Ranga N. Parthasarathy

Properties and expression of human tartrate-resistant acid phosphatase isoform 5a by monocyte-derived cells

Human serum tartrate‐resistant acid phosphatase exists as two enzyme isoforms (TRACP 5a and 5b), derived by differential, post‐translational processing of a common gene product. Serum TRACP 5b is from bone‐resorbing osteoclasts (OC) and becomes elevated in diseases of increased bone resorption. TRACP 5a is secreted by macrophages (MΦ) and dendritic cells (DC) and is increased in many patients with rheumatoid arthritis. Our purpose was to fully characterize the properties of human TRACP isoforms and to produce an antibody specific to TRACP 5a for use as a biomarker in chronic inflammatory diseases. Partially purified, natural serum TRACP isoforms and recombinant TRACP 5a (rTRACP 5a) were compared with respect to specific activity and subunit structure and presence of sialic acid. Mice were immunized with rTRACP 5a, and resulting hybridomas were screened for monoclonal antibody to serum TRACP 5a. One antibody, 220, was tested for its epitope specificity and use in various immunological techniques. rTRACP 5a had properties identical to serum TRACP 5a. Antibody 220 was specific for the trypsin‐sensitive epitope in the loop peptide, present only in TRACP 5a. Antibody 220 was effective for specific immunoprecipitation, immunoassay, and immunoblot of TRACP 5a. Intact TRACP was present in MΦ, DC, and OC. TRACP 5a was the predominant isoform secreted by MΦ and DC, whereas TRACP 5b was the predominant isoform secreted by OC. TRACP isoforms 5a and 5b may have different functions inside and outside of monocyte‐derived cells. Antibody 220 is an important resource for studies of the biosynthetic relationship among TRACP isoforms and of the significance of serum TRACP 5a as a marker in diseases of bone metabolism and inflammation.

Identification of myo-inositol 3-phosphate synthase isoforms: Characterization, expression and putative role of a 16 kDa γc isoform

myo-Inositol is an important constituent of membrane phospholipids and is a precursor for the phosphoinositide signaling pathway. It is synthesized from glucose 6-phosphate by myo-inositol-3-phosphate synthase (IP synthase), a homotrimer composed of a 68-kDa polypeptide in most mammalian tissues. It is a putative target for mood-stabilizing drugs such as lithium and valproate. Here, we show that the rat gene (Isyna1) encoding this enzyme generates a number of alternatively spliced transcripts in addition to the fully spliced form that encodes the 68-kDa subunit (the α isoform). Specifically, we identify a small 16-kDa subunit (the γc isoform) derived by an intron retention mechanism and provide evidence for its existence in rat tissues. The γc isoform is highly conserved in mammals, but it lacks the catalytic domain while retaining the NAD+ binding domain. Both α and γc isoforms are predominantly expressed in many rat tissues and display apparent stoichiometry in purified enzyme preparations. An IP synthase polyclonal antibody not only detects the α and γc isoforms but also several other isoforms in pancreas, intestine, and testis suggesting that the holoenzyme is composed of unique subunits in various tissues. Interestingly, the α isoform is not expressed in the intestine. IP synthase activity assays using purified α and γc isoforms indicate that the latter negatively modulates α isoform activity, possibly by competing for NAD+ molecules. Our findings have important ramifications for understanding the mood stabilization process and suggest that inositol biosynthesis is a highly regulated and dynamic process.

Reduction of ischemic cell death in cultured Islets of Langerhans by the induction of cytoglobin.

Despite the source or mechanism of origin of islets of Langerhans or islet β-cells, all suffer significant cell loss from ischemia after isolation, thereby reducing the surviving islet mass available for study or transplantation. Methods to reduce beta cell death after islet isolation and transplantation must be developed if islet transplantation is to become an accepted treatment for diabetes. In order to enhance intracellular oxygen delivery and utilization, islets were transfected with a plasmid encoding cytoglobin, an intracellular oxygen binding protein. Oxygen consumption, insulin secretion, and the degree of central islet necrosis were measured in untreated and transfected islets to test the effects of cytoglobin on islet survival and function in vitro. The presence of cytoglobin reduced islet cell loss by reducing hypoxia related central islet necrosis and increased insulin secretion as compared with untreated islets. Cytoglobin treated islets maintained a normal rate of oxygen consumption, while untreated islets increased the rate of oxygen consumption caused by a shift to anaerobic metabolism and increased reactive oxygen specie synthesis. The induction of cytoglobin in islets may reduce cell loss from chronic hypoxia and may be a useful adjunct to islet transplantation.

Mammalian Inositol 3-phosphate Synthase: Its Role in the Biosynthesis of Brain Inositol and its Clinical Use as a Psychoactive Agent

Latha K. Parthasarathy, L., Ratnam S. Seelan, Carmelita Tobias, Manuel F. Casanova, and Ranga N. Parthasarathy Molecular Neuroscience and Bioinformatics Laboratories and Autism Research Unit, Mental Health, Behavioral Science and Research Services, VA Medical Center (151), Louisville, Kentucky, 40206, USA. Departments of Psychiatry, Biochemistry and Molecular Biology, University of Louisville, Kentucky, 40202, USA.

Rat brain myo-inositol 3-phosphate synthase is a phosphoprotein

The therapeutic effects of lithium in bipolar disorder are poorly understood. Lithium decreases free inositol levels by inhibiting inositol monophosphatase 1 and myo-inositol 3-phosphate synthase (IPS). In this study, we demonstrate for the first time that IPS can be phosphorylated. This was evident when purified rat IPS was dephosphorylated by lambda protein phosphatase and analyzed by phospho-specific ProQ-Diamond staining and Western blot analysis. These techniques demonstrated a mobility shift consistent with IPS being phosphorylated. Mass spectral analysis revealed that Serine-524 (S524), which resides in the hinge region derived from exon 11 of the gene, is the site for phosphorylation. Further, an antibody generated against a synthetic peptide of IPS containing monophosphorylated-S524, was able to discriminate the phosphorylated and non-phosphorylated forms of IPS. The phosphoprotein is found in the brain and testis, but not in the intestine. The intestinal IPS isoform lacks the peptide bearing S524, and hence, cannot be phosphorylated. Evidences suggest that IPS is monophosphorylated at S524 and that the removal of this phosphate does not alter its enzymatic activity. These observations suggest a novel function for IPS in brain and other tissues. Future studies should resolve the functional role of phospho-IPS in brain inositol signaling.

Maintenance of aerobic metabolism increases immunoisolated islet survival.

The progress of immunoisolation as a treatment for diabetes has been hampered by the diminished long term viability of islets within the immunoisolation device. Chronic hypoxia is greatly responsible for islet cell death within an immunoisolation device and remains an obstacle to the success of this form of islet transplantation. In order to address this problem, isolated rat islets were transfected with a plasmid encoding cytoglobin, an intracellular oxygen binding protein. Untreated or transfected islets were placed in polyacrylonitrile-polyvinychloride hollow fiber and implanted beneath the hepatic capsule in streptozotocin-diabetic rats. Fasting blood glucose was used as an indicator of islet survival and function. Rats receiving fibers containing transfected islets remained normoglycemic through the 60 day trial. Untreated islets failed within two weeks after implantation resulting in elevated blood glucose in the recipient. The fibers were recovered and tested for insulin content. Cytoglobin promoted islet cell survival and insulin synthesis and secretion. The induction of cytoglobin in islets may reduce cell loss from chronic hypoxia and may be a useful method to improve the feasibility of immunoisolation as an islet transplantation modality.

Applications and performance of monoclonal antibodies to human tartrate resistant acid phosphatase

BACKGROUND Tartrate-resistant acid phosphatase (TRACP) is an enzyme common to cells of the mononuclear phagocyte system and a clinically relevant biomarker for osteoclasts and inflammatory macrophages. The purpose was to assess applications and performance of six anti-TRACP monoclonal antibodies. METHODS Mab9C5, 14G6, 162, 203, 220, and 89 were used as capture and detection antibodies in quantitative immunoassay, and for western blot (WB), immunoprecipitation, and immunohistochemistry of paraffin sections containing chronic inflammatory infiltrates. The clinical performance of mab14G6 for immunoassay of serum TRACP5b activity was compared to two commercial kit methods. RESULTS Mab9C5 is useful for WB and immunohistochemistry methods only. Mab14G6, 162, and 203 are useful for quantitative immunoassay and immunoprecipitation, however, mab203 causes inactivation of enzymatic activity. Mab220 and 89 are specific for TRACP5a and useful in all applications. Mab14G6 has similar clinical sensitivity and specificity as two commercial methods. CONCLUSIONS TRACP is an important marker in osteoimmunology. Specific antibodies with unique specificity for TRACP isoforms and defined applications will be valuable for clinical evaluation of bone metabolic, inflammatory and autoimmune diseases and will aid in basic research of TRACP biochemistry and biology.

Proteomic analysis of rat prefrontal cortex after chronic valproate treatment

Valproic acid (VPA) is commonly used to treat bipolar disorder (BD), but its therapeutic role has not been clearly elucidated. To gain insights into VPAs mechanism of action, proteomic analysis was used to identify differentially expressed proteins in the rat prefrontal cortex (PFC), a region particularly affected in BD, after 6 weeks of VPA treatment. Proteins from PFCs of control and VPA‐treated rats were separated by 2D‐DIGE and identified by mass spectrometry. Among the 2,826 protein spots resolved, the abundance of 19 proteins was found to be significantly altered in the VPA‐treated group (with the levels of three proteins increasing and 16 decreasing). Seven proteins whose levels were significantly altered after chronic VPA exposure were quantified by Western blot analysis. The 19 identified proteins represent potential new targets for VPA action and should aid in our understanding of the role of VPA in BD.