Receptor Kinase Interactions In Bacterial Chemotaxis Revealed By Pulsed Dipolar Esr Spectroscopy

Bacterial chemotaxis refers to the movement of bacteria under influence of attractants and repellents. This signal transduction pathway is characterized by incredible gain, sensitivity and co-operativity. The extra-cellular domains of receptors transmit the signal from outside the cell into the cytoplasm where it is further processed by the signaling complex of histidine kinase CheA, coupling protein CheW and cytoplasmic domains of receptors. Structural determination of individual protein components and their protein complexes is necessary to gain insight into the mechanism. In this work, we have used site-directed spin labeling and long-range distance restraints from Pulsed dipolar ESR spectroscopy to predict the structure of a ternary complex formed by CheA, CheW and the signaling domain of the receptor. We have developed a novel method to refine the structures of protein complexes from the distance restraints provided by pulsed dipolar ESR. Apart from determining the position of receptor in the ternary complex, our efforts have been directed towards understanding the change in orientations of CheA and CheW in the presence of receptor. On a macroscopic scale, CheA, CheW and receptors form dense clusters at the poles of cell. We observed that CheA also self-associates in solution to some extent. We speculate that this self-association property of CheA may play a crucial role in clustering of chemotaxis proteins in cell. We have successfully identified this binding interface with disulphide cross-linking and novel application of pulsed dipolar ESR signals that report on local spin concentrations

Fluorescence monitoring of pH dependent complexation of chlorin p6 with surfactants

We have observed the pH-dependent formation of electrostatic complexes of chlorin p6 with surfactants in submicellar concentrations. Successive protonation of the ionizable groups cause a progressively increasing positive charge on the fluorophore as the pH decreases. Complexes are formed with the cationic CTAB at pH 7 and 5 and with the anionic SDS at pH 3, as manifested in a decrease in fluorescence quantum yield and radiative lifetime at low surfactant concentrations. The fluorescence quantum yield and lifetime are observed to increase beyond certain concentrations of the surfactants and saturate before the critical micellar concentrations are reached. The variation in the fluorescence parameters is rationalized by a competitive model involving pH-induced and surfactant-induced aggregates.© Elsevie

RSM optimization of microwave pyrolysis parameters to produce OPS char with high yield and large BET surface area

Microwave pyrolysis is a very effective technique to convert organic waste into energy rich products. In the present work micro porous and carbonaceous OPS char was synthesized by microwave pyrolysis technique. OPS char yield and its BET surface area was investigated as a function of process parameters to make the microwave pyrolysis of OPS more efficient. Both of these quantities were found to be affected by the process parameters. With the rise in microwave power (MWP) and radiation time (RT), OPS char yield was lowered while increase N2 flow rate (NFR) increased the OPS char yield slightly. Extreme values of either of the process parameter was seen to lower the BET surface area of OPS char. Response Surface Methodology (RSM), was used to optimize the process parameters for maximized OPS char yield and its BET surface area. Regression models for the OPS char yield and its BET surface area were also developed in this study. These regression models were having high Fischer test value & lower p-value which ensure its reliability and applicability. ANOVA analysis of the experimental data provided the process parameters to achieve maximized OPS char yield (60.93%) and BET surface area (250.03 m2/g). The predicted results were validated and it was found that the experimental data varied only by 5.99% in yield and 6.34% in BET surface area from the predicted values. © 2020 Elsevier Lt

BBA, a Derivative of 23-Hydroxybetulinic Acid, Potently Reverses ABCB1-Mediated Drug Resistance <i>in Vitro</i> and <i>in Vivo</i>

23-<i>O</i>-(1,4′-Bipiperidine-1-carbonyl)­betulinic acid (BBA), a synthetic derivative of 23-hydroxybetulinic acid (23-HBA), shows a reversal effect on multidrug resistance (MDR) in our preliminary screening. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCG2, and ABCC1 has been reported in recent studies to be a major factor contributing to MDR. Our study results showed that BBA enhanced the cytotoxicity of ABCB1 substrates and increased the accumulation of doxorubicin or rhodamine123 in ABCB1 overexpressing cells, but had no effect on non ABCB1 substrate, such as cisplatin; what’s more, BBA slightly reversed ABCG2-mediated resistance to SN-38, but did not affect the ABCC1-mediated MDR. Further studies on the mechanism indicated that BBA did not alter the expression of ABCB1 at mRNA or protein levels, but affected the ABCB1 ATPase activity by stimulating the basal activity at lower concentrations and inhibiting the activity at higher concentrations. In addition, BBA inhibited the verapamil-stimulated ABCB1 ATPase activity and the photolabeling of ABCB1 with [¹²⁵I] iodoarylazidoprazosin in a concentration-dependent manner, indicating that BBA directly interacts with ABCB1. The docking study confirmed this notion that BBA could bind to the drug binding site(s) on ABCB1, but its binding position was only partially overlapping with that of verapamil or iodoarylazidoprazosin. Importantly, BBA increased the inhibitory effect of paclitaxel in ABCB1 overexpressing KB-C2 cell xenografts in nude mice. Taken together, our findings suggest that BBA can reverse ABCB1-mediated MDR by inhibiting its efflux function of ABCB1, which supports the development of BBA as a novel potential MDR reversal agent used in the clinic