Sesamol as a potential radioprotective agent: in vitro studies

Protection against radiation-induced DNA strand breaks is an important aspect in the design and development of a radioprotector. In this study, the radioprotective efficacy of sesamol, a natural antioxidant, was investigated in aqueous solution of plasmid DNA (pBR322) and compared with that of melatonin, a known antioxidant-based radioprotector. Thermal denaturation studies on irradiated calf thymus DNA were also carried out with sesamol and melatonin. Sesamol demonstrated greater radioprotective efficacy in both plasmid DNA and calf thymus DNA. To assess the radical scavenging capacity of sesamol and melatonin, 2-deoxyribose degradation, DPPH and ABTS assays were performed. Sesamol exhibited more scavenging capacity compared to melatonin. In vitro studies with V79 cells showed that sesamol is 20 times more potent than melatonin. It is proposed that the greater radioprotective efficacy of sesamol could be due to its greater capacity for scavenging of free radicals compared to melatonin. The results will be helpful in understanding the mechanisms and development of sesamol as a radioprotector.     

Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant ��-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization

A Bacillus sp., isolated from sludge and sediments of pulp and paper mill, was found to produce xylanase in a synthetic culture media containing oat spelt xylan (1% w/v) and 10% black liquor as inducers along with 2.5% (w/v) sucrose as additional carbon source. The purified enzyme was highly thermostable with half-life of 10 min at 90 °C and pH 8. The enzyme was stable over a broad range of pH (pH 6-10) and showed good thermal stability when incubated at 70 °C. Chemicals like EDTA, Hg²⁺, Cu²⁺ and solvents like glycerol and acetonitrile completely inhibited enzyme activity at high concentration. The molecular weights of the purified enzyme, determined by matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF/MS) analysis was analogous to the results obtained from SDS-PAGE, i.e. 55 kDa. Kinetic parameters were determined by using oat spelt xylan as substrate. The K_M and V_max values of the enzyme were 4.4 mg/ml and 287 U/mg respectively. At high xylan concentrations (>70 mg/ml) a substrate inhibition phenomenon of the enzyme was observed. In addition, crude xylanase showed enormous potential for decolorization of various recalcitrant dyes.     

Dephosphorization of LD slag by phosphorus solubilising bacteria

Phosphorus is one of the major nutrients, and microbial solubilisation of insoluble mineral phosphate in soil is an important process in natural ecosystem and in agricultural soil. Many soil microorganisms display the ability to solubilize many insoluble inorganic phosphates. They are generally referred as phosphorus solubilising microorganisms (PSM). In this study an attempt was made to look into the phosphorus solubilisation efficiency of some commonly available soil bacteria and their possible application in bio-beneficiation of metallurgical waste like LD Slag. Linz -Donawitz (LD) slag is produced in large quantities (200 kg LD slag per ton of hot metal) and poses a substantial disposal problem in the iron and steel making industry. LD slag contains around 29% Ca, 21% Fe, and 5% Mg. Its phosphorus content is about 1.5-6%. Due to presence of high amount of Ca, it can be used as flux in blast furnace, but presence of high amount of phosphorus in the LD slag makes them unsuitable for industrial application. Removal of phosphorus with the help of phosphorus solubilising microorganisms may be a great advantage in biotechnological applications. Two gram positive bacteria belonging to genus Bacillus and two gram negative bacteria belonging to genus Pseudomonas were selected in this study. Phosphorus solubilisation efficiency was studied initially with tricalcium phosphate as model insoluble phosphate compound at different sugar concentration, NaCl concentration and at different initial pH of the medium. About 35% of ‘P’ could be solubilized from LD slag by Pseudomonas aeruginosa at 2% solid content.     

Study on reaction mechanism of bioleaching of nickel and cobalt from lateritic chromite overburdens

Depletion of high-grade ores and presence of significant quantities of metals in low-grade oxide ores has enforced to utilize the overburdens (COB) and wastes (low-grade ores) generated during mining operations. The impact of ore mineralogy and mineral–microbe interaction during bioleaching could not be ignored. Seeking to the need, a systematic study was performed to establish the reaction mechanism involved for recovery of nickel and cobalt from chromite overburden (COB), Sukinda, Orissa using pure culture of Aspergillus niger. Mineralogical analysis reveals a complete conversion of goethite into hematite phase leading to exposure of nickel particles into the micro-pores and cracks developed in the matrix which was initially found to be intertwined in the goethite lattice. As a result, it became more susceptible to attack by the fungal bio acids which in turn accelerate the dissolution rate. Organic acids like oxalic and citric acids were detected in the culture filtrate using HPLC. TEM analysis of the leached samples shows that nickel dissolute into the solution leaving a porous space in the matrix of the hematite by forming nickel oxalate or nickel citrate. Kinetics of the nickel bioleaching was studied to support the mechanism of the reaction. It was observed that the initial rate of reaction follows the chemical control dissolution reaction where as the later part fits to shrinking core model. 18% of nickel and 37.8% of cobalt was recovered from pre-treated COB at 2.5% pulp-density with 10% (v/v) fungal inoculum at 30 °C within 25 days in shake flask while 32.5% of nickel and 86% of cobalt was recovered in bioreactor.     

Antigen-Antibody docking reveals the molecular basis for cross-reactivity of the 1918 and 2009 Influenza A/H1N1 pandemic viruses

To understand the reported cross-reactivity of the 2009 H1N1 and the 1918 H1N1 pandemic viruses we docked the crystal structure of 2D1, an antibody derived from a survivor of the 1918 pandemic, to the structures of hemaglutinin (HA) of the 2009 strain and seasonal H1 vaccine strains. Our studies revealed that 2D1 binds to the 2009 HA at antigenic site 'Sa', with stabilizing contacts, similar to that in an available co-crystal structure of 2D1-1918 HA. However, 2D1 failed to bind to the known antigenic sites in the HAs of seasonal strains. Our study thus reveals the molecular basis for pre-existing immunity in elderly people to the 2009 pandemic virus.     

Identification of EPAC (Exchange Protein Activated by cAMP) bioinformatically as a potential signalling biomarker in Cardiovascular Disease (CVD) and its molecular docking by a lead molecule

The present work delineates the combinatorial approach of firstly, creation of a centralized data-set comprising signalling proteins identified on the basis of altered expression, such as over-expression or repression of a set of signalling protein(s) leading to the cause of the disease, which is based on published reports screened through Pubmed and secondly, in the in silico creation of novel lead (drug) molecules and docking of identified signalling biomarkers using such drugs to investigate possibility of their future application in the model systems eventually. EPAC (Exchange Protein Activated by cAMP) emerges as a signalling biomarker in cases studied presently. Brefeldin, the known inhibitor of EPAC, though the mechanism yet unexplored, has been the molecule used as the pharmacophore for creation of lead drug molecule. Various modifications have been incorporated into the pharmacophore to increase the hydrophobic interactions for increasing the binding efficiency of the generated lead molecule. Side-chain modifications of the pharmacophore and refinement of data through firedock upon docking of EPAC with the modified pharmacophore yielded best results on the bases of atomic contact energy, van der Waal and partial electrostatic interactions as well as additional estimations of the binding free energy. Modifications of CH3 at C15 with COOH and H at C2 with OH in brefeldin showed the best docking results on the basis of protein-drug interaction parameters. The present work provides a clue in rational design of EPAC inhibitors which could be developed as drug lead in combating CVD.     

Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides

Recently, attention has been focused on pharmacological treatments that increase HDL cholesterol to prevent coronary artery disease. Despite three decades of extensive research of human apolipoprotein A-I (apoA-I), the major protein component of HDL, the molecular basis for its antiatherogenic and anti-inflammatory functions remain elusive. Another protein component of HDL, apoA-II, has structural features similar to those of apoA-I but does not possess atheroprotective properties. To understand the molecular basis for the effectiveness of apoA-I, we used model synthetic peptides. We designed analogs of the class A amphipathic helical motif in apoA-I that is responsible for solubilizing phospholipids. None of these analogs has sequence homology to apoA-I, but all are similar in their lipid-associating structural motifs. Although all of these peptide analogs interact with phospholipids to form peptide:lipid complexes, the biological properties of these analogs are different. Physical-chemical and NMR studies of these peptides have enabled the delineation of structural requirements for atheroprotective and anti-inflammatory properties in these peptides. It has been shown that peptides that interact strongly with lipid acyl chains do not have antiatherogenic and anti-inflammatory properties. In contrast, peptides that associate close to the lipid head group (and hence do not interact strongly with the lipid acyl chain) are antiatherogenic and anti-inflammatory. Understanding the structure and function of apoA-I and HDL through studies of the amphipathic helix motif may lead to peptide-based therapies for inhibiting atherosclerosis and other related inflammatory lipid disorders.     

Liposomes as adjuvant for combination vaccines

In the present study tetanus toxoid (TT) loaded liposomes and diphtheria toxoid (DT) loaded liposomes were prepared by reverse phase evaporation method and after combining these two vaccines the potential advantages were investigated. Prepared systems were characterized for the size, shape and entrapment efficiency. SDS-PAGE analysis of TT and DT was also performed. The selected liposomal formulations were administered subcutaneously to Balb/c mice and their immune responses were determined using ELISA after 15, 30, 45 days. After boosting the maximum immune response was observed after 45 days and was found to be 0.831 and 0.749 for TT loaded liposome and DT loaded liposomes respectively. When the mice were immunized subcutaneously with the physical mixture of TT loaded liposomes and DT loaded liposomes the immune response for the combination vaccine was found to be 1.44 and 0.741 for the TT and DT respectively. The result showed that the immune response of TT increased when it was combined with DT in liposomes. This confirms adjuvantcity of DT vis-a-vis immunogenicity. Thus, carrier mediated cocktail vaccination holds promise for clinical applications.     

Role of a putative polysaccharide locus in Bordetella biofilm development

Bordetellae are gram-negative bacteria that colonize the respiratory tracts of animals and humans. We and others have recently shown that these bacteria are capable of living as sessile communities known as biofilms on a number of abiotic surfaces. During the biofilm mode of existence, bacteria produce one or more extracellular polymeric substances that function, in part, to hold the cells together and to a surface. There is little information on either the constituents of the biofilm matrix or the genetic basis of biofilm development by Bordetella spp. By utilizing immunoblot assays and by enzymatic hydrolysis using dispersin B (DspB), a glycosyl hydrolase that specifically cleaves the polysaccharide poly-beta-1,6-N-acetyl-D-glucosamine (poly-beta-1,6-GlcNAc), we provide evidence for the production of poly-beta-1,6-GlcNAc by various Bordetella species (Bordetella bronchiseptica, B. pertussis, and B. parapertussis) and its role in their biofilm development. We have investigated the role of a Bordetella locus, here designated bpsABCD, in biofilm formation. The bps (Bordetella polysaccharide) locus is homologous to several bacterial loci that are required for the production of poly-beta-1,6-GlcNAc and have been implicated in bacterial biofilm formation. By utilizing multiple microscopic techniques to analyze biofilm formation under both static and hydrodynamic conditions, we demonstrate that the bps locus, although not essential at the initial stages of biofilm formation, contributes to the stability and the maintenance of the complex architecture of Bordetella biofilms.     

ApoA-I mimetic peptides with differing ability to inhibit atherosclerosis also exhibit differences in their interactions with membrane bilayers

Two homologous apoA-I mimetic peptides, 3F-2 and 3F(14), differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404-51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 microg/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 +/- 1000 microm(2), n = 29), whereas 3F(14) does not (lesion area 20,400 +/- 1000 microm(2), n = 26) compared with control saline administered (19,900 +/- 1400 microm(2), n = 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F(14) preferentially associates with apoB-containing particles. After intraperitoneal injection of (14)C-labeled peptides, 3F(14) reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F(14). In contrast, only 3F(14) affects the terminal methyl group of the acyl chain, decreasing the (2)H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F(14) can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.