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.     

Application of plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus

Papaya ringspot virus (PRSV-W) and Tomato chlorotic spot virus (TCSV) are responsible for severe losses in cucurbits and tomato production in south Florida and other regions in the USA. Traditional chemicals are not effective to control these viruses. Using plant growth-promoting rhizobacteria (PGPR) may present an alternative to control these viruses. Results from this study demonstrated that applying mixtures of PGPR strains is more efficient to control PRSV-W and TCSV compared to individual PGPR strain only. The application method significantly affected the efficiency of PGPR to control PRSV-W and TCSV. The highest reduction in disease severity of both PRSV-W and TCSV occurred in case of soil drenching with PGPR, followed by root dipping and seed coating treatments. Application of PGPR mixtures of (IN937a & SE34) or (IN937a &, SE34 & T4) were the most efficient methods to control these viral diseases.     

Co-translational Folding Intermediate Dictates Membrane Targeting of the Signal Recognition Particle Receptor

Much of our knowledge on the function of proteins is deduced from their mature, folded states. However, it is unknown whether partially synthesized nascent protein segments can execute biological functions during translation and whether their premature folding states matter. A recent observation that a nascent chain performs a distinct function, co-translational targeting in vivo, has been made with the Escherichia coli signal recognition particle receptor FtsY, a major player in the conserved pathway of membrane protein biogenesis. FtsY functions as a membrane-associated entity, but very little is known about the mode of its targeting to the membrane. Here we investigated the underlying structural mechanism of the co-translational FtsY targeting to the membrane. Our results show that helices N_2–4, which mediate membrane targeting, form a stable folding intermediate co-translationally that greatly differs from its fold in the mature FtsY. These results thus resolve a long-standing mystery of how the receptor targets the membrane even when deleted of its alleged membrane targeting sequence. The structurally distinct targeting determinant of FtsY exists only co-translationally. Our studies will facilitate further efforts to seek cellular factors required for proper targeting and association of FtsY with the membrane. Moreover, the results offer a hallmark example for how co-translational nascent intermediates may dictate biological functions.     

Selection and application of antibodies modifying the function of beta-lactamase

Nine monoclonal antibodies directed against class A beta-lactamases were detected and selected by a novel screening procedure based on assaying the modifications in the catalytic and stability properties of beta-lactamase in solution. Unlike conventional screening, e.g., ELISA or immunoprecipitation, the present method does not depend on firm binding and thus favors detection of low affinity antibodies. Individual antibodies were found to affect the enzymatic activity in various ways including stimulation, neutralization, protection and stabilization. Class A beta-lactamases show only 20% among members of this class. In contrast, two of our monoclonal antibodies cross-reacted with different beta-lactamases and thus demonstrate the presence of shared structural epitopes in this class of enzymes. One of the cross-reacting antibodies was elicited by sequential immunization with two different beta-lactamases. Taken together, our findings stress the importance of the screening method in antibody selection and illustrate the use of 'functional' monoclonal antibodies in the study of the structure-function relationship in an enzyme.     

Calreticulin is crucial for calcium homeostasis mediated adaptation and survival of thick ascending limb of Henle's loop cells under osmotic stress

The thick ascending limb of Henle's loop (TALH) is normally exposed to variable and often very high osmotic stress and involves different mechanisms to counteract this stress. ER resident calcium binding proteins especially calreticulin (CALR) play an important role in different stress balance mechanisms. To investigate the role of CALR in renal epithelial cells adaptation and survival under osmotic stress, two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry and functional proteomics were performed. CALR expression was significantly altered in TALH cells exposed to osmotic stress, whereas renal inner medullary collecting duct cells and interstitial cells exposed to hyperosmotic stress showed no significant changes in CALR expression. Moreover, a time dependent downregulation of CALR was accompanied with continuous change in the level of free intracellular calcium. Inhibition of the calcium release, through IP3R antagonist, prevented CALR expression alteration under hyperosmotic stress, whereas the cell viability was significantly impaired. Overexpression of wild type CALR in TALH cells resulted in significant decrease in cell viability under hyperosmotic stress. In contrast, the hyperosmotic stress did not have any effect on cells overexpressing the CALR mutant, lacking the calcium-binding domain. Silencing CALR with siRNA significantly improved the cell survival under osmotic stress conditions. Taken together, our data clearly highlight the crucial role of CALR and its calcium-binding role in TALH adaptation and survival under osmotic stress.     

Determinants of substrate recognition by the Escherichia coli multidrug transporter MdfA identified on both sides of the membrane

The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 (cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteine-less MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (Deltamicro(H(+)), inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.     

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.