the essential common protein of the inclusions in both diseases is an SOD1 protein. This finding, together with the immunoelectron microscopy finding that the abnormal granule-coated fibrils comprising the inclusions are positive for SOD1, indicates that these granule-coated fibrils containing SOD1 are important evidence for mutant SOD1-linked disease in human and mouse. For immunoelectron microscopy, the granule-coated fibrils are modified by advanced glycation endproducts (AGE) such as N(epsilon)-carboxymethyl lysine, pyrraline and pentosidine (Maillard reaction). Based on the fact that AGE themselves are insoluble molecules with direct cytotoxic effects, the granule-coated fibrils and granular materials are not digested by the lysosomal and ubiquitin systems. The neurons and astrocytes of the normal individuals and non-transgenic mice show no significant immunoreactivity for AGE. Considered with the mutant-SOD1 aggregation toxicity, a portion of the SOD1 comprising both types of the inclusion is modified by the AGE, and the formation of the AGE-modified SOD1 (probably AGE-modified mutant SOD1) is one of the mechanisms responsible for the aggregation (i.e. granule-coated fibril formation).

An optical resonator with a high-efficient output coupler was developed for the JAERI far-infrared free- electron laser. The optical resonator is symmetrical near-concentric geometry with an insertable scraper output coupler. As a result of the development of the optical resonator, the JAERI-FEL has been successfully lased with averaged power over 1kW. Performance of the optical resonator with the output coupler was evaluated at optical wavelength of 22 μm by using an optical mode calculation code. The output coupling and diffractive loss with a dominant eigen-mode of the resonator were calculated using an iterative computation called Fox-Li procedure. An efficiency factor of the optical resonator was introduced for the evaluation of the optical resonator performance. The efficiency factor was derived by the amount of the output coupling and diffractive loss of the optical resonator. It was found that the optical resonator with the insertable scraper coupler was the most suitable to a high-power and high-...

Carboxymethyllysine (CML) is currently recognized as a major advanced glycation end product and a marker for glycoxidation. Plasma CML levels are increased in patients with chronic renal failure (CRF). However, significance and mechanism of CML accumulation in these patients are poorly understood. The objective of the present study was to analyze CML in soluble and collagen-binding fractions of the dermis to investigate CML deposition and formation and collagen damage related to CML accumulation in patients with CRF. Skin samples (among them autopsy samples) were obtained from 33 subjects: 8 nondiabetic CRF patients, 7 diabetic predialysis patients with CRF (CRF-DM), 7 hemodialysis patients, and 11 control subjects without either CRF or DM. The dermal samples were extracted sequentially by phosphate-buffered normal saline, pepsin, and collagenase. The extracts were referred to as the soluble fraction and the proteinase-extracted fraction (including pepsin-extracted and collagenase-extracted fractions). Our ELISA assay for CML in dermal collagen from predialysis patients with CRF (CRF and CRF-DM groups) demonstrated that the levels of CML in both the soluble fraction (containing soluble CML which was mainly determined by serum clearance) and the structural collagen-binding proteinase-extracted fraction (in which high CML levels could be a strong indication of in situ formation) were increased and could not be completely reduced after hemodialysis in CRF-DM and CRF groups. These results suggest that accumulation of CML may be due to both a low serum clearance and/or increased in situ CML formation in CRF. CML contents in the proteinase extracted fraction inversely correlated with the susceptibility of collagen to extraction by proteinases (n = 33, r = –0.59, p < 0.001). Our results provide the first biochemical evidence that CML level is increased in both the soluble and collagen-binding fractions and that increased CML level resulted in increased fractions of proteinase-resistant collagen in dermal extracts of patients with CRF.

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 Nepsilon-(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.

Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGEs) that are recognized by AGE receptors. Glyoxal, glycolaldehyde (GA), and methylglyoxal are potential intermediates for the formation of AGE structures such as Nomega-(carboxymethyl)lysine (CML). We evaluated the contribution of these aldehydes to the formation of AGE structure(s), particularly the structure important for the receptor-mediated endocytic uptake of AGE proteins by macrophages. GA-modified bovine serum albumin (BSA), methylglyoxal-modified BSA (MG-BSA), and glyoxal-modified BSA (GO-BSA) were prepared, and their physicochemical, immunological, and biologic properties were compared with those of glucose-derived AGE-BSA. CML contents were high in GO-BSA and low in GA-modified BSA (GA-BSA) but did not exist in MG-BSA. The fluorescence patterns of GA-BSA and MG-BSA were similar to those of glucose-derived AGE-BSA but were weak in GO-BSA. Immunochemically, the antibody against non-CML structures of glucose-derived AGE-BSA reacted strongly with GA-BSA and weakly with GO-BSA but did not react with MG-BSA. The negative charge of these ligands increased to a similar extent. However, GA-BSA, but not MG-BSA or GO-BSA, underwent receptor-mediated endocytosis by the macrophage-derived cell line RAW 264.7, which was effectively inhibited by glucose-derived AGE-BSA, acetylated LDL, and oxidized LDL, which are well-known ligands for the macrophage type I and type II class A scavenger receptors (MSR-A). The endocytic uptake of GA-BSA by mouse peritoneal macrophages was also significant, but that by peritoneal macrophages from MSR-A-deficient mice was markedly reduced. Our results suggest that GA serves as an important intermediate for the generation of AGE structure(s) responsible for recognition by MSR-A.

Although there have been suggestions that the glycation and oxidation of low density lipoprotein (LDL) might increase its atherogenic potential, little is known about the presence of glycoxidative LDL in human atherosclerotic lesions. We developed specific antibodies against different immunological epitopes of AGE structures, including N o -(carboxymethyl)lysine-protein adduct (CML), a glycoxidation product, and structure(s) other than CML (nonCML), and a monoclonal antibody against oxidized phosphatidylcholine (oxPC), as an epitope of oxidized LDL. Immunohistochemical analysis demonstrated that the CML- and oxPC-epitopes were accumulated mainly in macrophage-derived foam cells in atherosclerotic lesions, including fatty streaks and atherosclerotic plaques. On the other hand, the nonCML-epitope and apolipoprotein B were localized mainly in extracellular matrices of atherosclerotic lesions. The CML- and oxPC-epitopes were characterized by a model antigen-generating system using the copper ion-induced peroxidation and:or glucose-induced glycation of LDL. The glycoxidation of LDL caused the formation of CML-epitope with increasing concentrations of copper ion and glucose. It was also formed to some extent in LDL incubated with high concentrations (500 mM) of glucose. However, no CML-epitope was observed in oxidized LDL induced by copper ion alone. On the other hand, the formation of oxPC-epitope in LDL was dependent on copper ion-induced peroxidation, but independent of glucose-induced glycation. The addition of chelators, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, reduced the increase in electrophoretic mobility and TBARS caused by the peroxidation and glycoxidation of LDL, but had no effects on the formation of fructosamine caused by the glycation and glycoxidation of LDL. Chelators as well as aminoguanidine protected the formation of CML-epitope in glycated or glycoxidative LDL. Although the formation of oxPC-epitope was completely inhibited by the addition of chelators, it was partially protected by aminoguanidine. These in vitro results suggest that the glycoxidative modification of LDL may occur in the arterial intima, and may contribute to the development of human atherosclerotic lesions. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

A high-average power free-electron laser driven by a superconducting linac has been developed in Japan Atomic Energy Research Institute (JAERI), and stable laser output over 0.1 kW in infrared region is now available. For further increasing of FEL output power, installing energy-recovery transport has been planned. The lattice design for the energy-recovery transport is discussed in the present paper. It is found that a recirculation transport, which fulfills the requirements for energy acceptance and isochronicity, can be realized by adding another triple-bend arc to the existing beam line.