60-Hz electric field exposure inhibits net apparent H(+)-ion excretion from excised roots of Zea mays L

Plant root model cell systems have provided insight into the biophysical mechanism by which extremely low frequency electric fields (EF; f less than or equal to 100 Hz) affect nonexcitable eukaryotic cells. The evidence indicates that the plasma membrane is the site of interaction with applied extremely low frequency EF, and that cells respond to field exposure via a sensing mechanism involving the induction of extremely low frequency membrane potentials (Vim). We suggest a mechanism by which Vim may be transduced into EF-induced root growth inhibition. Suspensions of excised Zea root tips were used to test the hypothesis that growth-inhibiting extremely low frequency EF exposures inhibit net H+ excretion from protoplasts, a process mediated by a plasma membrane H(+)-ATPase which is intimately involved in cellular extension. Rates of acidification of root tip suspensions were measured as an analog for net H+ efflux. The experimental results support this hypothesis. At the apparent threshold for inhibition of H+ excretion, the associated 60-Hz EF strength was about 220 V.m-1 (root mean square). Estimates of Vim associated with inhibition of net H+ excretion are in agreement with those known to affect Na+/K+ transport in human erythrocytes.     

Proportionality of 60-Hz electric field bioeffect severity to average induced transmembrane potential magnitude in a root model system

The postulate that electric field-induced bioeffects in the root model system are related to the induction of 60-Hz transmembrane potentials (Vim) was quantitatively tested. Root segment growth rate data and the calculated mean 60-Hz Vim which would arise in the cortical cells of a segment under specified exposure conditions were subjected to regression analysis. Statistically significant correlations between segmental growth rate and segmental-average Vim were obtained using data analyzed (1) within species at a constant applied field strength, (2) within species and pooled across field exposures, and (3) pooled across both species and exposures. In C. sativus roots, segmental growth is inhibited when segmental-average Vim attain a value of 3.4-3.6 mV. In C. maxima roots, growth inhibition occurs when Vim attain or exceed 2.3-2.7 mV. Segmental growth cessation is predicted to occur when segmental-average Vim exceed 7-9 mV.     

Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25 pM with the linearity of the standard curve spanned to 2500 pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.     

Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.