The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles—Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive—was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics.

We have characterized the expression and secretion of the acute kidney injury (AKI) biomarkers insulin-like growth factor binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in human kidney epithelial cells in primary cell culture and tissue. We established cell culture model systems of primary kidney cells of proximal and distal tubule origin and observed that both proteins are indeed expressed and secreted in both tubule cell types in vitro. However, TIMP-2 is both expressed and secreted preferentially by cells of distal tubule origin, while IGFBP7 is equally expressed across tubule cell types yet preferentially secreted by cells of proximal tubule origin. In human kidney tissue, strong staining of IGFBP7 was seen in the luminal brush-border region of a subset of proximal tubule cells, and TIMP-2 stained intracellularly in distal tubules. Additionally, while some tubular colocalization of both biomarkers was identified with the injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, both biomarkers could also be seen alone, suggesting the possibility for differential mechanistic and/or temporal profiles of regulation of these early AKI biomarkers from known markers of injury. Last, an in vitro model of ischemia-reperfusion demonstrated enhancement of secretion of both markers early after reperfusion. This work provides a rationale for further investigation of these markers for their potential role in the pathogenesis of acute kidney injury.

Azobenzenes can function as molecular switches driven by their unusual cis <--> trans photoisomerization properties. The stability of an azobenzene-based switch depends on its rate of thermal relaxation, which is known to depend on the solvent environment, but few kinetic studies in aqueous media have been reported. We use nanosecond UV laser flash photolysis-transient absorption spectroscopy to measure thermal cis --> trans isomerization rates for mono- and disubstituted p-aminoazobenzenes and p-hydroxyazobenzenes in water at 23 degrees C over the pH range of 4 to 11. Observed absorption transients are fit to first-order relaxation rate constants between 10(5) and 10(1) s(-1), which is generally much faster than in nonpolar solvents, and the relaxation rates vary systematically and predictably with pH as the equilibrium shifts to ionized forms of the dyes that isomerize much more rapidly. Acid ionization constants for these dyes determined from our kinetic mechanism are compared with the pH dependence of their equilibrium UV-vis spectra. New kinetics results may enable pH control of azobenzene-based molecular switching times.

Using fluorescence microscopy we have tracked the cellular binding, surface motion, and internalization of polyarginine and polyethylenimine, cationic ligands used for gene and protein delivery. Each ligand was complexed with a quantum dot to provide a photostable probe. Transfection with exogenous DNA was used to relate the observed motion to gene delivery. Cell surface motion was independent of sulfated proteoglycans, but dependent on cholesterol. Cellular internalization required sulfated proteoglycans and cholesterol. These observations suggest that sulfated proteoglycans act as cellular receptors for the cationic ligands, rather than only passive binding sites. Understanding the interaction of polyarginine and polyethylenimine with the plasma membrane may assist in designing more efficient gene delivery systems.

Improved measurements of the radiative lifetimes of NO A Σ2+(v′=0,1,2) are presented and used to update the absolute electronic transition moment for the NO γ bands. The pressure-dependent fluorescence decay rate was measured in a low-pressure, room-temperature, flow cell containing dilute mixtures of NO in N2 using time-resolved laser-induced fluorescence excited with a picosecond laser and detected with a microchannel-plate photomultiplier tube. Fluorescence decay rates were determined using an analysis procedure that accounted for the electronic response of the detection system and measurement noise. Radiative lifetimes were determined from an extrapolation of the measured decay rates to zero pressure. In comparison with prior measurements of these radiative lifetimes, the improved experimental approach and analysis procedure result in a significant improvement in the measurement precision. The accuracy of the fluorescence decay-rate measurements was confirmed by independent measurements using time-correlated single-photon counting and time-resolved probing of laser-excited population in A Σ2+ using 266 nm photoionization and charge detection. The measured radiative lifetimes are 192.6±0.2 ns for v′=0, 186.2±0.4 ns for v′=1, and 179.4±0.7 ns for v′=2. The measured lifetimes are shown to be in outstanding agreement with those predicted by an electronic transition moment that is identical in form to the function recommended by Luque and Crosley [J. Chem. Phys. 111, 7405 (1999)] after appropriate rescaling. This rescaling does not affect the agreement of the transition moment function with the previously reported vibrational branching ratios and improves agreement with previously reported absolute oscillator strength measurements. Based on the rescaled transition moment, updated values of absolute transition probabilities in the NO A Σ2+−X Π2 system are presented.