High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

Heat Shock Protein 90 as a Drug Target against Protozoan Infections Biochemical characterization of HSP90 From plasmodium falciparum and trypanosoma evansi and evaluation of its inhibitor as a candidate drug

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

High-quality annotation of promoter regions for 913 bacterial genomes

Motivation: The number of bacterial genomes being sequenced is increasing very rapidly and hence, it is crucial to have procedures for rapid and reliable annotation of their functional elements such as promoter regions, which control the expression of each gene or each transcription unit of the genome. The present work addresses this requirement and presents a generic method applicable across organisms. Results: Relative stability of the DNA double helical sequences has been used to discriminate promoter regions from non-promoter regions. Based on the difference in stability between neighboring regions, an algorithm has been implemented to predict promoter regions on a large scale over 913 microbial genome sequences. The average free energy values for the promoter regions as well as their downstream regions are found to differ, depending on their GC content. Threshold values to identify promoter regions have been derived using sequences flanking a subset of translation start sites from all microbial genomes and then used to predict promoters over the complete genome sequences. An average recall value of 72% (which indicates the percentage of protein and RNA coding genes with predicted promoter regions assigned to them) and precision of 56% is achieved over the 913 microbial genome dataset.

Taking Multiple Infections of Cells and Recombination into Account Leads to Small Within-Host Effective-Population-Size Estimates of HIV-1

Whether HIV-1 evolution in infected individuals is dominated by deterministic or stochastic effects remains unclear because current estimates of the effective population size of HIV-1 in vivo, N-e, are widely varying. Models assuming HIV-1 evolution to be neutral estimate N-e similar to 10(2)-10(4), smaller than the inverse mutation rate of HIV-1 (similar to 10(5)), implying the predominance of stochastic forces. In contrast, a model that includes selection estimates N-e>10(5), suggesting that deterministic forces would hold sway. The consequent uncertainty in the nature of HIV-1 evolution compromises our ability to describe disease progression and outcomes of therapy. We perform detailed bit-string simulations of viral evolution that consider large genome lengths and incorporate the key evolutionary processes underlying the genomic diversification of HIV-1 in infected individuals, namely, mutation, multiple infections of cells, recombination, selection, and epistatic interactions between multiple loci. Our simulations describe quantitatively the evolution of HIV-1 diversity and divergence in patients. From comparisons of our simulations with patient data, we estimate N-e similar to 10(3)-10(4), implying predominantly stochastic evolution. Interestingly, we find that N-e and the viral generation time are correlated with the disease progression time, presenting a route to a priori prediction of disease progression in patients. Further, we show that the previous estimate of N-e>10(5) reduces as the frequencies of multiple infections of cells and recombination assumed increase. Our simulations with N-e similar to 10(3)-10(4) may be employed to estimate markers of disease progression and outcomes of therapy that depend on the evolution of viral diversity and divergence.

Detection of nif genes in nitrogen-fixing bacterial isolate from tomato (Lycopersicon esculentum Mill cv Pusa ruby)

Nitrogen-fixing bacterial isolate from the intercellular spaces of tomato root cortical cells was studied for the location of nif genes on the chromosomal or plasmid DNA. The bacterial isolate showed two plasmids of approximate molecular sizes of 220 and 120 kb. Klebsiella pneumoniae nif HDK probe hybridized with the chromosomal DNA and not with the plasmid DNA thereby showing that nif genes are localised on the chromosomal DNA.

Role of bacterial growth conditions and adhesion in the bioleaching of chalcopyrite by Thiobacillus ferrooxidans

The role of growth conditions and adhesion of Thiobacillus ferrooxidans on the leaching of chalcopyrite was investigated. Thiobacillus ferrooxidans grown on sulfur, thiosulfate and ferrous ion substrates was used in this comparative study. Growth on sulfur, a solid substrate, requires bacterial adhesion unlike that required in the presence of soluble thiosulfate and ferrous ion in a mineral-salts medium. Solid substrate-grown cells showed higher rates of leaching than those grown in liquid media. An initial lag period noticed during leaching by solution-grown cells was absent when solid substrate-grown cells were used. Such a behavior is attributed to the presence of an inducible proteinaceous cell-surface appendage on the sulfur-grown cells. This appendage aids in bacterial adhesion onto the mineral surfaces. Such an appendage is absent in solution-grown cells, as substantiated by electrophoretic measurements. The importance of bacterial adhesion and the direct mechanism in leaching by Thiobacillus ferrooxidans are demonstrated.

Autocalibration of dynamic bacterial growth model for water distribution system using GA

The presence of residual chlorine and organic matter govern the bacterial regrowth within a water distribution system. The bacterial growth model is essential to predict the spatial and temporal variation of all these substances throughout the system. The parameters governing the bacterial growth and biodegradable dissolved organic carbon (BDOC) utilization are difficult to determine by experimentation. In the present study, the estimation of these parameters is addressed by using simulation-optimization procedure. The optimal solution by genetic algorithm (GA) has indicated that the proper combination of parameter values are significant rather than correct individual values. The applicability of the model is illustrated using synthetic data generated by introducing noise in to the error-free measurements. The GA was found to be a potential tool in estimating the parameters controlling the bacterial growth and BDOC utilization. Further, the GA was also used for evaluating the sensitivity issues relating parameter values and objective function. It was observed that mu and k(cl) are more significant and dominating compared to the other parameters. But the magnitude of the parameters is also an important issue in deciding the dominance of a particular parameter. GA is found to be a useful tool in autocalibration of bacterial growth model and a sensitivity study of parameters.

Bacterial transformation using micro-shock waves

Shock waves are one of the most competent mechanisms of energy dissipation observed in nature. We have developed a novel device to generate controlled micro-shock waves using an explosive-coated polymer tube. In this study, we harnessed these controlled micro-shock waves to develop a unique bacterial transformation method. The conditions were optimized for the maximum transformation efficiency in Escherichia coli. The maximum transformation efficiency was obtained when we used a 30 cm length polymer tube, 100 mu m thick metal foil, 200 mM CaCl(2), 1 ng/mu l plasmid DNA concentration, and 1 x 10(9) cell density. The highest transformation efficiency achieved (1 x 10(-5) transformants/cell) was at least 10 times greater than the previously reported ultrasound-mediated transformation (1 x 10(-6) transformants/cell). This method was also successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonella typhimurium. This novel method of transformation was shown to be as efficient as electroporation with the added advantage of better recovery of cells, reduced cost (40 times cheaper than a commercial electroporator), and growth phase independent transformation. (C) 2011 Elsevier Inc. All rights reserved.

Surface chemical and adsorption studies using Thiobacillus ferrooxidans with reference to bacterial adhesion to sulfide minerals

Adhesion of Thiobacillus ferrooxidans to pyrite and chalcopyrite in relation to its importance in bioleaching and bioflotation has been studied. Electrokinetic studies as well as FT-IR spectra suggest that the surface chemistry of Thiobacillus ferrooxidans depends on bacterial growth conditions. Sulfur-,Pyrite- and chalcopyrite-grown Thiobacillus ferrooxidans were found to be relatively more hydrophobic. The altered surface chemistry of Thiobacillus ferrooxidans was due to secretion of newer and specific proteinaceous compounds. The adsorption density corresponds to a monolayer coverage in a horizontal orientation of the cells. The xanthate flotation of pyrite in presence of Thiobacillus ferrooxidans is strongly depressed where as the cells have insignificant effect on chalcopyrite flotation. This study demonstrate that: (a)Thiobacillus ferrooxidans cells can be used for selective flotation of chalcopyrite from pyrite and importantly at natural pH values. (b)Sulfur-grown cells exhibits higher leaching kinetics than ferrous ion-grown cells.

Deorphanization of Malonyl CoA:ACP Transacylase Drug Target in Plasmodium falciparum (PfFabD) Using Bacterial Antagonists: A Piggyback' Approach for Antimalarial Drug Discovery

Quest for new drug targets in Plasmodium sp. has underscored malonyl CoA:ACP transacylase (PfFabD) of fatty acid biosynthetic pathway in apicoplast. In this study, a piggyback approach was employed for the receptor deorphanization using inhibitors of bacterial FabD enzymes. Due to the lack of crystal structure, theoretical model was constructed using the structural details of homologous enzymes. Sequence and structure analysis has localized the presence of two conserved pentapeptide motifs: GQGXG and GXSXG and five key invariant residues viz., Gln109, Ser193, Arg218, His305 and Gln354 characteristic of FabD enzyme. Active site mapping of PfFabD using substrate molecules has disclosed the spatial arrangement of key residues in the cavity. As structurally similar molecules exhibit similar biological activities, signature pharmacophore fingerprints of FabD antagonists were generated using 0D-3D descriptors for molecular similarity-based cluster analysis and to correlate with their binding profiles. It was observed that antagonists showing good geometrical fitness score were grouped in cluster-1, whereas those exhibiting high binding affinities in cluster-2. This study proves important to shed light on the active site environment to reveal the hotspot for binding with higher affinity and to narrow down the virtual screening process by searching for close neighbors of the active compounds.

Moderate intensity static magnetic field has bactericidal effect on E. coli and S. epidermidis on sintered hydroxyapatite

The application of electromagnetic field in the context of bacteria associated infections on biomaterial surfaces has not been extensively explored. In this work, we applied a moderate intensity static magnetic field (100 mT) to understand the adhesion and growth behavior of both gram positive (S. epidermidis) and gram negative bacteria (E. coli) and also to investigate bactericidal/bacteriostatic property of the applied electromagnetic field. An in-house built magnetometer was used to apply static homogeneous magnetic field during a planned set of in vitro experiments. Both the sintered hydroxyapatite (HA) and the control samples seeded with bacteria were exposed to the magnetic field (100 mT) for different timescale during their log phase growth. Quantitative analysis of the SEM images confirms the effect of electromagnetic field on suppressing bacterial growth. Furthermore, cell integrity and inner membrane permeabilization assays were performed to understand the origin of such effect. The results of these assays were statistically analyzed to reveal the bactericidal effect of magnetic field, indicating cell membrane damage. Under the investigated culture conditions, the bactericidal effect was found to be less effective for S. Epidermidis than E. coli. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2012:100B:12061217, 2012.