Molecular Resolution of Gram Positive Bacteria Cell Wall using AFM

Abstract

To determine which of P-gp's two binding sites interact with oligomeric Ab, a competition binding assay was performed. Rhodamine 123 (R123) and Hoechst 33342 (H33342) are fluorescent compounds with well-described alternate P-gp binding sites. The transport of each R123 and H33342 is evidenced by a quenching of fluorescence; this transport is impeded by the presence of a compound that binds to the same site. Inverted vesicles are incubated in the presence of both oligomeric Ab and either R123 or H33342 for identification of the binding site. This study demonstrates that the size of Ab aggregate species plays a crucial role in Ab binding to P-gp for transport and identifies the probable P-gp binding site of Ab. Fast uncaging of low affinity competitive receptor antagonists can in principle measure the timing and concentration dependence of transmitter action at receptors during synaptic transmission, distinguishing fast, independent contacts from diffuse multisite transmission. MNI-caged g-D-glutamyl-glycine combines the fast photolysis and hydrolytic stability of nitroindoline cages with the fast-equilibrating competitive glutamate receptor antagonist g-DGG. Its suitability as a caged probe was tested at climbing fiber-Purkinje cell (CF-PC) synapses. MNI-caged g-DGG applied at 5 mM permitted release of up to 2 mM g-DGG in 1 ms in wide-field flashlamp photolysis. In steady-state conditions, photoreleased g-DGG at 1 mM inhibited the CF first and second paired EPSCs by 30% and 60% respectively, similar to bath applied g-DGG. Photolysis of the L-isomer MNI-caged g-L-glutamylglycine was ineffective. The time-course of receptor activation was investigated by photolysis of MNI-caged-g-DGG at defined intervals following CF stimulation in the second EPSC of a paired pulse protocol. Photolysis prior to the stimulus and up to 3 ms after showed strong inhibition similar to steady-state; photolysis at 4 ms post-stimulus produced weaker and highly variable block, suggesting transmitter-receptor interaction occurs mainly in the interval 3-4 ms post-stimulus. This indicates an unexpected delay between stimulus and release. The data show a late component of inhibition when g-DGG was released at 5 ms or 6 ms post stimulus, near the peak of the CF-PC EPSC, or after the peak at 10 ms. This indicates competition for receptor activation during the late phase of the EPSC, due to delayed transmitter release or persistence of glutamate in the synapse. MNI-cagedg-DGG appears suitable as a synaptic probe at high concentration, and its photolysis can resolve timing and extent of receptor activation to investigate synaptic independence.