An inhibitory substance of glyceraldehyde 3-phosphate dehydrogenase in urine of diabetic patients

In urine of diabetics, a significantly great inhibitory activity of glyceraldehyde 3-phosphate dehydrogenase (GAP-Dehyd) was observed compared with that of normal subjects. The 0.2 ml of urine from 41 patients with diabetes mellitus inhibited 0.5 units of GAP-Dehyd by 27.2 +/- 3.0% (mean +/- S.E.M.), while that from 17 normal volunteers inhibited only by 9.0 +/- 1.0% (P less than 0.05). This inhibitory substance was extracted by 90% ethanol from diabetic urine and partially purified by anion exchange chromatography using Dowex-1 (HCOO type). The molecular weight of this substance was confirmed to be 100--300 daltons by an analysis on Biogel P-4 gel filtration chromatography. And analysis by thin layer chromatography using silicagel plate showed that this inhibitor was a ninhydrin reactive substance which has not been reported previously. From the above facts, it was assumed that the inhibitory substance of GAP-Dehyd in urine of diabetics was a new acidic compound of low molecular weight containing an amino residue in the molecule.</p

Slipping through the Cracks: Linking Low Immune Function and Intestinal Bacterial Imbalance to the Etiology of Rheumatoid Arthritis

Autoimmune diseases (ADs) are considered to be caused by the host immune system which attacks and destroys its own tissue by mistake. A widely accepted hypothesis to explain the pathogenic mechanism of ADs is “molecular mimicry,” which states that antibodies against an infectious agent cross-react with a self-antigen sharing an identical or similar antigenic epitope. However, this hypothesis was most likely established based on misleading antibody assay data largely influenced by intense false positive reactions involved in immunoassay systems. Thus reinvestigation of this hypothesis using an appropriate blocking agent capable of eliminating all types of nonspecific reactions and proper assay design is strongly encouraged. In this review, we discuss the possibility that low immune function may be the fundamental, common defect in ADs, which increases the susceptibility to potential disease causative pathogens located in the gastrointestinal tract (GI), such as bacteria and their components or dietary components. In addition to these exogenous agents, aberrations in the host’s physical condition may disrupt the host defense system, which is tightly orchestrated by “immune function,” “mucosal barrier function,” and “intestinal bacterial balance.” These disturbances may initiate a downward spiral, which can lead to chronic health problems that will evolve to an autoimmune disorder

A new rapid assay method of collagenase activity and its clinical applications Part 2. Determination of collagenase activity in human leucocytes and synovial fluids by the newly developed method

A newly developed collagenase assay method using (14)C-labeled soluble collagen as substrate has been applied to the determination of collagenase activity in human leucocytes and synovial fluids. The results indicated that collagenase activity in the both samples could be measured by this method as observed with tadpole collagenase, except that 35 % dioxane was preferable for the extraction of the enzyme digestion products in the case of synovial fluids. Collagenase activity determined by the present method was well correlated with that obtained by the conventional assay method using reconstituted collagen fiblils as substrate (r=0.963 and 0.829 (p<0.01) respectively). In preliminary experiments with leucocytes from patients with diabetes mellitus (DM), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), it was observed that collagenase activity in leucocytes increased significantly in SLE and from some patients with RA, while no appreciable differences were observed in diabetics, compared to that in normals. In synovial fluids from patients with RA and osteoarthritis (OA), the accumulation of large amount of inactive (latent) form of collagenase was demonstrated by the treatment of synovial fluids with 3M KI. And it was varied significantly in one patient at different time while no appreciable amount of the active form was observed during the period

A new rapid assay method of collagenase activity and its clinical applications Part 1. A rapid assay method of collagenase activity using (14)C-labeled soluble collagen as substrate

A rapid assay method of collagenase activity has been developed using (14)C-labeled soluble collagen as substrate. This method is based on following three parts; first, carrying out the enzyme reaction with (14)C-labeled collagen in solution containing 0.25M glucose to prevent collagen fiblil formation at 35℃ under neutral condition, second, denaturation of the enzyme digestion products at 35℃ for 1hr after stopping the reaction by adding o-phenanthroline and third, selective extraction of the denatured (14)C-products into dioxane at a final concentration of 50 % and counting (14)C-activity in the surpernatant. The rate of the reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fiblils as substrate and relationship between enzyme activity and enzyme concentration was linear over a wider range regardless of the purity of collagenase

A new rapid assay method of collagenase activity and its clinical applications Part 1. A rapid assay method of collagenase activity using (14)C-labeled soluble collagen as substrate

A rapid assay method of collagenase activity has been developed using (14)C-labeled soluble collagen as substrate. This method is based on following three parts; first, carrying out the enzyme reaction with (14)C-labeled collagen in solution containing 0.25M glucose to prevent collagen fiblil formation at 35℃ under neutral condition, second, denaturation of the enzyme digestion products at 35℃ for 1hr after stopping the reaction by adding o-phenanthroline and third, selective extraction of the denatured (14)C-products into dioxane at a final concentration of 50 % and counting (14)C-activity in the surpernatant. The rate of the reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fiblils as substrate and relationship between enzyme activity and enzyme concentration was linear over a wider range regardless of the purity of collagenase

An ELISA protocol to improve the accuracy and reliability of serological antibody assays

To assay serum antibodies by indirect ELISA, it is critical to eliminate a variety of false positive and negative reactions attributed to the principle. These include 1) the background (BG) noise reaction caused by hydrophobic binding of immunoglobulin components in sample specimens to solid surfaces, 2) false positive reaction caused by non-specific binding of immunoglobulins to target-antigens by protein-protein interactions, and 3) other false positive and negative reactions caused by buffer components. No current blocking agents can prevent these false positive and negative reactions, and antibody assay results vary significantly depending on the buffer system used. To address these fundamental problems, we investigated all types of non-specific reactions involved in indirect ELISAs, and the blocking efficacy of current buffer systems and a newly developed ELISA buffer, ChonBlock™. The accuracy and reliability of these assay results were examined in detail by inhibition tests in individual buffer systems. Based on these studies, we are providing a definitive ELISA protocol for all users to improve ELISA technique and obtain accurate, reliable, and reproducible assay data against a variety of antigens

New Studies of Pathogenesis of Rheumatoid Arthritis with Collagen-Induced and Collagen Antibody-Induced Arthritis Models: New Insight Involving Bacteria Flora

Much public research suggests that autoimmune diseases such as rheumatoid arthritis (RA) are induced by aberrant “self” immune responses attacking autologous tissues and organ components. However, recent studies have reported that autoimmune diseases may be triggered by dysbiotic composition changes of the intestinal bacteria and an imbalance between these bacteria and intestinal immune systems. However, there are a few solid concepts or methods to study the putative involvement and relationship of these inner environmental factors in RA pathogenesis. Fortunately, Collagen-Induced Arthritis (CIA) and Collagen Antibody-Induced Arthritis (CAIA) models have been widely used as animal models for studying the pathogenesis of RA. In addition to RA, these models can be extensively used as animal models for studying complicated hypotheses in many diseases. In this review, we introduce some basic information about the CIA and CAIA models as well as how to apply these models effectively to investigate relationships between the pathogenesis of autoimmune diseases, especially RA, and the dysbiosis of intestinal bacterial flora