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Voltage clamp recordings were made from visually identified CA1 pyramidal neurons and synaptic currents were evoked in the Schaffer collateral pathway. For UV photolysis of caged glutamate, direct current responses were measured by uncaging Entinostat glutamate directly over the pyramidal cell body UV power was calibrated to give an initial current amplitude of between 150 and 200 pA. The recombinant mycobacterial strains were grown in the presence of 0. 012% MMS and SEM observation was carried out as described in Materials and Methods. Representative images are shown. The images were taken at 80006 magnification.

Bars, 2 mm. Figure 4. Effects of MsTAG and its co expression with MsParA on mycobacterial growth and morphology. A portion of an alignment of 3 methyladenine DNA glycosylase is shown with conserved catalytic residues Glu indicated by an arrow. Comparative CUDC-101 growths of E. coli overexpressing the Tag gene b3459 and M. smegmatis strain overexpressing MsTAG on 7H10 agar plates with or without 0. 012% MMS at 37uC. Co IP assays for the interaction between the MsTAG HSP mutant and MsParA. MMS sensitivity assays. Growth of M. smegmatis strains overexpressing MsTAG or its mutant variant and those co expressing MsTAG and MsParA in 7H9 medium with and without 0. 012% MMS were compared. Aliquots were taken at the indicated times and the OD600 was measured as described in Materials and Methods.

Each analysis was performed in triplicate. Representative growth curves are shown. Scanning electron microscopy assay of cell morphology. The experiment was carried out as described in Materials and Methods. The recombinant mycobacterial strains were grown in 7H9 medium supplemented with 0. 012% MMS. Representative images are shown. The images were taken at 80006 magnification. Bars, 2 mm. The ortholog of M. smegmatis MsTAG in M. tuberculosis is Rv1210 . In the above assays, we had shown that MtTAG interacted with MtParA . Here we used a co IP assay and further confirmed the cross species interaction between the M. smegmatis MsParA and MtTAG, which was expressed using a pMind recombinant plasmid in M. smegmatis. Taken together, our results show that M.

tuberculosis MtTAG can cross interact CP-690550 with M. smegmatis MsTAG and inhibit its ATPase activity. Moreover, overexpression of MtTAG had a similar effect as MsTAG on the growth rate and cell morphology of M. smegmatis. Figure 5. MsTAG regulates the ATPase activity of COX Inhibitors . ATPase activity was determined as described under Materials and Methods. Reactions were performed in a volume of 50 mL and were terminated by the addition of 50 mL malachite green reagent. Absorbance was measured at 630 nm for the color reactions. A calibration curve was constructed using 0 25 mmol inorganic phosphate standards and samples were normalized for acid hydrolysis of ATP by the malachite green reagent. Time course ATPase activity assays for ParA and its mutant K78A. Monitoring of growth of the M.

smegmatis wildtype , MsParA deletion strain and K78A complementation strain in 7H9 medium by CFU analysis as described under Materials and Methods. Effects of MsTAG on MsParA ATPase activity.