Tetsuhiko Ohba

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: Evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis

Acyl‐lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl‐lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step‐wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri‐ and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.

Incorporation of impurity to a tetragonal lysozyme crystal

Abstract Concentration of a phosphor-labeled impurity (ovalbumin) incorporated into protein (hen egg white lysozyme) crystals during growth was measured by fluorescence.This technique enabled us to measure the local impurity concentration in a crystal quantitatively. Impurity concentration increased with growth rate, which could not be explained by two conventional models (equilibrium adsorption model and Burton–Prim–Slichter model); a modified model is proposed. Impurity concentration also increased with the pH of the solution. This result is discussed considering the electrostatic interaction between the impurity and the crystallizing species.

Microscopic Imaging of Phase Separation in Giant Liposome by Using Laurdan at Video Rate

Laurdan is a useful fluorescence dye for observation of physical properties of membranes. An optical system was constructed in order to image generalized polarization (GP), that characterizes the change of the spectrum of laurdan. The fluorescence images through the optical system were recorded on a digital video recorder and the images were processed to GP images, which made it possible to measure the spatial distribution of membrane fluidity quantitatively at video rate. Using this imaging instrument, the change of membrane fluidity at every moment of phase transition was observed in a giant liposome of pure dimyristoylphosphatidylcholine and the dynamics of phase separation was observed in a giant liposome composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine after an annealing process. A clear phase separation was observed in the giant liposome in the cooling process from 43 to 40°C. The GP value of the phase-separated region on the liposome was related to the shape of the same region. The linear part of the liposome had a high GP value and the hinge part had a low GP value.

Flow cytometric quantitation of EpCAM-positive extracellular vesicles by immunomagnetic separation and phospholipid staining method

Extracellular vesicles (EV) have attracted attention as circulating biomarkers for many diseases, particularly cancer. Conventional immunofluorescence staining has been used for the detection of target antigens on EV by flow cytometry. However, the staining intensity depends on the amount of antigen expressed on the vesicles and is often only around the noise level. Instead of immunofluorescence, we combined immunomagnetic separation using nanosize MACS® MicroBeads with phospholipid staining of EV (IMS‐PS method). EpCAM‐positive EV were prepared from the culture supernatants of OVCAR3 (EpCAM‐high), A431 (EpCAM‐low) or Colon‐26 (non‐human control) cells as cancer models and were examined by the IMS‐PS method using EpCAM mAb‐coated MicroBeads. By employing Polaric‐500c6F as the dye for staining EV phospholipids and using appropriate flow cytometry settings, autofluorescence was excluded, whereas pretreatment of the MicroBeads with conventional blocking agents reduced nonspecific binding to non‐target vesicles. These modifications resulted in a linear relation between the number of EV detected and the sample volume, regardless of the level of EpCAM expression on the vesicles. A431 EV spiked into healthy volunteer plasma were enumerated with good accuracy. The IMS‐PS method may be useful for clinical evaluation of EV with low levels of antigen expression that are difficult to detect by conventional immunofluorescence.