The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized.

BackgroundTubercle bacilli are thought to persist in a dormant state during latent tuberculosis (TB) infection. Although little is known about the host factors that induce and maintain Mycobacterium tuberculosis (M. tb) within latent lesions, O2 depletion, nutrient limitation and acidification are some of the stresses implicated in bacterial dormancy development/growth arrest. Adaptation to hypoxia and exposure to NO/CO is implemented through the DevRS/DosT two-component system which induces the dormancy regulon.Methodology/Principal FindingsHere we show that vitamin C (ascorbic acid/AA) can serve as an additional signal to induce the DevR regulon. Physiological levels of AA scavenge O2 and rapidly induce the DevR regulon at an estimated O2 saturation of <30%. The kinetics and magnitude of the response suggests an initial involvement of DosT and a sustained DevS-mediated response during bacterial adaptation to increasing hypoxia. In addition to inducing DevR regulon mechanisms, vitamin C induces the expression of selected genes previously shown to be responsive to low pH and oxidative stress, triggers bacterial growth arrest and promotes dormancy phenotype development in M. tb grown in axenic culture and intracellularly in THP-1 cells.Conclusions/SignificanceVitamin C mimics multiple intracellular stresses and has wide-ranging regulatory effects on gene expression and physiology of M. tb which leads to growth arrest and a ‘dormant’ drug-tolerant phenotype, but in a manner independent of the DevRS/DosT sytem. The ‘AA-dormancy infection model’ offers a potential alternative to other models of non-replicating persistence of M. tb and may be useful for investigating host-‘dormant’ M. tb interactions. Our findings offer a new perspective on the role of nutritional factors in TB and suggest a possible role for vitamin C in TB.

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Viral fusion proteins of classes I and II differ radically in their initial structures but refold toward similar conformations upon activation. Do fusion pathways mediated by alphavirus E1 and influenza virus hemagglutinin (HA) that exemplify classes II and I differ to reflect the difference in their initial conformations, or concur to reflect the similarity in the final conformations? Here, we dissected the pathway of low pH–triggered E1-mediated cell–cell fusion by reducing the numbers of activated E1 proteins and by blocking different fusion stages with specific inhibitors. The discovered progression from transient hemifusion to small, and then expanding, fusion pores upon an increase in the number of activated fusion proteins parallels that established for HA-mediated fusion. We conclude that proteins as different as E1 and HA drive fusion through strikingly similar membrane intermediates, with the most energy-intensive stages following rather than preceding hemifusion. We propose that fusion reactions catalyzed by all proteins of both classes follow a similar pathway.