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K. Sandeep Prabhu

Centre for Molecular Toxicology and Carcinogenesis
Centre of Molecular Immunology and Infectious Disease
Department of Veterinary and Biomedical Sciences
115 Henning Building
THE PENNSYLVANIA STATE UNIVERSITY
UNIVERSITY PARK, PA 16802

Phones: (office) 1-814-863-8976/(lab)1-814-863-8329

Fax: 1-814-863-6140

E-Mail: ksp4@psu.edu
The Pennsylvania State University 1997

The Pennsylvania State University 1997Expertise

* Cyclodextrin-based Host-Guest Complex Formation
* Enzyme Catalysis of Hydrophobic Substrates-Role of Cyclodextrin as a Substrate Delivery Agent
* Protein Purification
* Molecular Cloning and Heterologous Expression of Proteins
* Ligand-Dependent Activation of Nuclear Receptors
* Synthesis of Oxidized Fatty Acid Derivatives
The Pennsylvania State University 1997

The Pennsylvania State University 1997Current Projects

1. Cloning, Expression and Inhibition Kinetics of Human Cyclooxygenases
2. Purification, Molecular Cloning and Expression of a Unique Microsomal Glutathione S-Transferase from Sheep & Human Liver Tissues
3. Crystal Structure of the Microsomal GST Isozyme
4. Purification, Molecular Cloning and Characterization of myo-Inositol Oxygenase and its Crystal Structure (X-ray crystallography with Dr. Focco Van den Akker, Cleveland Clinic Foundation, Cleveland, OH)

5. Regulation of the Microsomal Ya-GST Expression in HepG2 Cells by Planar Aromatics and Prooxidants
6. Role of Selenium in the Regulation of Pro-inflammatory Gene(s) (such as COX2 and iNOS) Expression via the NF-kappaB Pathway in RAW264.7 Macrophages
The Pennsylvania State University 1997

The Pennsylvania State University 1997 About My Current Projects:

CYCLOOXYGENASE-2: Cyclooxygenase (COX), first purified in 1976 and cloned in 1988, is the key enzyme in the synthesis of prostaglandins (PGs) from arachidonic acid. In 1991, several laboratories identified a product from a second gene with COX activity and called it COX-2. However, COX-2 was inducible, and the inducing stimuli included pro-inflammatory cytokines and growth factors, implying a role for COX-2 in both inflammation and control of cell growth. The two isoforms of COX are almost identical in structure but have important differences in substrate and inhibitor selectivity and in their intracellular locations. Protective PGs, which preserve the integrity of the stomach lining and maintain normal renal function in a compromised kidney, are synthesized by COX-1. In addition to the induction of COX-2 in inflammatory lesions, it is present constitutively in the brain and spinal cord, where it may be involved in nerve transmission, particularly that for pain and fever. PGs made by COX-2 are also important in ovulation and in the birth process. The discovery of human COX-2 (hCOX-2) has made possible the design of drugs that reduce inflammation without removing the protective PGs in the stomach and kidney made by COX-1. These highly selective COX-2 inhibitors may not only be anti-inflammatory but may also be active in colon cancer and Alzheimer's disease.

MICROSOMAL GLUTATHIONE S-TRANSFERASES: Cellular oxidative stress reactions are known to produce a wide range of oxidized species including fatty acid hydroperoxides. These lipid peroxidation products are substrates for the glutathione peroxidases. A Ya-size subunit of glutathione-S-transferase (Ya-GST or Microsomal GSTA1-1) with significant non-selenium glutathione-dependent peroxidase (Non-SeGPX) activity towards fatty acid hydroperoxides from sheep liver microsomes has been cloned and expressed in BL21(DE3) cells. The microsomal Ya-GST cDNA shows that the enzyme is made up of 222 amino acid residues and shares about 73-83% sequence homology with GSTs also from different species. Sequence analysis shows that the enzyme has a high molar percentage of Leu and Lys that makes it hydrophobic and basic in nature. The Ya-GST is a cationic isozyme with a pI of 9.2 and has a subunit molecular mass of 25.2 kDa. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, R., Guthenberg, C. and DePierre, J. W. 1982, Eur J. Biochem. 128, 243-248]. Even though the microsomal Ya-GST closely resembles the cytosolic GSTs in terms of sequence homology, the substrate specificity profiles are not comparable to any of the cytosolic isoforms previously reported from this laboratory. Electrospray ionization mass spectral and reverse-phase h.p.l.c. studies indicate that the microsomal Ya-GST isozyme has a mass of 25611.3 Da and is not similar to any one of the cytosolic GSTs in that respect as well. In addition, Western immunoblot analysis of the microsomes treated with trypsin, alkaline Na2CO3, and EDTA following Sepharose S-300 washing of microsomes indicate that Ya-GST is intrinsic to the microsomes. The Km and Vmax values with 1-chloro-2,4-dinitrobenzene were 38 microM and 31 micromol/min/mg protein, respectively. In addition to catalyzing the conjugation of 4-hydroxynonenal, a lipid peroxidation product, with reduced glutathione, the enzyme also exhibits significant Non-SeGPX activity towards physiologically relevant fatty acid hydroperoxides such as linoleic and arachidonic acid hydroperoxides as well as phosphatidylcholine hydroperoxide, but not with hydrogen peroxide. In summary, it appears that the microsomal Ya-GST isozyme plays an important role in the protection of biological membranes against oxidative damage.

REDOX REGULATION OF PRO-INFLAMMATORY GENES BY CELLULAR SELENIUM STATUS: Selenium (Se) is an essential micronutrient for all mammalian species and is associated with a variety of physiological functions, notably immune system, in the form of selenoproteins. Inadequate Se nutrition has been linked to various diseases, including rheumatoid arthritis, cardiomyopathy and cancer. Important to this discussion is that cyclooxygenase-2 (COX-2) is over-expressed in all the aforesaid pathologies; however, a causal relationship between Se status and COX-2 expression remains to be established. The present study is based on the hypothesis that oxidant stress, a consequence of Se deficiency, lowers the activation potential of the redox-sensitive transcription factor, NF-kB, and that the activated NF-kB is required for the altered expression of COX-2. To test this hypothesis, we have investigated the relationship between Se status and COX-2 expression in response to LPS (a bacterial endotoxin) stimulation in RAW 264.7, a macrophage-like cell line. In Se-deficient cells, the Se-dependent glutathione peroxidase activity (Se-GPx), a measure of Se status, was markedly reduced and the overall oxidative stress was significantly higher than Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation, we found 2-3 folds higher COX-2 protein expression as well as higher prostaglandin E2 (PGE2) levels in Se-deficient cells than Se-supplemented cells. In comparison, COX-1 protein expression was not affected by either LPS stimulation or Se status. Following LPS stimulation, the nuclear localization of NF-kB was significantly increased in Se-deficient macrophages, thereby leading to increased expression of COX-2. This is the first report demonstrating an inverse relationship between Se status and the expression of COX-2.

myo-INOSITOL OXYGENASE AND ITS ROLE IN DIABETES: myo-Inositol oxygenase (MIOX) catalyses the first committed step in the only pathway of myo-inositol catabolism, which occurs predominantly in the kidney. The enzyme is a non-haem-iron protein that catalyses the ring cleavage of myo-inositol with the incorporation of a single atom of oxygen. A full-length cDNA was isolated from a pig kidney library with an open reading frame of 849 bp and a corresponding protein subunit molecular mass of 32.7 kDa. The cDNA was expressed in a bacterial pET expression system and an active recombinant MIOX was purified from bacterial lysates to electrophoretic homogeneity. The purified enzyme displayed the same catalytic properties as the native enzyme with K(m) and k(cat) values of 5.9 mM and 11 min(-1) respectively. The pI was estimated to be 4.5. Preincubation with 1 mM Fe(2+) and 2 mM cysteine was essential for the enzyme's activity. D-chiro-Inositol, a myo-inositol isomer, is a substrate for the recombinant MIOX with an estimated K(m) of 33.5 mM. Both myo-inositol and D-chiro-inositol have been implicated in the pathogenesis of diabetes. Thus an understanding of the regulation of MIOX expression clearly represents a potential window on the aetiology of diabetes as well as on the control of various intracellular phosphoinositides and key signalling pathways.

The Pennsylvania State University 1997Research Group:

Dr. C. Channa Reddy, Dr. Ryan J. Arner, Hemalatha Vunta, Venkatesh Krishnan, Vikrant Yerram, Dr. Uma Maheshwari, Stephanie Larson
The Pennsylvania State University 1997

The Pennsylvania State University 1997 Education:


Ph.D. (Biochemistry/Enzymology): Vittal Mallya Scientific Research Foundation, University of Mysore, India (1998)

M.Sc (Biochemistry):University of Mysore, India (1992)

B.Sc (Biochemistry): University of Mysore, India (1990)

The Pennsylvania State University 1997

The Pennsylvania State University 1997PROFESSIONAL EXPERIENCE:


2004- Graduate Faculty, Pathobiology Program

2002-present: Senior Research Associate/Research Assistant Professor, Dept. of Veterinary Science, Pennsylvania State University, University Park.

1998-2002: Post-Doc, Dept. of Veterinary Science, Pennsylvania State University, University Park

The Pennsylvania State University 1997

The Pennsylvania State University 1997 Honors & Awards:

1. Junior & Senior Research Fellowships of the Council of Scientific & Industrial Research, India
2. Recipient of the Professor of Biochemistry Gold Medals in Biochemistry, University of Mysore

The Pennsylvania State University 1997

The Pennsylvania State University 1997Publications:

The Pennsylvania State University 1997Conferences and Symposia

R. J. Arner, K. S. Prabhu, and C. C. Reddy (2004). Characterization of a novel protein complex that catabolizes myo-inositol. FASEB J. 18, 71.15


K. S. Prabhu, R. J. Aner, N. V. Hegde, and C. C. Reddy (2004) Characterization of the myo-inositol oxygenase gene promoter. FASEB J. 15, 71.14.


K. S. Prabhu, J. Stahle, and C. C. Reddy (2004) Expression of apolipoprotein A-I is regulated by cellular selenium status. FASEB J. 18, 37.22


K. S. Prabhu, F. Davis, D. Bhat, and C. C. Reddy (2004) Selenium suppresses LPS-induced NF-kB activation of cyclooxygenase-2 by inhibiting IkBa phosphorylation in a mouse macrophage cell line. FASEB J. 18 37.23


K. S. Prabhu, P. V. Reddy, E. C. Jones, J. T. Thompson, A. D. Liken, R. J. Arner and C. C. Reddy. Isolation and characterization of a microsomal glutathione S-transferase with glutathione-peroxidase activity from human liver FASEB J 15 (4): A536-A536 Part 1 MAR 7 2001


R. J. Arner, K. S. Prabhu, J. T. Thompson and C. C. Reddy. Cloning, expression, and characterization of myo-inositol oxygenase from hog kidney. FASEB J 15 (4): A535-A535 Part 1 MAR 7 2001


F. Zamamiri-Davies, J. Stewart, J. Thompson, K. S. Prabhu, L. M. Sordillo and C. C. Reddy. Up regulation of the inducible nitric oxide synthase is mediated by NF-kappa B in macrophages during selenium deficiency. FASEB J 15 (4): A199-A199 Part 1 MAR 7 2001


F. Zamamiri-Davis, Y. Lu, J. Thompson, K. S. Prabhu, L.M. Sordillo and C. C. Reddy. Up regulation of cyclooxygenase-2 is mediated by NF-kappa B in macrophages during selenium deficiency. Mol. Biol. Cell 11: 49 Suppl. S DEC 2000


K. S. Prabhu, P. V. Reddy, H.P. Yennawar, G. P. Farber and C. C. Reddy (2000) Molecular cloning, expression and crystal structure of a unique sheep liver microsomal glutathione-S-transferase. Abstracts of the Meeting of Federation of American Societies for Experimental Biology, Boston, MA, June 3-8, 2000. FASEB J. 14, Suppl. #804


P. V. Reddy, K. S. Prabhu, E. Gumpricht, G. R. Hildenbrandt, R. W.Scholz and C. C. Reddy. Identification of a Unique Microsomal Glutathione S-Transferase (GST) from Sheep Liver. Abstracts of the Meeting of Federation of American Societies for Experimental Biology, San Francisco, CA, May 16-20, 1999. FASEB J. 13: A9 (late abstracts)


K. S. Prabhu and C. S. Ramadoss. Hydrolysis of N-benzoyl-L-tyrosine ethyl ester anchored in CD by a-chymotrypsin and subtilisin. In Proceedings of the 8th International Cyclodextrin Symposium, Budapest, Eds Szente, L and Szejtli, J. 281-284. (1996)


The Pennsylvania State University 1997Peer Reviewed Journals

Gong Xing, Y Diao, L. M. Hoffart, E. W. Barr, K. S. Prabhu, R. J. Arner, C. C. Reddy, K. Krebs, J. M. Bollinger (2006) Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase. Proc. Natl. Acad. Sci. (USA). 103: 6130-6135.


G. Xing, L. M. Hoffart, Y. Diao, E. W. Barr, K. S. Prabhu, R. J. Arner, C. C. Reddy, K. Krebs, J. M. Bollinger (2006) A coupled dinuclear iron cluster that is perturbed by substrate binding in myo-inositol oxygenase. Biochemistry; Apr08; [Epub ahead of print]


G. Xing, E. W. Barr, L. M. Hoffart, K. S. Prabhu, R. J. Arner, C. C. Reddy, K. Krebs, J. M. Bollinger (2006) Oxygen activation by a mixed-valent, diiron (II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase. Biochemistry, Apr 08; [Epub ahead of print]


D. J. Kim, K. S. Prabhu, F. J. Gonzalez and J.M. Peters (2006) Inhibition of chemically-induced skin carcinogenesis by sulindac is independent of peroxisome proliferator-activated receptor-beta/delta. Carcinogenesis. Jan 16; [Epub ahead of print]


R. J. Arner, K. S. Prabhu, V. Krishnan, M. Johnson, and C. C. Reddy (2006) Expression of myo-inositol oxygenase in tissues susceptible to diabetic complications. Biochem. Biophys. Res. Commun. 339: 816-820.


K. Sandeep Prabhu, Ryan J. Arner, Hema Vunta, and C. Channa Reddy. Upregulation of human myo-inositol oxygenase by hyperosmotic stress in renal proximal tubular epithelial cells. J Biol Chem. 2005 Mar 18; [Epub ahead of print]


Ryan J. Arner, K. Sandeep Prabhu,and C. Channa Reddy. Molecular cloning, expression, and characterization of myo-inositol oxygenase from mouse, rat, and human kidney. Biochem Biophys Res Commun. 2004 Nov 26;324(4):1386-92


K. Sandeep Prabhu, Padala V. Reddy, Emily C. Jones, Andrew D. Liken, and C. Channa Reddy. Characterization of a class alpha glutathione S-transferase with glutathione peroxidase activity in human liver microsomes. Arch. Biochem. Biophys. (2004) In press.

K. Sandeep Prabhu, Padala V. Reddy, Andrew D. Liken, Emily C. Jones, Hemant P. Yennawar, and C. Channa Reddy. Identification, characterization, and properties of a class alpha microsomal glutathione S-transferase. Adv. Prostaglandins Leukotriene and Other Bioactive Lipid Res. (Chapter 40; Z. Yazici Ed.) (2003)


P. Reddanna, K. Sandeep Prabhu, J. Whelan, and C. Channa Reddy. Direct conversion of Leukotriene C4 to Leukotriene F4 by carboxypeptidase A. Arch. Biochem. Biophys.413:158-163 (2003)


K. S. Nagabhushana, S. Umamaheshwari, F. E. Tocoli, K. S. Prabhu, I. R. Green, and C. S. Ramadoss. Inhibition of Soybean and Potato Lipoxygenases by Bhilawanols from Bhilawan (Semecarpus anacardium) Nut Shell Liquid and some Synthetic Salicylic Acid Analogues. J. Enzyme Inhibition Med. Chem. 17(4): 255-259 (2002)


K. S. Prabhu, F. Zamamiri-Davis, J. Stewart, J. T. Thompson, L. M. Sordillo and C. C. Reddy. Selenium deficiency increases the expression of inducible nitric oxide synthase (iNOS) in RAW264.7 macrophages: role of nuclear factor-kappa B in up regulation. Biochem. J. 366: 203-209 (2002) PDF File (e-publ ahead of print)


F. Zamamiri-Davis, Y. Lu, J. T. Thompson, K. S. Prabhu, P. V. Reddy, L. M. Sordillo and C. C. Reddy. Nuclear factor-kappaB mediates over-expression of cyclooxygenase-2 during activation of RAW264.7 macrophages in selenium deficiency. Free Radic. Biol. Med. 32(9):890-97 (2002)


K. S. Prabhu, P. V. Reddy, E. Gumpricht, G. R. Hildenbrandt, R. W.Scholz and C. C. Reddy. Microsomal class alpha glutathione S-transferase with glutathione peroxidase activity from sheep liver: Molecular cloning, expression and characterization. Biochem. J. 360(2): 345-354 (2001) PDF File


R. J. Arner, K. S. Prabhu, J. T. Thompson and C. C. Reddy. Myo-Inositol Oxygenase: Molecular Cloning and Expression of a Unique Enzyme that Oxidizes myo-Inositol and D-chiro-inositol. Biochem J. 360(2): 313-320 (2001) PDF File


K. S. Prabhu and C. S. Ramadoss. Penicillin acylase catalyzed synthesis of Penicillin-G from cyclodextrin anchored substrates. Ind. J. Biochem. Biophys. 37:6-12. (2000)


T. Shivashankar, R. S. Annadurai, M. Srinivas, G. Preethi, T. B. Sharada, R. Paramashivappa, A. Srinivasa Rao, K.S. Prabhu, C. S. Ramadoss, G. K. Veeresh and P. V. Subba Rao. Control of coconut black-headed caterpillar (Opisina arenosella Walker) by systemic application of "Soluneem"-a new water-soluble neem insecticide formulation. Curr. Sci (India) 78:176-178. (2000)


K. S. Prabhu and C. S. Ramadoss. Subtilisin catalysis of substrate anchored in cyclodextrin. Appl. Biochem. Biotechnol. 69:69-77. (1998)


K. S. Prabhu and C. S. Ramadoss. Alfa-Chymotrypsin catalyzed hydrolysis of N-benzoyl-L-tyrosine ethyl ester anchored in beta-cyclodextrin. Biocatal. Biotransform. 12:281-291. (1995)


The Pennsylvania State University 1997Patents

K. S. Prabhu, S. R. Annadurai, M. Srinivas, A. Srinivasa Rao, C. S. Ramadoss and P. V. Subba Rao. Process for Preparing Cyclodextrin Inclusion Complex. US Patent # 6635757


K. S. Prabhu, R. S. Annadurai, M. Srinivas, A. Srinivasa Rao, C. S. Ramadoss and P. V. Subba Rao. Process for the preparation of cyclodextrin inclusion complex. European Patent # 1191851


K. S. Prabhu, et al. A process for the preparation of water-soluble cyclodextrin inclusion complex of neem seed kernel extract containing Azadirachtin-A. Indian Patent # 187645


The Pennsylvania State University 1997 Membership:

Amercian Association for the Advancement of Science

The Pennsylvania State University 1997

The Pennsylvania State University 1997Interesting Links:

The Pennsylvania State University 1997 Science
The Pennsylvania State University 1997 Nature
The Pennsylvania State University 1997 THE JOURNAL OF BIOLOGICAL CHEMISTRY
The Pennsylvania State University 1997 Proceedings of the National Academy of Sciences (USA)
US Patent and Trade Office
The Pennsylvania State University 1997 Annual Reviews (Biochemistry,Genetics and more..)
The Pennsylvania State University 1997 WebCutter v2.0-Restriction digestion of your DNA sequence
The Pennsylvania State University 1997 Funding Sources in Biological Sciences
BRENDA Database-The Comprehensive Enzyme Information System
The Pennsylvania State University 1997 NCBI-PUBMED DATABASE
The Pennsylvania State University 1997 Signal Scan
The Pennsylvania State University 1997 Human BAC Resource
The Pennsylvania State University 1997 Peptide Cutter
The Pennsylvania State University 1997San Diego Supercomputer Center's Biology WorkBench
The Pennsylvania State University 1997Gene Expression Omnibus (GEO)-Gene expression and hybridization array data repository
The Pennsylvania State University 1997Gene Expression Atlas by Genomics Institute of the Novartis Research Foundation, San Diego
The Pennsylvania State University 1997 Instructions to Authors in the Health Sciences

The Pennsylvania State University 1997

The Pennsylvania State University 1997 Lab Alumni

Faith Z. Davis (PhD 2002)
Ryan J. Arner (PhD 2002)
Andrew Liken (PhD 2002)
Jennifer Stewart (BS 2002)
Emily Jones (BS 2002)
Sasha Snyder (BS 1999)
Katie L. Miro (BS (Hons) 2004)
Kiran Manne (BS (Hons) 2004)
Deepa Bhat (BS (Hons) 2004)
Jessica A. Stahle (BS (Hons) 2005)

ksp4@psu.edu
The Pennsylvania State University 1997

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The Pennsylvania State University 1997


Copyright 2006 Sandeep K. Prabhu