http://dkr.cdri.res.in:8080/xmlui/handle/123456789/745 2026-04-19T13:56:03Z http://dkr.cdri.res.in:8080/xmlui/handle/123456789/1194 Influence of vehicles used for oral dosing of test molecules on the progression of Mycobacterium tuberculosis infection in mice Singh, Shubhra; Dwivedi, Richa; Chaturvedi, Vinita Preclinical evaluation of drug-like molecules requires their oral administration to experimental animals using suitable vehicles. We studied the effect of oral dosing with corn oil, carboxymethyl cellulose, dimethyl sulphoxide and Polysorbate-80, on the progression of Mycobacterium tuberculosis infection in mice. Infection was monitored by physical (survival time, body weight) and bacteriological (viable counts in lungs) parameters. As compared with water, corn oil significantly improved both the parameters whereas the other vehicles affected only physical parameters 2012-01-01T00:00:00Z http://dkr.cdri.res.in:8080/xmlui/handle/123456789/1084 E6AP, an E3 ubiquitin ligase negatively regulates granulopoiesis by targeting transcription factor C/EBPα for ubiquitin mediated proteasome degradation Pal, Pooja; Lochab, Savita; Kanaujiya, J K; Kapoor, Isha; Sanyal, Sabyasachi; Behre, Gerhard; Trivedi, A K CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is an important transcription factor involved in granulocytic differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase targets C/EBPα for ubiquitin mediated proteasome degradation and thereby negatively modulates its functions. Wild type E6AP promotes ubiquitin dependent proteasome degradation of C/EBPα, while catalytically inactive E6AP-C843A rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (over expressed HEK293T) and myeloid cells. We show that E6AP mediated degradation of C/EBPα protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild type p42C/EBPα and mutant isoform p30C/EBPα, this may explain perturbed p42C/EBPα/p30C/EBPα ratio often observed in AML. We show that over expression of catalytically inactive E6AP-C843A in C/EBPα inducible K562- p42C/EBPα-ER cells inhibits E2 induced C/EBPα degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2 induced activation of C/EBPα in C/EBPα stably transfected cells (K562-C/EBPα-p42-ERcells) was further substantiated by siE6AP mediated knock down of E6AP in both K562-C/EBPα-p42-ER and 32dcl3 cells. Taken together, our data suggest that E6AP targeted C/EBPα protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBPα often experienced in myeloid leukemia 2013-01-01T00:00:00Z http://dkr.cdri.res.in:8080/xmlui/handle/123456789/1079 E3 Ubiquitin Ligase E6AP Negatively Regulates Adipogenesis by Downregulating Proadipogenic Factor C/EBPalpha Pal, Pooja; Lochab, Savita; Kanaujiya, J K; Kapoor, Isha; Sanyal, Sabyasachi; Behre, Gerhard; Trivedi, A K CCAAT/Enhancer Binding Protein Alpha (C/EBPa) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPa protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPa protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6APC843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPa expression by targeting it to ubiquitin-proteasome pathway for degradation. 2013-01-01T00:00:00Z http://dkr.cdri.res.in:8080/xmlui/handle/123456789/1069 SOLiDTM Sequencing of Genomes of Clinical Isolates of Leishmania donovani from India Confirm Leptomonas Co-Infection and Raise Some Key Questions Singh, Neeloo; Chikara, Surendra; Sundar, Shyam Background: Known as ‘neglected disease’ because relatively little effort has been applied to finding cures, leishmaniasis kills more than 150,000 people every year and debilitates millions more. Visceral leishmaniasis (VL), also called Kala Azar (KA)or black fever in India, claims around 20,000 lives every year. Whole genome analysis presents an excellent means to identify new targets for drugs, vaccine and diagnostics development, and also provide an avenue into the biological basis of parasite virulence in the L. donovani complex prevalent in India. Methodology/Principal Findings: In our presently described study, the next generation SOLiDTM platform was successfully utilized for the first time to carry out whole genome sequencing of L. donovani clinical isolates from India. We report the exceptional occurrence of insect trypanosomatids in clinical cases of visceral leishmaniasis (Kala Azar) patients in India. We confirm with whole genome sequencing analysis data that isolates which were sequenced from Kala Azar (visceral leishmaniasis) cases were genetically related to Leptomonas. The co-infection in splenic aspirate of these patients with a species of Leptomonas and how likely is it that the infection might be pathogenic, are key questions which need to be investigated. We discuss our results in the context of some important probable hypothesis in this article. Conclusions/Significance: Our intriguing results of unusual cases of Kala Azar found to be most similar to Leptomonas species put forth important clinical implications for the treatment of Kala Azar in India. Leptomonas have been shown to be highly susceptible to several standard leishmaniacides in vitro. There is very little divergence among these two species viz. Leishmania sp. and L. seymouri, in terms of genomic sequence and organization. A more extensive perception of the phenomenon of co-infection needs to be addressed from molecular pathogenesis and eco-epidemiological standpoint 2013-01-01T00:00:00Z