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By using standard analytical tools of cellular and molecular biology he has elucidated the molecular mode of action of an anti-diabetic candidate BMOV on the key components of insulin signaling cascades like IR Kinase (IRK) and IRS complexes including gross tyrosine phosphorylation of IRK and IRSs and site specific tyrosine phosphorylation of IRK like Tyr972 of NPEY motif, and Tyr1158, Tyr1162, Tyr1163 of PT Kinase activation segment of IR (Journal of Molecular Endocrinology, 38, 2007, 627–649) The insulin signaling induction by this compound was also seen on Thr308 and Ser473 phosphorylation of Akt kinase in association with Ser9 phosphorylation of GSK-3ß kinase. He found that this signaling also regulates the Ser259 phosphorylation of Raf Kinase. Besides, he has developed MALDI-TOF mass spectrometric technique for the identification and analysis of glycated insulin (Biochimica et Biophysica Acta, 1725, 2005, 269-282). Using this method he elucidated the molecular mechanism of insulin glycation, which might be considered as an important contribution to the diabetes research. He has also developed sensitive cationic liposomes (CLs)-based technique for identification and characterization of apoptotic cells (Analytical Biochemistry. 331, 385–394, 2004). Accordingly, control cells showed negligible and irregular binding patterns of CLs, whereas apoptotic cells revealed a strongly augmented staining of their plasma membrane. While, by employing fluorescence anisotropy, CD spectrophotometry, confocal fluorescence microscopy. FRET, giant liposomes and standard cell biology tools he elucidated the molecular mechanism of action of antimicrobial peptide Plantaricin A (pIA) in human leukemic T-cells (Biochimica et Biophysica Acta, (Biomembranes), 1758, 1461-1474, 2006). Using the similar techniques he also established a model that was used to elucidate the molecular mechanism of histone H1 mediated apoptosis in cancerous cells (Biochemistry (USA), 43, 10192-10202, 2004). By utilizing standard hematological technique he has elucidated the mode of action of toxic metal on blood platelet aggregation (Biomed.Environ.Sci. (USA), 9, 26-36). Moreover, by applying radioreceptorassay. radioimmunoassay, atomic absorption spectrometry and standard cellular and biochemical tools he demonstrated that Hg2+ binds specifically to Na+/ K+ - ATPase in rat liver plasma membrane leading to a cascade of molecular events which is linked with the induction of MT gene expression (BioMetals (UK), 10, 157-162). He has developed an efficient method for antibody immobilization on amino terminal magnetic beads that strongly favors proper orientation of the immunoglobulin with minimal chances of nonspecific binding due to removal of Fc region. He has also developed mass spectrometry based immunoassay technique on glyco-affinity MALDI-TOF plate that can be adopted as a useful method for characterization and profiling of biomolecules including biomarkers. The presented posters (also as abstracts in Mol. Cell Proteomics) are: 1) SOriented immobilization of monovalent antibody on magnetic beads: An approach for a proteomics tool. 6th Annual Congress, Human Proteomics Organization (HUPO), October 6–10, 2007, COEX, Seoul, Korea. 2) Immunoassay with glyco-specific affinity MALDI plates. 6th Annual Congress, Human Proteomics Organization (HUPO), October 6–10, 2007, COEX, Seoul, Korea.

He performed extensive research on metallothionein (MT), a low molecular weight cysteine rich protein which binds metals avidly and eventually detoxifies them. He has found that MT level is increased several fold in the vital detoxifying organs of both mammals and teleosts due to exposure to the metals, in keeping with previous reports. However, the precise mechanism by which metals induce the synthesis of MT is largely unknown. The area of research is relatively new and very little information is available on the exact signals raised by cell membrane - metal interaction for the induction of the MT gene transcription. To understand the major molecular events of metal induced MT biosynthesis, he established different in vitro models using the plasma membrane preparations from different sources. It has been found that binding of inorganic mercury to fish oocyte membrane is very specific while rat erythrocyte plasma membrane shows multiple binding sites for this metal. He also revealed that calcium or calcium dependent pathway like calcium-calmodulin systems have no regulatory role in transduction of signal raised by membrane-metal interaction for induced biosynthesis of MT in rat liver. He showed that metals like Cd2+ has a direct effect on Na+/Ca2+ exchange phenomenon in which Na+ is linked with a higher rate of MT synthesis. Cd2+ enters through the Ca2+ channel and simultaneously deregulates Na+ homeostasis. Furthermore, he demonstrated that Hg2+ binds specifically to Na+/ K+ - ATPase in rat liver plasma membrane leading to a cascade of molecular events which is linked with the induction of MT gene expression. In another study, he elucidated the response of blood platelets to mercury exposure. Although platelets are primarily concerned with the processes of thrombosis and hemostasis, it also plays a critical part in almost all responses of blood to injury including the response to the xenobiotics introduced in the blood stream. He showed that in vivo treatment with mercury in rat caused dose- and time -dependent inhibition of the agonist mediated aggregation of platelets where the effect was more drastic in ADP-induced rather than adrenaline -induced aggregation. In the in vitro experiments he found that low doses of Hg2+ does not require the presence of Ca2+ in the medium to induce the aggregation. At high concentration the metal binds directly to membrane thiols and inhibits platelet aggregation while at low concentration mercury acts indirectly to cause aggregation by stimulating cyclooxygenase and inhibiting cAMP-PDE pathway. (1) BioMetals (UK), 10, 157-162. (2) Biomed. Environ. Sci. (USA), 7, 232-240. (3) Biomed. Environ. Sci. (USA), 9, 26-36. (4) Mercury binding to plasma membrane of rat erythrocyte. In Environmental Toxicology in South East Asia (B. Widianarko, K.Vink and N. N. Van Straaten, Eds.), VU University Press, Amsterdam, Section 3.9, pp. 215-219. Besides, he has also studied the in vitro responses of both mammalian and piscine acetylcholinesterase (AChE) enzyme to toxic metals. 1) Inhibition of fish brain acetylcholinesterase by cadmium and mercury : Interaction with selenium. In Enzymes of the Cholinesterase Family (D.M.Quinn, A.S. Balasubramhanian, B.P Doctor and P.Taylor, Eds.), Plenum Publication, USA, pp. 369-374.

He has elucidated the molecular mechanism of action of antimicrobial peptide Plantaricin A (pIA) in human leukemic T-cells (Biochimica et Biophysica Acta, (Biomembranes), 1758, 1461-1474, 2006). His study suggests that an increase in membrane spontaneous negative curvature may affect the mode of association of this peptide with lipid bilayer of the cell membrane. He showed that at micromolar concentrations, plA destroyed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA forms supramolecular protein–lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, which suggests a possible mechanistic connection between fibril formation and the cytotoxicity of plA. He has also developed sensitive cationic liposomes (CLs)-based technique for identification and characterization of apoptotic cells (Analytical Biochemistry. 331, 385–394, 2004). Accordingly, control cells showed negligible and irregular binding patterns of CLs, whereas apoptotic cells revealed a strongly augmented staining of their plasma membrane. Besides, morphological observations and comparison with standard procedures for detecting apoptotic cells further demonstrated the binding of CLs to be intense for cells undergoing apoptosis. In addition, some apoptotic cells with higher caspase-3 activity also revealed more pronounced staining by CLs. His data suggest that the binding of CLs to apoptotic cells is mediated through an electrostatic interaction between the positively charged head group of cationic lipid and the translocated anionic phospholipid phosphatidylserine (PS) in the cell membrane. He also established a model that was used to elucidate the molecular mechanism of histone H1 mediated apoptosis in cancerous cells (Biochemistry (USA), 43, 10192-10202, 2004). Accordingly, comparison of the behavior of H1 in giant liposomes to that in cultured leukemic T cells demonstrated very similar patterns. More specifically, using fluorescence microscopy he revealed the binding of H1 to the plasma membrane as lateral segregated microdomains, followed by translocation into the cell. H1 also triggered membrane blebbing and fragmentation of the nuclei of these cells, thus suggesting induction of apoptosis. His findings also indicate that histone H1 and acidic phospholipids form supramolecular aggregates in the plasma membrane of T cells, subsequently resulting in major rearrangements of cellular membranes. Moreover, he demonstrate that the minimal requirement for the interaction of histone H1 with the leukemia cell plasma membrane is reproduced by giant liposomes composed of unsaturated phosphatidylcholine and phosphatidylserine, the latter being mandatory for the observed changes in the secondary structure of H1 as well as the macroscopic consequences of the H1-PS interactions. In another study he has identified the molecular mechanism of vanadate-induced insulin signaling in insulin-target 3T3-L1 cells and non-target IM9 cells that express insulin receptor on the plasma membrane. This can be considered as the basic platform to design and develop more appropriate insulinomimetic candidate for controlling type-II diabetes, especially in patients showing insulin resistance (Journal of Molecular Endocrinology, 38, 2007, 627–649).

Currently he is involved in major segments of diabetes research, such as insulin signaling, insulin resistance, insulin glycation, and inhibition of protein tyrosine phosphatase 1B (PTP 1B). He has focused on the development of a new therapeutic strategy for controlling diabetes, especially emphasizing the state of insulin resistance. Major works were carried out on two important areas, PTP IB inhibition and insulin glycation. He has identified the molecular mechanism of vanadate-induced insulin signaling in insulin-target cells 3T3 L1 & non-target cells like IM9. More specifically, he has elucidated the effect of anti-diabetic candidate BMOV on the key components of insulin signaling cascades like IR Kinase (IRK) and IRS complexes including gross tyrosine phosphorylation of IRK and IRSs and site specific tyrosine phosphorylation of IRK like Tyr972 of NPEY motif, and Tyr1158, Tyr1162, Tyr1163 of PT Kinase activation segment of IR (Journal of Molecular Endocrinology, 38, 2007, 627–649). The insulin signaling induction by this compound was also seen on Thr308 and Ser473 phosphorylation of Akt kinase in association with Ser9 phosphorylation of GSK-3ß kinase. He also demonstrated that this signaling could regulate the Ser259 phosphorylation of Raf Kinase. On the other hand It has been proposed that glycation of insulin may also contribute to insulin resistance. To understand this more deeply, he has developed MALDI-TOF mass spectrometric technique for the identification and analysis of glycated insulin (Biochimica et Biophysica Acta, 1725, 2005, 269-282). Using this technique he has elucidated the molecular mechanism of insulin glycation, which might be considered as an important area of diabetes research. Moreover, he has elucidated the molecular mechanism of action of antimicrobial peptide Plantaricin A (pIA) in human leukemic T-cells (Biochimica et Biophysica Acta, (Biomembranes), 1758, 1461-1474, 2006). His study suggests that an increase in membrane spontaneous negative curvature may affect the mode of association of this peptide with lipid bilayer of the cell membrane. He showed that at micromolar concentrations, plA destroyed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein–lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, which suggests a possible mechanistic connection between fibril formation and the cytotoxicity of plA. He also established a model that was used to elucidate the molecular mechanism of histone H1 mediated apoptosis in cancerous cells (Biochemistry (USA), 43, 10192-10202, 2004). Accordingly, comparison of the behavior of H1 in giant liposomes to that in cultured leukemic T cells demonstrated very similar patterns. More specifically, using fluorescence microscopy he revealed the binding of H1 to the plasma membrane as lateral segregated microdomains, followed by translocation into the cell. In his study H1 also triggered membrane blebbing and fragmentation of the nuclei of these cells, thus suggesting induction of apoptosis. In the New Drug Discovery Research Centre of the major Pharmaceutical industries he was involved in pharmacological and toxicological evaluation and screening of the candidate drug molecules, therapeutic proteins like erythropoietin, vaccine like HbsAg and new formulations at preclinical level using various animals and in vitro models.

He is involved in project dealing with major therapeutic segments in diabetes, such as insulin signaling, insulin resistance, insulin glycation, and inhibition of protein tyrosine phosphatase 1B (PTP 1B). He has focused on the development of a new therapeutic strategy for controlling diabetes, especially emphasizing the state of insulin resistance. Major works were carried out on two important areas, PTP IB inhibition and insulin glycation. He has identified the molecular mechanism of vanadate-induced insulin signaling in insulin-target & non-target cells. This finding can be considered as the basic platform to design and develop more appropriate insulinomimetic candidate for controlling type-II diabetes, especially in patients showing insulin resistance. Recently he has published an article on this topic entitled ‘Molecular mechanism of BMOV-induced insulin signaling in 3T3-L1 and IM9 cells: Impact of dexamethasone’ (Journal of Molecular Endocrinology, 38, 2007, 627–649) as an outcome of his research on PTP 1B inhibition. This is based on the elucidation of the molecular mode of action of an anti-diabetic candidate BMOV on the key components of insulin signaling cascades like IR Kinase (IRK) and IRS complexes including gross tyrosine phosphorylation of IRK and IRSs and site specific tyrosine phosphorylation of IRK like Tyr972 of NPEY motif, and Tyr1158, Tyr1162, Tyr1163 of PT Kinase activation segment of IR. The insulin signaling induction by this compound was also seen on Thr308 and Ser473 phosphorylation of Akt kinase in association with Ser9 phosphorylation of GSK-3ß kinase. He found that this signaling also regulates the Ser259 phosphorylation of Raf Kinase. The whole study was conducted elaborately using two models namely 3T3 L1, and IM9 cells. On the other hand It has been proposed that glycation of insulin may also contribute to insulin resistance. To understand this more deeply, he has developed MALDI-TOF mass spectrometric technique for the identification and analysis of glycated insulin (Biochimica et Biophysica Acta, 1725, 2005, 269-282). Using this method he elucidated the molecular mechanism of insulin glycation, which might be considered as an important contribution to the diabetes research. Moreover, in one of studies he demonstrated the mode of action of mercury, a group-IIB toxic metal on the fish oocytes. Using radioreceptorassay he showed that this metal can bind specifically to the membrane of oocyte in which sulfhydryl groups play a major role. “Fish oocyte membrane binds inorganic mercury in a in vitro system.” The Second Congress of The Asia and Oceania Society of Comparative Endocrinology, New Delhi, India, 1991.

Currently he is engaged in research dealing with major therapeutic segments in diabetes, such as insulin signaling, insulin resistance, insulin glycation, and inhibition of protein tyrosine phosphatase 1B (PTP 1B). He has focused on the development of a new therapeutic strategy for controlling diabetes, especially emphasizing the state of insulin resistance. Major works were carried out on two important areas, PTP IB inhibition and insulin glycation. He has identified the molecular mechanism of vanadate-induced insulin signaling in insulin-target & non-target cells. This finding can be considered as the basic platform to design and develop more appropriate insulinomimetic candidate for controlling type-II diabetes, especially in patients showing insulin resistance. Recently he has published an article on this topic entitled ‘Molecular mechanism of BMOV-induced insulin signaling in 3T3-L1 and IM9 cells: Impact of dexamethasone’ (Journal of Molecular Endocrinology, 38, 2007, 627–649) as an outcome of his research on PTP 1B inhibition. This is based on the elucidation of the molecular mode of action of an anti-diabetic candidate BMOV on the key components of insulin signaling cascades like IR Kinase (IRK) and IRS complexes including gross tyrosine phosphorylation of IRK and IRSs and site specific tyrosine phosphorylation of IRK like Tyr972 of NPEY motif, and Tyr1158, Tyr1162, Tyr1163 of PT Kinase activation segment of IR. The insulin signaling induction by this compound was also seen on Thr308 and Ser473 phosphorylation of Akt kinase in association with Ser9 phosphorylation of GSK-3ß kinase. He found that this signaling also regulates the Ser259 phosphorylation of Raf Kinase. The whole study was conducted elaborately using two models namely 3T3 L1, and IM9 cells. On the other hand It has been proposed that glycation of insulin may also contribute to insulin resistance. To understand this more deeply, he has developed MALDI-TOF mass spectrometric technique for the identification and analysis of glycated insulin (Biochimica et Biophysica Acta, 1725, 2005, 269-282). Using this method he elucidated the molecular mechanism of insulin glycation, which might be considered as an important contribution to the diabetes research. On the other hand he also developed affi-MADI-TOF plates and functional as well as affi-magnetic beads oriented proteomic tools that would have potential future applications in mass spectrometry based biological and pharmaceutical applications, especially for identification of biomarkers for cancers and diabetes. Other future applications of those tools include cell biology, immunology, and glycobiology, protein purification etc. These are based on ELISA based techniques as well as analysis through MALDI-TOF Mass Spectrometry. Some of the products are on the process of patenting and commercialization, while others are under development. The presented posters (also as abstracts in Mol. Cell Proteomics) are: 1) Oriented immobilization of monovalent antibody on magnetic beads: An approach for a proteomics tool. 6th Annual Congress, Human Proteomics Organization (HUPO), October 6–10, 2007, COEX, Seoul, Korea. 2) Immunoassay with glyco-specific affinity MALDI plates. 6th Annual Congress, Human Proteomics Organization (HUPO), October 6–10, 2007, COEX, Seoul, Korea.

He has elucidated the impact of toxic metal on blood platelet aggregation. Although platelets are primarily concerned with the processes of thrombosis and hemostasis, it also plays a critical part in almost all responses of blood to injury including the response to the xenobiotics introduced in the blood stream. He found that in vivo treatment with mercury in rat causes dose- and time dependent inhibition of the agonist mediated aggregation of platelets where the effect was more drastic in ADP-induced rather than adrenaline induced aggregation. In the in vitro experiments he has demonstrated that low doses of Hg2+ does not require the presence of Ca2+ in the medium to induce the aggregation. He has shown that mercury interferes with normal platelet function at both high and low concentrations. At high concentration the metal binds directly to membrane thiols and inhibits platelet aggregation while at low concentration mercury acts indirectly to cause aggregation by stimulating cyclooxygenase and inhibiting cAMP-PDE pathway (Biomed.Environ.Sci. (USA), 9, 26-36). He also elucidated the probable mechanism of metal induced biosynthesis of metallothionein (MT), a protein that plays a major role in binding and detoxification of metals. The area of research is relatively new and very little information is available on the exact signals raised by cell membrane - metal interaction for the induction of the MT gene transcription. The main objective of his study was to understand the molecular events by which metal exposure induces the synthesis of MT protein in mammalian liver and have made much progress in this area of research. To understand the exact molecular phenomena in membrane-metal interaction he used different in vitro models using the membrane preparations from different sources. It has been found that binding of inorganic mercury to fish oocyte membrane is very specific while rat erythrocyte plasma membrane shows multiple binding sites for this metal. In one of his studies he revealed that calcium or calcium dependent pathway like calcium-calmodulin system have no regulatory roles in transduction of signal originated by membrane-metal interaction for induced biosynthesis of MT in rat liver. Cd2+ has direct effect on Na+/Ca2+ exchange phenomenon in which Na+ is linked with a higher rate of MT synthesis. Cd2+ enters through the Ca2+ channel and simultaneously deregulates Na+ homeostasis. Moreover, he demonstrated that Hg2+ binds specifically to Na+/ K+ - ATPase in rat liver plasma membrane leading to a cascade of molecular events which is linked with the induction of MT gene expression. (1) BioMetals (UK), 10, 157-162. (2) Biomed. Environ. Sci. (USA), 7, 232-240. (3) Biomed. Environ. Sci. (USA), 9, 26-36. (4) Mercury binding to plasma membrane of rat erythrocyte. In Environmental Toxicology in South East Asia (B. Widianarko, K.Vink and N. N. Van Straaten, Eds.), VU University Press, Amsterdam, Section 3.9, pp. 215-219. Besides he also established that C-reactive protein (CRP) can be utilized as an important parameter to monitor severe acute poisoning by environmental pollutants. Moreover, in the New Drug Discovery Research Centre of the major Pharmaceutical industries he was involved in pharmacological and toxicological evaluation and screening of the candidate drug molecules, therapeutic proteins like erythropoietin, vaccine like HbsAg and new formulations at preclinical level using various animals and in vitro models.

He has identified the molecular mechanism of vanadate-induced insulin signaling in insulin-target 3T3 L1 & non-target IM9 cells. This finding can be considered as the basic platform to design and develop more appropriate insulinomimetic candidate for controlling type-II diabetes, especially in patients showing insulin resistance. Recently he has published an article on this topic entitled ‘Molecular mechanism of BMOV-induced insulin signaling in 3T3-L1 and IM9 cells: Impact of dexamethasone’ (Journal of Molecular Endocrinology, 38, 2007, 627–649) as an outcome of his research on PTP 1B inhibition. This is based on the elucidation of the molecular mode of action of an anti-diabetic candidate BMOV on the key components of insulin signaling cascades like IR Kinase (IRK) and IRS complexes including gross tyrosine phosphorylation of IRK and IRSs and site specific tyrosine phosphorylation of IRK like Tyr972 of NPEY motif, and Tyr1158, Tyr1162, Tyr1163 of PT Kinase activation segment of IR. The insulin signaling induction by this compound was also seen on Thr308 and Ser473 phosphorylation of Akt kinase in association with Ser9 phosphorylation of GSK-3ß kinase. He found that this signaling also regulates the Ser259 phosphorylation of Raf Kinase. On the other hand It has been proposed that glycation of insulin may also contribute to insulin resistance. To understand this more deeply, he has developed MALDI-TOF mass spectrometric technique for the identification and analysis of glycated insulin (Biochimica et Biophysica Acta, 1725, 2005, 269-282). Using this method he elucidated the molecular mechanism of insulin glycation, which might be considered as an important contribution to the diabetes research. On the other hand he has developed affi-MADI-TOF plates and functional as well as affi-magnetic beads oriented proteomic tools that would have potential future applications in mass spectrometry based biological and pharmaceutical applications, especially for identification of biomarkers for cancers and diabetes. Briefly, he has developed an efficient method for antibody immobilization on amino terminal magnetic beads that strongly favors proper orientation of the immunoglobulin with minimal chances of nonspecific binding due to removal of Fc region. He has also developed mass spectrometry based immunoassay technique on glyco-affinity MALDI-TOF plate that can be adopted as a useful method for characterization and profiling of biomolecules including biomarkers. Other future applications of those tools include cell biology, immunology, and glycobiology, protein purification etc. Accordingly, the assay conditions were based on ELISA oriented techniques as well as analysis through MALDI-TOF Mass Spectrometry. Some of the products are on the process of patenting and commercialization, while others are under development. The presented posters (also as abstracts in Mol. Cell Proteomics) are: 1) Oriented immobilization of monovalent antibody on magnetic beads: An approach for a proteomics tool. 6th Annual Congress, Human Proteomics Organization (HUPO), October 6–10, 2007, COEX, Seoul, Korea. 2) Immunoassay with glyco-specific affinity MALDI plates. 6th Annual Congress, Human Proteomics Organization (HUPO), October 6–10, 2007, COEX, Seoul, Korea.