Immuno-chemistry of hydroxyl radical modified GAD-65: A possible role in experimental and human diabetes mellitus.

The repertoire of known auto-antigens is limited to a very small proportion of all human proteins, and the reason why only some proteins become auto-antigens is unclear. The 65 kDa isoform of the enzyme glutamic acid decarboxylase (GAD-65) is a major auto-antigen in type I diabetes, and in various neurological diseases. Most patients with type I diabetes (70-80%) have auto-antibodies against GAD-65, which often appear years before clinical onset of the autoimmune diabetes. Thus, the aim of the study is to focus on the immunogenicity of GAD65 and its reactive oxygen species (ROS) conformer in STZ-induced diabetic rats and on human diabetic patients. In the present study, GAD-65 was modified by hydroxyl radical following Fenton's reaction. The modifications in the structure of the GAD-65 are supported by UV-vis and fluorescence spectral studies. Immunogenicity of both native and hydroxyl radical modified GAD-65 (ROS-GAD-65) was studied in experimental rabbits and was confirmed by inducing type I diabetes in experimental male albino rats using streptozotocin (45 mg/kg). We found that ROS-GAD-65 was a better immunogen as compared to the native GAD-65. A considerable high binding to ROS-GAD-65 was observed as compared to native GAD-65 in both the serum antibodies from diabetes animal models and as well as in the serum samples of type I diabetes. Hydrogen peroxide under the exposure of UV light produces hydroxyl radical (·OH) which is most potent oxidant, and could cause protein damage (GAD-65) to the extent of generating neo-epitopes on the molecule, thus making it immunogenic.

Amadori glycated proteins: role in production of autoantibodies in diabetes mellitus and effect of inhibitors on non-enzymatic glycation.

Nonenzymatic glycation of macromolecules, especially proteins leading to their oxidation is increased in diabetes mellitus due to hyperglycemia and play an important role in associated complications of the disease. The glycation primarily occurs at intrachain lysine residues of proteins and results in the formation of an early stage stable product as Amadori-lysine which undergo further irreversible chemical reactions to form advanced glycation endproducts. This review deals with the role of Amadori modified proteins in pathogenesis of diabetes. We aim to explain immunogenicity of Amadori-glycated proteins, which might be involve in production of serum autoantibodies in the diabetic patients, and effect of inhibitors on the glycation process.

Biochemical Studies on Methylglyoxal-Mediated Glycated Histones: Implications for Presence of Serum Antibodies against the Glycated Histones in Patients with Type 1 Diabetes Mellitus.

Reactive carbonyl species (RCS) mainly reacts with lysine and arginine residues of proteins to form advanced glycation end products (AGEs). Histone was glycoxidated with glyoxal and methylglyoxal. It was characterized by polyacrylamide gel electrophoresis and quenching studies involving penicillamine and aminoguanidine as carbonyl scavengers. Further characterization of histone modified with methylglyoxal was done by UV, fluorescence, and IR spectrophotometry. Spectral analysis of the protein clearly demonstrates structural perturbation in the histone by methylglyoxal. Methylglyoxal-induces cross-linking in the protein leading to aggregation. Role of methylglyoxal mediated glycoxidation of histone in type 1 diabetes was also undertaken. Antibodies were detected against glycoxidated histone in sera of type 1 diabetes patients by solid-phase enzyme immunoassay. The findings indicate that as a result of structural perturbation in histone by methylglyoxal, the modified histone may be involved in production of serum antibodies in the diabetes patients.

Detecting phenotype-specific interactions between biological processes from microarray data and annotations.

High throughput technologies enable researchers to measure expression levels on a genomic scale. However, the correct and efficient biological interpretation of such voluminous data remains a challenging problem. Many tools have been developed for the analysis of GO terms that are over- or under-represented in a list of differentially expressed genes. However, a previously unexplored aspect is the identification of changes in the way various biological processes interact in a given condition with respect to a reference. Here, we present a novel approach that aims at identifying such interactions between biological processes that are significantly different in a given phenotype with respect to normal. The proposed technique uses vector-space representation, SVD-based dimensionality reduction, differential weighting, and bootstrapping to asses the significance of the interactions under the multiple and complex dependencies expected between the biological processes. We illustrate our approach on two real data sets involving breast and lung cancer. More than 88 percent of the interactions found by our approach were deemed to be correct by an extensive manual review of literature. An interesting subset of such interactions is discussed in detail and shown to have the potential to open new avenues for research in lung and breast cancer.

Glycated lysine residues: a marker for non-enzymatic protein glycation in age-related diseases.

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

Preferential recognition of Amadori-rich lysine residues by serum antibodies in diabetes mellitus: role of protein glycation in the disease process.

This study analyzes effect of glycation on proteins rich in lysine residues as hyperglycemia induced protein glycation has been mainly reported in diabetes mellitus at the intrachain lysine residues leading to the formation of Amadori modified proteins. We have studied the effect of glucose on poly-l-lysine (PLL), a homopolymer of lysine residues. Levels of Amadori products in the glycated PLL were evaluated by fructosamine assay and the presence of 5-hydroxymethylfurfural (HMF) in the glycated PLL was analyzed by thiobarbituric acid assay. Fluorescence and FT-IR spectroscopy were applied to characterize the modified PLL. Binding characteristics of experimentally induced antibodies against glycated PLL and the presence of antibodies against glycated PLL in the sera of diabetes patients was evaluated by solid phase enzyme immunoassays. The fructosamine assay showed significantly high yield of early glycation (Amadori) products in the glycated PLL, which was confirmed by increased yield of HMF from Amadori products of glycated PLL. Loss in fluorescence intensity and appearance of a new band corresponding to Amadori products were observed in FT-IR spectrum of the glycated PLL. Glycated PLL was found to be highly immunogenic in rabbits as compared to the native form. Serum antibodies from diabetes patients showed appreciably high recognition of the glycated PLL. The results conclusively show the glycation induced damage to the lysine molecules and specific recognition of Amadori-lysine residues by serum antibodies from diabetes patients. The glycated lysine residues may serve as a diagnostic biomarker for early glycation process in diabetes mellitus.

Immunoglobulin heavy and light chain isotypes in multiple myeloma patients.

The frequency of expression of immunoglobulin (Ig) light and heavy chain isotypes was analyzed in myeloma proteins (M-proteins) from sera of 40 Indian patients with clinically established multiple myeloma. Patients samples were screened by a combination of electrophoresis, immunoelectrophoresis (IEP) and ELISA techniques in this study. We found that majority of the myeloma proteins (58%) were of the IgG isotype followed by IgA (24%) and biclonal gammopathy associated with IgG and IgA (5%). Both kappa and lambda light chains were associated with the heavy chain isotypes. We recommend the triangular combination for detection of M-proteins and biclonal gammopathy of cancerous plasma cells as biomarkers for diagnosis of myeloma.