Lipid vesicle-mediated alterations of membrane cholesterol levels: Effects on Na+ and K+ currents in squid axon

Summary We demonstrate that cholesterol can exchange from sonicated lipid vesicles to a perfused squid giant axon membrane and that vesicles with varying cholesterol/phospholipid (C/P) mole ratios can be used to achieve either net loading or net depletion of axon membrane cholesterol. Two types of evidence were obtained which show that net loading or depletion of cholesterol was achieved: (i) changes in the cholesterol/phospholipid (C/P) mole ratios of axons, and (ii) visualization of cholesterol depleted from the preparation by cholesterol-free vesicles by thin-layer chromatography. The C/P mole ratios indicate that cholesterol levels in the preparation were increased or decreased by 30–40%. Increasing or decreasing membrane cholesterol levels were ineffective in altering the Na⁺ or K⁺ occurrents in voltage-clamped axons. In addition, we determined that cholesterol flip-flop across the axonal membrane occurred with at ^1/2 of 7.3 to 15.3 min.     

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group^1-3. JoVE video articles describing the method are available^1,4. The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group^5,6. The other two have recently become available and are included here for completeness. An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions.     

Ca−K Bi-ionic action potential in squid giant axons

Summary Under intracellular perfusion with a solution containing K⁺ as the sole cation species, squid giant axons were found to be capable of developing all-or-none action potentials when immersed in a medium in which CaCl₂ was the only electrolyte. The adequate range of ion concentration for demonstrating this capability was mentioned. The reversal potential level measured by the voltage-clamp technique varied directly with the logarithm of the concentration of extracellular Ca-ion; the proportionality constant was close toRT/2F. The action potential observed under this Ca–K bi-ionic condition could not be suppressed by addition of tetrodotoxin or saxitoxin to the external medium. The external Ca-ion could be replaced with Co- or Mn-ion without eliminating the capability of the axons to develop action potentials. D-600 could not suppress the inward current observed under the voltage-clamp condition, but 4-aminopyridine could suppress it. The experimental findings were interpreted based on the current channel hypothesis and on the macromolecular theory.     

Use of a LightCycler gyrA mutation assay for identification of ciprofloxacin-resistant Campylobacter coli

An outbreak caused by dried processed squids contaminated with Salmonella Oranienburg occurred in Japan in 1999. Isolates obtained from the causative food were resistant to NaCl osmotic stress, but isolates from the patients were sensitive to NaCl. Although strains from both sources were almost identical in their virulence in mice, a NaCl-resistant strain from food (Sa9911T) became NaCl-sensitive after passage in mice and a NaCl-sensitive strain from one patient (Sa99004) retained NaCl sensitivity after such passage. When dried squid was contaminated experimentally with both strains during processing, only Sa9911T was recovered directly from the final product. Nevertheless, the viability of the Sa99004 cells was over 90% found by fluorescent staining. We suggested that Sa99004 might become viable but non-culturable (VNC) by NaCl stress. This hypothesis was confirmed by resuscitation by efficient enrichment. We concluded that VNC S. Oranienburg would be potentially dangerous contaminants of NaCl-preserved foods and that measures to ensure their detection should be taken at the time of food inspection.