Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.     

Selective activation of antitumor activity of macrophages by the delivery of muramyl dipeptide using a novel polynucleotide-based carrier recognized by scavenger receptors

We have shown that muramyl dipeptide (MDP) conjugated to a 10-mer polyguanylic acid (PolyG) is specifically internalized by macrophages through scavenger receptor (SCR)-mediated endocytosis. Macrophages activated by PolyG-MDP displayed about 20-fold higher cytotoxic activity against nonmacrophage tumor cells compared to that elicited by free MDP. The PolyG-MDP was found to trigger the secretion of higher levels of interleukin-6, interleukin-1alpha, TNF-alpha, and nitric oxide in comparison to free MDP. Addition of antibodies directed against IL-6 and TNF-alpha to macrophage culture completely abrogated the tumoricidal response of PolyG-MDP, indicating that these two cytokines are primarily responsible for bioefficacy. This general approach of PolyG as a vehicle may find wide application in the delivery of genes and antisense oligonucleotides to macrophages.     

Antioxidant associated chemoprevention by selenomethionine in murine tumor model

Effectiveness of selenium in different forms like sodium selenite, selenocysteine and selenomethionine has been compared in four different doses, namely 4, 6, 8 and 10 ppm of each, in terms of their bioavailability and prolongation of survival of Dalton's lymphorna (DL) bearing mice. Selenomethionine, at a dose of 8 ppm, was found to be the most bioavailable and least cytotoxic form that was capable of increasing the life span of the tumour bearing hosts maximally (almost two-fold). Benef iciality of selenomethionine has also been studied by observing continuous changes brought about by this compound on the glutathione (GSH) level, glutathione peroxidase (GPx) activity and extent of lipid peroxidation in the hepatic tissue of the tumour bearing hosts, which are indispensable for a cell to function normally and are found to exhibit significantly altered behaviour in neoplastic cells. Selenomethionine caused the maintenance of high steady state GSH level and a normal GPx activity during the fist phase of tumour growth. It also controlled lipid peroxidation during the first 15-20 days following tumour transplantation. These conditions helped in the maintenance of intracellular redox balance, cellular integrity and metabolic rhythms of cells in DL bearing mice receiving selenomethionine.Affiliation     

Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis

BACKGROUND: Plant-derived flavonoids, which occur abundantly in our daily dietary intake, possess antitumor, antibacterial, and free radical scavenging properties. They form active constituents of a number of herbal and traditional medicines. Several flavonoids have been shown to exert their action by interacting with DNA topoisomerases and promoting site-specific DNA cleavage. Therefore, flavonoids are potential candidates in drug design. We report here that, although the flavonoids luteolin and quercetin are potent antileishmanial agents, luteolin has great promise for acting as a lead compound in the chemotherapy of leishmaniasis, a major concern in developing countries. MATERIALS AND METHODS: Kinetoplast DNA (kDNA) minicircle cleavage in drug-treated parasites was measured by electrophoresis of the total cellular DNA, followed by Southern hybridization using 32P labeled kDNA as a probe. Cell cycle progression and apoptosis were measured by flow cytometry using propidium iodide and fluorescein isothiocyanate (FITC)-labeled Annexin V. RESULTS: Luteolin and quercetin inhibited the growth of Leishmania donovani promastigotes and amastigotes in vitro, inhibited DNA synthesis in promastigotes, and promoted topoisomerase-II-mediated linearization of kDNA minicircles. The IC50 values of luteolin and quercetin were 12.5 microM and 45.5 microM, respectively. These compounds arrest cell cycle progression in L. donovani promastigotes, leading to apoptosis. Luteolin has no effect on normal human T-cell blasts. Both luteolin and quercetin reduced splenic parasite burden in animal models. CONCLUSION: Luteolin and quercetin are effective antileishmanial agents. Quercetin has nonspecific effects on normal human T cells, but luteolin appears nontoxic. So, luteolin can be a strong candidate for antileishmanial drug design.     

A scanning tunnelling microscopy study of Clostridium pasteurianum rubredoxin

Scanning tunnelling microscopy (STM), which can provide 'direct' and 'non-averaged' information on molecular structure in three dimensions, has been used to achieve sub-molecular resolution in a 'single molecule' of rubredoxin, an important iron-sulphur protein, at the gold (111)/water interface. The metal-ligand site [Fe(III)-Cys4] appears distinct because of an enhancement of the tunnelling current over this region compared to the surrounding protein structure.     

Bactericidal Activity of Curcumin I Is Associated with Damaging of Bacterial Membrane

Curcumin, an important constituent of turmeric, is known for various biological activities, primarily due to its antioxidant mechanism. The present study focused on the antibacterial activity of curcumin I, a significant component of commercial curcumin, against four genera of bacteria, including those that are Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa). These represent prominent human pathogens, particularly in hospital settings. Our study shows the strong antibacterial potential of curcumin I against all the tested bacteria from Gram-positive as well as Gram-negative groups. The integrity of the bacterial membrane was checked using two differential permeabilization indicating fluorescent probes, namely, propidium iodide and calcein. Both the membrane permeabilization assays confirmed membrane leakage in Gram-negative and Gram-positive bacteria on exposure to curcumin I. In addition, scanning electron microscopy and fluorescence microscopy were employed to confirm the membrane damages in bacterial cells on exposure to curcumin I. The present study confirms the broad-spectrum antibacterial nature of curcumin I, and its membrane damaging property. Findings from this study could provide impetus for further research on curcumin I regarding its antibiotic potential against rapidly emerging bacterial pathogens.     

Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain

Histone deacetylases (HDAC’s) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa–specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([¹⁸F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [¹⁸F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [¹⁸F]DFAHA and previously reported [¹⁸F]FAHA. PET imaging with [¹⁸F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [¹⁸F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.