Rapid and reliable protein structure determination via chemical shift threading

Protein structure determination using nuclear magnetic resonance (NMR) spectroscopy can be both time-consuming and labor intensive. Here we demonstrate how chemical shift threading can permit rapid, robust, and accurate protein structure determination using only chemical shift data. Threading is a relatively old bioinformatics technique that uses a combination of sequence information and predicted (or experimentally acquired) low-resolution structural data to generate high-resolution 3D protein structures. The key motivations behind using NMR chemical shifts for protein threading lie in the fact that they are easy to measure, they are available prior to 3D structure determination, and they contain vital structural information. The method we have developed uses not only sequence and chemical shift similarity but also chemical shift-derived secondary structure, shift-derived super-secondary structure, and shift-derived accessible surface area to generate a high quality protein structure regardless of the sequence similarity (or lack thereof) to a known structure already in the PDB. The method (called E-Thrifty) was found to be very fast (often?<?10 min/structure) and to significantly outperform other shift-based or threading-based structure determination methods (in terms of top template model accuracy)—with an average TM-score performance of 0.68 (vs. 0.50–0.62 for other methods). Coupled with recent developments in chemical shift refinement, these results suggest that protein structure determination, using only NMR chemical shifts, is becoming increasingly practical and reliable. E-Thrifty is available as a web server at http://ethrifty.ca.     

Industrial wastewater treatment in internal circulation bioreactor followed by wetlands containing emergent plants and algae

Wastewater treatment based on ecological principles is a low cost and highly desirable solution for the developing countries like Pakistan. The present study evaluated the effectiveness of biological treatment systems including Internal Circulation (IC) anaerobic bioreactor and constructed wetlands (CWs) containing macrophytes and mixed algal cultures for industrial wastewater treatment. The IC bioreactor reduced COD (52%), turbidity (89%), EC (24%) of the industrial wastewater. However, the effluents of IC bioreactor did not comply with National Environmental Quality Standards (NEQS) of Pakistan. Post-treatment of IC bioreactor effluents was accomplished in CW containing macrophytes (Arundo donax and Eichhornia crassipes) and mixed algal culture. The CWs planted with macrophytes lowered the concentrations of COD (89%) and turbidity (99%). CWs with algal biomass were not effective in further polishing the effluent. Inhibition of algal biomass growth was observed due to physicochemical characteristics of wastewater. The integrated treatment system consisting of IC bioreactor and macrophytes was found more suitable option for industrial wastewater treatment.     

Theoretical insights into thermal cyclophanediene to dihydropyrene electrocyclic reactions; a comparative study of Woodward Hoffmann allowed and forbidden reactions

The thermally allowed electrocyclic reaction syn-cyclophanediene (CPD) to dihydropyrene (DHP) was compared with the disallowed thermal electrocyclic reaction in anti CPD through density functional theory (DFT) calculations at the B3LYP/6-31?+?G(d) level. Moreover, the results were also compared with the electrocyclization of 1,3,5 hexatriene to 1,3-cyclohexadiene . The Woodward-Hoffmann (W-H) allowed thermal reaction in syn CPD 11 has a calculated activation barrier of 6.23 kcal mol^?1, compared with 29 kcal mol^?1 for the electrocyclization of 1,3,5 hexatriene to 1,3-cyclohexadiene. The enhanced acceleration of electrocyclization is believed to arise from geometrically enforced spatially aligned termini of the hexatriene. Substituents at the electrocyclic terminus of cyclophanediene significantly affected (up to three fold) the activation barriers. Mono-substitution of CPD has substituent dependent acceleration or deceleration whereas di-substitution always increased the activation barrier. The activation barrier for electrocyclization in 33 is 4.44 kcal mol^?1, which is the lowest activation barrier for any thermal electrocyclic reaction. Cyclophanedienes (CPDs) substituted with electron-rich substituents cyclized with high activation barriers and vice versa, a phenomenon significantly different from electrocyclic reaction of 1,3,5-hexatriene where no such trend is traceable. Comparison of W-H allowed and forbidden electrocyclization in syn and anti CPDs, respectively, revealed quite similar electronic demand, although the transition states are different in nature. The transition state for a W-H forbidden reaction is biradicaloid, with most of the spin density at the electrocyclic termini; however, the transition state for a W-H allowed reaction has no such contribution. We also believe that this is the first study of its type, where W-H allowed and forbidden reactions are compared on a similar set of molecules, and compared for electronic effect through substituents.     

Lipophilic cations: a group of model substrates for the multidrug-resistance transporter.

The possibility that simple lipophilic cations such as tetraphenylphosphonium (TPA+), triphenylmethylphosphonium (TPMP+), and diphenyldimethylphosphonium (DDP+) are substrates for the multidrug-resistance transport protein, P-glycoprotein, was tested. Hamster cells transfected with and overexpressing mouse mdr1 or mouse mdr3 exhibit high levels of resistance to TPP+ and TPA+ (20-fold) and somewhat lower levels of resistance to TPMP+ and DDP+ (3-12-fold). Transfected cell clones expressing mdr1 or mdr3 mutants with decreased activity against drugs of the MDR spectrum (e.g., Vinca alkaloids and anthracyclines) also show reduced resistance to lipophilic cations. Studies with radiolabeled TPP+ and TPA+ demonstrate that increased resistance to cytotoxic concentrations of these lipophilic cations is correlated quantitatively with a decrease in intracellular accumulation in mdr1- and mdr3-transfected cells. This decreased intracellular accumulation is shown to be strictly dependent on intact intracellular nucleotide triphosphate pools and is reversed by verapamil, a known competitive inhibitor of P-glycoprotein. Taken together, these results demonstrate that lipophilic cations are a new class of substrates for P-glycoprotein and can be used to study its mechanism of action in homologous and heterologous systems.     

Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions

In plants, peroxisomes are the primary site of fatty acid β-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4(Col) and acx1acx3acx4(Col) mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial β-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.     

In vitro antituberculosis activities of the constituents isolated from Haloxylon salicornicum

In vitro antituberculosis activities of fractions and pure compounds (1–20) including seven triterpenes, two alkaloids, two cycloheximide derivatives, two coumarins six sterol derivatives and a long chain alcohol, respectively, isolated from Haloxylon salicornicum were determined against Mycobecterium tuberculosis H37Rv. Actively growing cultures were tested by rapid colorimetric method while the stationary phase cultures were tested by drug exposure methods for bactericidal activity. The MIC values were found to be 50 μg/ml for compounds 15, 19 and 20 where as rest of the compounds invariably showed MIC value of 100 μg/ml against the logarithmic phase culture. These were compare to Isoniazid as a control drug. The compounds exhibited no activity against the stationary phase culture of M. tuberculosis H37Rv up to 200 μg/ml. Further studies are required to investigate the in vivo efficacies and activities of the compounds in combination with antimicrobials that are already being used for TB therapy.     

A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA

The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.