| Development of site-specific drug delivery systems |
Ph D Students The
programme aims at developing systems for intracellular delivery of drugs or
pharmacologically active agents selectively to specific cell types by
exploiting the efficiency of the process of receptor-mediated endocytosis. The
objectives of the project are (i) exploration of heme acquisition mechanism in
Leishmania donovani, (ii) drug targeting for overcoming multidrug
resistance in cancer chemotherapy, (iii) modulation of macrophage metabolism
through receptor-mediated delivery of biological response modifiers and (iv)
therapeutic strategies based on scavenger receptor-mediated delivery of
antisense oligonucleotides Exploration
of heme acquisition mechanism in L.donovani In the reporting year, we worked on molecular cloning of the gene for the hemoglobin-binding protein (HBP) from Leishmania based on a strategy of exploiting the partial amino acid sequencing data of seventeen distinct peptide fragments obtained by limited proteolysis of the purified HBP. BLAST searches revealed substantial homology of several of these peptide sequences from the purified HBP with the reported hexokinase sequence of Trypanosoma (1416 bp; 472 aa). Initially, eight of these seventeen peptide sequences from the purified HBP could be aligned with the hexokinase sequence of Trypanosoma to determine the position of the peptides. Several degenerate primers against the obtained peptide sequences were synthesized which were used in RT-PCR to amplify different segments of the HBP gene of Leishmania. A 400 bp fragment covering the putative 5’ end of the HBP gene of Leishmania was generated by RT-PCR using a forward primer against the mini-exon of Leishmania and a reverse primer against the peptide # 17 that maps at positions 120-126 on the amino acid sequence of Trypanosoma hexokinase. The segment contiguous to this fragment was amplified by RT-PCR using a forward primer against the peptide #17 and a reverse primer against the peptide # 15 which maps at positions 269-276 on the Trypanosoma hexokinase sequence which produced an expected 470 bp fragment. Similarly, the adjacent fragment was amplified by RT-PCR using a forward primer against the peptide #15 and a reverse primer against the peptide # 14 of HBP which maps at amino acid positions 455-462 on the Trypanosoma hexokinase sequence, close to the 3’ of the gene. This generated the expected 570 bp adjacent to the 3’ end of the HBP gene of Leishmania. These three fragments were cloned and sequenced. All the seventeen peptide sequences obtained from the purified protein by limited proteolysis could be found in the amino acid sequence deduced from the nucleotide sequence of the cloned fragments. Studies are in progress to confirm the sequence of the 3’ end of the putative HBP gene using 3’ RACE. Publications Original
peer-reviewed articles
Reviews/Proceedings
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