Increased toll-like receptor 2 expression in peptidoglycan-treated blood monocytes is associated with insulin resistance in patients with nondiabetic rheumatoid arthritis.

The close relationship between increased TLR-2 expression in blood monocytes and insulin resistance in RA patients is shown in this study. Traditional risk factors for metabolic disorders, including the waist circumstance, body mass index (BMI), triglyceride (TG), and ratio of TG to high density lipoprotein (HDL) cholesterol, were closely correlated with HOMA (homoeostasis model assessment) index in patients with nondiabetic RA. Expressions of TLR2 in peripheral blood monocytes, following stimulation with peptidoglycan which is known as a TLR2 agonist, were closely correlated with the HOMA index, TNF-α, and IL-6 concentrations. Accordingly, TLR-2 receptor and its related inflammatory cytokines could be potential therapeutic targets in managing insulin resistance in RA patients.

Glycine N-methyltransferase affects the metabolism of aflatoxin B1 and blocks its carcinogenic effect.

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B(1) (AFB(1)) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB(1) treatment. Competitive enzyme immunoassays indicated that after AFB(1) treatment, the AFB(1)-DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB(1)-DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB(1) in the cultural supernatants of cells exposed to AFB(1) showed that the AFM(1) level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB(1). Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB(1). Automated docking experiments showed that AFB(1) binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT-AFB(1) interaction is 44.9 microM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB(1), resulting in reduced AFB(1)-DNA adducts formation and cell death.

Characterization of reduced expression of glycine N-methyltransferase in cancerous hepatic tissues using two newly developed monoclonal antibodies.

Glycine N-methyltransferase (GNMT) is a protein with multiple functions. Recently, two Italian siblings who had hepatomegaly and chronic elevation of serum transaminases were diagnosed to have GNMT deficiency caused by inherited compound heterozygosity of the GNMT gene with missence mutations. To evaluate the expression of GNMT in cell lines and tissues from hepatocellular carcinoma (HCC) patients, we produced two monoclonal antibodies (mAbs) 4-17 and 14-1 using two recombinant GNMT fusion proteins. M13 phage peptide display showed that the reactive epitopes of mAbs 4-17 and 14-1 were amino acid residues 11-15 and 272-276 of human GNMT, respectively. The dissociation constants of the binding between GNMT and mAbs were 1.7 x 10(-8) M for mAb 4-17 and 1.8 x 10(-9) M for mAb 14-1. Both mAbs can identify GNMT present in normal human and mouse liver tissues using Western blotting (WB) and immunohistochemical staining assay (IHC). In addition, WB with both mAbs showed that none of 2 hepatoblastoma and 5 HCC cell lines expressed GNMT. IHC demonstrated that 50% (13/26) of nontumorous liver tissues and 96% (24/25) of HCC tissues did not express GNMT. Therefore, the expression of GNMT was downregulated in human HCC.