Identification of prolargin expression in articular cartilage and its significance in rheumatoid arthritis pathology.

Qualitative 2D gel-electrophoresis (2DE) protein profiling for osteoarthritis (OA) and rheumatoid arthritis (RA) is challenging because of selective protein loss due to discrepancies in protein precipitation methodologies. Thus, we aimed at developing qualitative protein representation from OA/RA articular cartilage without protein precipitation towards identification of clinically relevant proteins. Chondroitinase digested human articular cartilages from RA patients were subjected to protein extraction using guanidinium hydrochloride (GuHCl) or 8 M urea with 10 or 2% ASB-14-4 or 0.45 M urea with 2% ASB-14-4 with cetylpyridinium chloride (CPC). The GuHCl extract is further protein precipitated with acetone or ammonium acetate-methanol or centricon-fractionated using 100 kDa cut filters and protein precipitated using ethanol. Processed extracts were subjected to 2DE to identify protein profiles. Poor proteins representations were observed in 2D gels with protein precipitated samples compared to qualitative protein representations seen in 2D gels of 0.45 M urea and 2%ASB-14-4 extraction procedure reproducibly. The strategy circumventing protein precipitation generated qualitative 2D gels. RA vs OA gel comparison showed elevated prolargin levels in RA with biglycan levels remaining unaltered. Up regulation of prolargin in RA suggests the likelihood of an adaptive mechanism to control the increased osteoclastogenesis in RA and may have therapeutic value in controlling the disease.

Expression and Purification of Quinine Dihydro Pteridine Reductase from astrocytes and its significance in the astrocyte pathology.

Quinine dihydropteridinereductase (QDPR) is involved in the synthesis of tetradihydrobiopteridine (BH4) that serve as cofactor for many aromatic hydroxylases including induced nitric oxide synthase (NOS) leading to NO production. Increased activity of QDPR has been associated with decrease levels of TGF-ß, a cytokine that regulates the immune response and that elevated levels of NO has been associated with neurodegenerative diseases. Thus, expression of QDPR in astrocytes is essential to study the pathological changes observed in many neurodegenerative disorders. We have expressed QDPR in astrocytes and generated stably expressing clones that overexpresses QDPR. We further verified the specificity of QDPR expression using immunofluorescence and immunoblotting. To further confirm, we purified QDPR using Ni-NTA column and subjected the purified fraction to immunoblotting using anti-QDPR antibody and identified two major protein products of QDPR resolving at 25 and 17 kDa as reported in the literature. In order to further assess the significance of QDPR expression, we verified the expression of iNOS in QDPR over expressing cells. We show for the first time statistically significant up regulation of iNOS in QDPR overexpressing astrocytes. Increased expression of iNOS associated with astrocyte pathology seen in many neurodegenerative disorders may have implications in autoimmune neurodegenerative disorders.

Significance of elevated Prohibitin 1 levels in Multiple Sclerosis patients lymphocytes towards the assessment of subclinical disease activity and its role in the central nervous system pathology of disease.

Multiple Sclerosis (MS) is an autoimmune-neurodegenerative disorder managed therapeutically by modulating lymphocytes activity which has potential in disease management. Prohibitin 1(PHB) that controls the reactive oxygen species (ROS) and present on the activated lymphocytes have significance in the therapy of MS as esters of fumaric acid that regulates ROS is in phase II/III clinical trials. Thus, we evaluated the expression levels of PHB1 in experimental autoimmune encephalomyelitis (EAE), the animal model of MS and on MS patient's lymphocytes. PHB levels in brain tissue of EAE animals were determined by immunoblotting and on blood lymphocytes from MS relapse, Remission, Optic Neuritis, Neurological controls and Healthy volunteers by FACS using anti-PHB and anti-CD45 antibodies. We observed significant elevation of PHB in EAE brains (91.0 ±â€¯17.59%) vs controls (29.8 ±â€¯12.9%) (p = 0.01) and on lymphocytes of MS patients in acute (73.5 ±â€¯11.20%) or relapsing (69.3 ±â€¯17.33%) phase compared to remission (45.9 ±â€¯8.08%) [p = 0.034 acute vs remission; p = 0.004 relapse vs remission]. Up regulation of PHB in relapsing vs remission MS patients imply the potential use of PHB to clinically evaluate subclinical disease status towards prognosis of an oncoming relapse. Elevated PHB levels in EAE brains signify the role of PHB in regulating ROS and implies PHB's role in oxidative stress.

Exploitation of detergent thermodynamics in the direct solubilization of myelin membrane proteins for two-dimensional gel electrophoresis for proteomic analysis.

Performing 2-DE of lipid-rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2-D-proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2-DE profiling of myelin proteins is very important for the detection of immuno-reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent-mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB-14-4 resulted in significant increase in protein representation of myelin in 2-DE compared with the addition of ASB-14-4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB-14-4 strategy than other strategies of myelin sample processing such as ASB-14-4 to myelin or ethanol or acetone or methanol-ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2-D gel is achieved.