The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids

Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC₅₀ ∼ 70 μM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a K_i of ≈20 μM.     

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope¹. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns². The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam^3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell^6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 µm/sec, typically displaying a directed motion along the microtubule network⁸.     

Substrate supply for calcite precipitation in Emiliania huxleyi: assessment of different model approaches

Over the last four decades, different hypotheses of Ca²⁺ and dissolved inorganic carbon transport to the intracellular site of calcite precipitation have been put forth for Emiliania huxleyi (Lohmann) Hay & Mohler. The objective of this study was to assess these hypotheses by means of mathematical models. It is shown that a vesicle‐based Ca²⁺ transport would require very high intravesicular Ca²⁺ concentrations, high vesicle fusion frequencies as well as a fast membrane recycling inside the cell. Furthermore, a kinetic model for the calcification compartment is presented that describes the internal chemical environment in terms of carbonate chemistry including calcite precipitation. Substrates for calcite precipitation are transported with different stoichiometries across the compartment membrane. As a result, the carbonate chemistry inside the compartment changes and hence influences the calcification rate. Moreover, the effect of carbonic anhydrase (CA) activity within the compartment is analyzed. One very promising model version is based on a Ca²⁺/H⁺ antiport, CO₂ diffusion, and a CA inside the calcification compartment. Another promising model version is based on an import of Ca²⁺ and HCO₃^? and an export of H⁺.     

Emission-wavelength-dependent decay of the fluorescent probe N-phenyl-1-naphthylamine

We have measured the fluorescence decay of $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ using the phase-modulation method, in several solvent systems and egg phosphatidylcholine vesicles. The decay is monoexponential in pure solvents (both polar and non-polar) of low viscosity. In polar viscous solvents or in non-polar solvents containing an added polar solute, the decay is heterogeneous and emission wavelength dependent. In such cases, dielectric relaxation and/or excited-state complexing give rise to a shift of the emission spectrum on the nanosecond time scale. Emission-wavelength-dependent decay was also observed when $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ was bound to egg phosphatidylcholine vesicles. From these results as well as the position of the emission spectral maximum, we conclude that $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ probes the ester-carbonyl region of the phospholipid acyl chains, where it undergoes an excited-state reaction. This result contradicts the often made assumption that $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ probes the deeper hydrocarbon region of the bilayer.     

Increased Na pump activity in the kidney cortex of the Milan hypertensive rat strain

The (Na+,K+)-ATPase activity from the kidney cortex of the Milan hypertensive rat strain (MHS) and the corresponding normotensive control (MNS) was measured both in active solubilized enzyme preparations and in isolated basolateral membrane vesicles. Kinetic analysis of the purified enzyme showed that the Vmax value was significantly higher in MHS rats. The difference between MHS and MNS was not linked to a different number of sodium pumps, but was related to the molecular activity of the enzyme. Using basolateral membrane vesicles, an increased ATP-dependent ouabain-sensitive sodium transport was also demonstrated in MHS rats. These results support the hypothesis that a higher tubular sodium reabsorption may be involved in the pathogenesis of hypertension in this rat strain.     

Transition of a liquid crystalline phosphatidylcholine bilayer to the gel phase in a vesicle reduces the internal aqueous volume

The liquid crystalline to gel phase transition in phospholipid bialyers is associated with a marked reduction in the area per phospholipid molecule. Geometric considerations based on published data suggest that this decrease in molecular area is accompanied by a reduction in the internal aqueous volume trapped within a unilamellar bilayer vesicle. This volume reduction, which depends upon the shape of the vesicle, is shown to be between 23 and 60 percent. We have observed a 25 to 30 percent reduction in the internal aqueous volume of unilamellar vesicles about 700 Å in diameter formed from dipalmitoylphosphatidylcholine using the self-quenching of 6-carboxyfluorescein trapped within this compartment.     

K+-dependent phenylalanine uptake in membrane vesicels isolated from the midgut of Philosamia cynthia larvae

Membrane vesicles prepared from the midguts of Philosamia cynthia larvae (Lepidoptera) show a concentrative uptake of phenylalanine in the presence of salt gradients. Unlike mammalian intestines, the highest accumulation of the amino acid occurs with a potassium salt gradient. Glucose is very poorly permeable across the vesicles.     

The effect of surface curvature on the head-group structure and phase transition properties of phospholipid bilayer vesicles

Proton nuclear magnetic resonance spectra at 360 MHz of small sonicated distearoyl phosphatidylcholine vesicles show easily distinguishable resonances due to choline N-methyl head-group protons located in the inner and outer bilayer halves. A study of the chemical shift of these resonances as a function of temperature reveals that the splitting between them increases below the phase transition. This occurs as a result of an upfield shift of the inner layer resonance at the phase transition. Consideration of the possible causes of this effect results in the conclusion that, at the phase transition, there is a change in the organization of the inner layer head-groups which does not occur for the outer layer head-groups.     

Determination of the electric potential at the external and internal bilayer-aqueous interfaces of the human erythrocyte membrane using spin probes

Partitioning of oppositely charged amphipathic spin probes indicates that the electric potential at the external bilayer-aqueous interface of the human erythrocyte is insignificant, and that protruding sialic acids do not contribute to this potential. This potential at the surface is distinguished from the electrokinetic potential due to all charged groups within the hydrodynamic surface of shear. By contrast, using inside-out erythrocyte membrane vesicles, a substantial potential is observed at the cytoplasmic membrane surface. This can be attributed to the asymmetric distribution of acidic phospholipids on the two sides of the erythrocyte membrane bilayer.