Jun Usami

Imidazolium crosslinks derived from reaction of lysine with glyoxal and methylglyoxal are increased in serum proteins of uremic patients: evidence for increased oxidative stress in uremia

Glyoxal (GO) and methylglyoxal (MGO) are reactive dicarbonyl compounds formed during autoxidation of both carbohydrates and lipids. They may react with lysine and arginine residues of proteins in Maillard or browning reactions, yielding advanced glycation or lipoxidation end products. Among these are the imidazolium crosslinks, N,N(‐di(N ϵ‐lysino))imidazolium (glyoxal‐lysine dimer, GOLD) and N,N(‐di(N ϵ‐lysino))‐4‐methyl‐imidazolium (methylglyoxal‐lysine dimer, MOLD). We have detected and measured GOLD and MOLD in human serum by electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS), using 15N4‐GOLD and 15N4‐MOLD as internal standards. In this report we show that levels of GOLD and MOLD are significantly elevated (3–4‐fold, P<0.01) in sera of non‐diabetic uremic patients, compared to age‐matched controls, and represent a major class of non‐enzymatic, Maillard reaction crosslinks in plasma proteins. These results provide strong evidence for increased non‐enzymatic crosslinking of tissue proteins by GO and MGO in uremia, implicating oxidative stress and resultant advanced glycation and lipoxidation reactions in tissue damage in uremia.

Mass spectrometric study on the protein chemical modification of uremic patients in advanced Maillard reaction

The Maillard reaction, initiated by the nonenzymatic reaction of reducing sugar with protein, is proposed to play a significant role in protein aging and the complications of aging and diabetes. In this study, we detected and quantified some advanced glycation endproducts (AGEs) in human serum proteins of control and uremic patients by a highly selective and specific assay, electrospray ionization liquid chromatography-mass spectrometry-mass spectrometry (ESI-LC-MS-MS). From our results, levels of each AGEs in serum of uremic patients were significantly elevated, compared to age-matched controls. These results provide the evidence for increased modifications of proteins by Maillard reaction in uremia.

Accelerated Progression of a Murine Retrovirus‐Induced Immunodeficiency Syndrome In Fas Mutant C57BL/6/lpr/lpr Mice

We reported previously that CD4+ T cells and B cells in mice with retrovirus‐induced murine acquired immunodeficiency syndrome (MAIDS) caused by LP‐BM5 murine leukemia virus (MuLV) mixtures increased the expression of Fas antigen (Fas) during progression of the disease. However, the contribution of the Fas/Fas ligand (Fas L) system to the pathogenesis of MAIDS remained unknown. Here, we examined the susceptibility of C57BL/6 (B6) lpr/lpr mice, which has been reported to be defective for the expression of Fas, to MAIDS. We found that the Thy 1.2− CD4 T cells and IgK dull B220+ cells, which are characteristic of MAIDS, increased after the inoculation of LP‐BM5 MuLV in B6 lpr/lpr mice. B22+ TCR αβ T cells, unique to lupus prone mice, also increased in the B6 lpr/lpr mice after infection. CD4+ B220+ TCR αβ T cells increased profoundly among the B220+ TCR αβ T cells from LP‐BM5 MuLV‐infected B6 lpr/lpr mice, while the B220+ TCR αβ T cells observed in non‐infected B6 lpr/lpr mice were largely of the CD4− CD8− phenotype. A DNA PCR analysis of the LP‐BM5 MuLV‐infected B6 lpr/lpr mice revealed the genome integration of defective LP‐BM5 virus, further confirming that MAIDS is inducible to B6 lpr/lpr mice. LP‐BM5 MuLV‐infected lpr/lpr mice died within 3 months, while MAIDS‐infected B6 +/+ mice usually died within 5 to 6 months, and B6 lpr/lpr mice not infected with LP‐BM5 MuLV lived more than 6 months. Taken together, these results suggest that MAIDS is inducible independently with functional Fas expression and the possibility of accelerated progression of murine AIDS and lpr‐associated autoimmune disease in B6 lpr/lpr mice infected with LP‐BM5 MuLV.

Involvement of oxidative stress in the accelerated formation of pentosidine in patients with end‐stage renal failure

SUMMARY: Advanced glycation end products (AGEs) have been found to accumulate in the amyloid deposits, skin and plasma of haemodialysis patients (HD), implicating the possible involvement of AGE‐modified protein in pathogenesis in dialysis‐related amyloidosis. Pentosidine, an AGE cross‐link, is a specific marker for AGEs. Plasma pentosidine levels in HD patients were increased dramatically. In the present study, plasma pentosidine, fructoselysine, advanced oxidation protein products (AOPP) and glutathione peroxidase (GSHPx) levels were measured to elucidate the role of oxidative stress in pentosidine formation in nondiabetic HD patients. Plasma pentosidine did not correlate with fructoselysine; plasma AOPP levels were significantly higher than those in normal subjects (201.45 ± 57.93 vs. 55.91 ± 6.57 μmol/L, P<0.001) and correlated positively with plasma pentosidine in HD patients (r=0.52, P<0.005); plasma GSHPx levels were significantly lower than those in normal subjects (168.40 ± 65.08 vs. 348.87 ± 86.10 U/I, P<0.001) and correlated negatively with plasma pentosidine (r=0.54, P<0.001) in HD patients. Decreased GSHPx levels may lead to the accumulation of hydrogen peroxide. These findings implicate the involvement of oxidative stress in the accelerated formation of pentosidine in uraemia and suggest that pentosidine could be considered as an oxidative stress biomarker to estimate the degree of oxidative‐stress‐mediated protein damage.

Th0-like CD4+ T cells protect mice with murine retrovirus-induced immunodeficiency syndrome (MAIDS) against co-infection with Listeria monocytogenes.

We examined the host defence mechanism against infection with Listeria monocytogenes, a facultative intracellular bacterium, in mice with murine acquired immunodeficiency syndrome (MAIDS) caused by LP‐BM5 murine leukaemia virus (MuLv) infection. Although LP‐BM5 MuLV infection in C57BL/6 mice leads to a stage of immunodeficiency characterized by severe compromise of cell‐mediated immunity, the mice with established MAIDS infected with LP‐BM5 8 weeks previously, showed resistance to an intraperitoneal infection with Listeria monocytogenes. These MAIDS mice also showed resistance to a lethal dose of secondary listerial challenge, while the delayed‐type hypersensitivity response to heat‐killed Listeria (HKL) was severely impaired in MAIDS mice. The resistance of MAIDS mice to listerial infection was mediated by CD4+αβ T cells but neither by γδ T cells nor natural killer (NK) cells. Interferon‐γ (IFN‐γ) and interleukin‐10 (IL‐10) were produced by CD4+ T cells from Listeria‐infected MAIDS mice in response to the in vitro stimulation with HKL, whereas IFN‐γ but not IL‐10 were produced by those from Listeria‐infected control mice. These results suggest that T‐helper 0 (Th0)‐like immune responses of CD4+ T cells occur and participate in host defence mechanisms against listerial infection in MAIDS mice.