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The methodology exploring the cardioprotective potential of the flavonoid Fisetin through its ability to modulate PPAR-γ was unraveled in the present study. Computational modelling through molecular docking based binding study of interactions between Fiestin and PPAR-γ revealed the potential role of Fisetin as an agonist of PPAR-γ. A murine model of cardiac ischemia-reperfusion injury was used to explore this further. Male Wistar Rats were randomly assigned to five groups. Fisetin (20 mg/kg; p. o) was administered for 28 days. Ischemia was induced for 45 min on the 29th day followed by 60 min of reperfusion. Fisetin pretreatment upregulated the expression of PPAR-γ in heart tissue significantly Cardioprotection was assessed by measurement of hemodynamic parameters, infarct size, ELISA for oxidative stress, immunohistochemistry and TUNEL assay for apoptosis, and western blot analysis for MAPK proteins and inflammation. PPAR-γ activation by fisetin led to significantly reduced infarct size, suppression of oxidative stress, reduction of cardiac injury markers, alleviation of inflammation, and inhibition of apoptosis The MAPK-based molecular mechanism showed a rise in a key prosurvival kinase, ERK1/ERK2 and suppression of JNK and p38 proteins. The aforementioned beneficial findings of fisetin were reversed on the administration of a specific antagonist of PPAR-γ. In conclusion, through our experiments, we have proved that fisetin protects the heart against ischemia-reperfusion injury and the evident cardioprotection is PPAR-γ dependant. In conclusion, our study has revealed a prime mechanism involved in the cardioprotective effects of fisetin. Hence, Fisetin may be evaluated in further clinical studies as a cardioprotective agent in patients undergoing reperfusion interventions.

Secretory signalling glycoprotein (SPX-40) from mammary gland is highly expressed during involution. This protein is involved in a programmed cell death during tissue remodelling which occurs at the end of lactation. SPX-40 was isolated and purified from buffalo (SPB-40) from the samples obtained during involution. One solution of SPB-40 was made by dissolving it in buffer containing 25 mM Tris-HCl and 50 mM NaCl at pH 8.0. Another solution was made by adding 25% ethanol to the above solution. The biological effects of SPB-40 dissolved in above two solutions were evaluated on MCF-7 breast cancer cell lines. Free SPB-40 indicated significant pro-apoptotic effects while ethanol exposed SPB-40 showed considerably reduced effects on the apoptosis. SPB-40 was crystallized in the native state. The crystals of SPB-40 were soaked in four separate solutions containing 25% acetone, 25% ethanol, 25% butanol and 25% MPD. Four separate data sets were collected and their structures were determined at high resolutions. In all the four structures, the molecules of acetone, ethanol, butanol and MPD respectively were observed in the hydrophobic binding pocket of SPB-40. As a result of which, the conformation of Trp78 was altered thus blocking the binding site in SPB-40 leading to the loss of activity.

DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 A resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.

Short peptidoglycan recognition protein (PGRP-S) is a member of the mammalian innate immune system. PGRP-S from Camelus dromedarius (CPGRP-S) has been shown to bind to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Its structure consists of four molecules A, B, C and D with ligand binding clefts situated at A-B and C-D contacts. It has been shown that LPS, LTA and PGN bind to CPGRP-S at C-D contact. The cleft at the A-B contact indicated features that suggested a possible binding of fatty acids including mycolic acid of Mycobacterium tuberculosis. Therefore, binding studies of CPGRP-S were carried out with fatty acids, butyric acid, lauric acid, myristic acid, stearic acid and mycolic acid which showed affinities in the range of 10(-5) to 10(-8) M. Structure determinations of the complexes of CPGRP-S with above fatty acids showed that they bound to CPGRP-S in the cleft at the A-B contact. The flow cytometric studies showed that mycolic acid induced the production of pro-inflammatory cytokines, TNF-α and IFN-γ by CD3+ T cells. The concentrations of cytokines increased considerably with increasing concentrations of mycolic acid. However, their levels decreased substantially on adding CPGRP-S.

Three COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs), etoricoxib, parecoxib, and nimesulide are widely prescribed against inflammatory conditions. However, their long term administration leads to severe conditions of cardiovascular complications and gastric ulceration. In order to minimize these side effects, C-terminal half (C-lobe) of colostrum protein lactoferrin has been indicated to be useful if co-administered with NSAIDs. Lactoferrin is an 80 kDa glycoprotein with two similar halves designated as N- and C-lobes. Since NSAID-binding site is located in the C-terminal half of lactoferrin, C-lobe was prepared from lactoferrin by limited proteolysis using proteinase K. The incubation of lactoferrin with serine proteases for extended periods showed that N-lobe was completely digested but C-lobe was resistant for more than 72 h indicating its long half life in the animal gut. The solution studies have shown that COX-2-specific NSAIDs bind to C-lobe with binding constants ranging from 10 −4 to 10 −5 M showing significant affinities for sequestering these compounds. In order to understand the mode of binding and sequestering properties, the complexes of C-lobe with all these three compounds, etoricoxib, parecoxib, and nimesulide were prepared and the structures of their complexes with C-lobe were determined at 2.2, 2.9, and 2.7 Ǻ resolutions, respectively. The analysis of the structures of complexes of C-lobe with NSAIDs clearly show that all the three compounds bind firmly at the same ligand-binding site in the C-lobe revealing the details of the interactions between C-lobe and NSAIDs. The mode of binding of COX-2-specific NSAIDs to C-lobe is similar to that of the binding of COX-2 non-specific NSAIDs to C-lobe.