A protein essential for recovering oxygen evolution in cholate-treated chloroplasts

A novel method for the reconstitution of oxygen evolution in cholate-extracted spinach thylakoid membranes was established and a protein essential for the reconstitution was purified from cholate extracts. Purification of the protein was accomplished by chromatography on a DEAE-Sephacel column. This protein (M_r 17 000) was reinserted into vesicular membranes reconstituted from cholate-extracted thylakoids in the presence of 25% glycerol to reactivate oxygen evolution.     

Chloride flux in bilayer membranes: chloride permeability in aqueous dispersions of single-walled, bilayer vesicles.

Aqueous dispersions of phosphatidylcholine vesicles were utilized to determine bilayer permeability to 36-Cl as a function of pH and temperature. These dispersions were comprised of single-walled vesicles, homogeneous in size, prepared by sonication of purified egg phosphatidylcholine under argon followed by fractionation on a molecular sieve. Permeability constants calculated from the inward flux of 36-Cl and the geometric parameters of these vesicles proved to be dependent on both pH and temperature. Analysis of these dependences leads to the conclusion that 36-Cl permeation in the presence of KCl is due principally to a carrier mediated exchange process involving a phospholipid-HCL complex. Net permeation by H-36-Cl may make a small contribution to the 36-Cl flux, however, studies carried out at very low chloride concentrations show that this flux is much smaller than the exchange flux. Thus chloride permeability for the exchange process is 1.5 times 10- minus 11 cmsec- minus 1 while the corresponding coefficient for the net flux of H-36-Cl is 1.0 times 10- minus 12 cm sec- minus 1 at pH 7. The activation energy for the 36-Cl exchange flux was found to be 19 plus or minus 2 kcal/mol. This value is similar to that obtained for the transbilayer "flip-flop" of phosphatidylcholine molecules in a similar system (Kornberg and McConnell, 1971). This correspondence together with the fact that the experimentally determined flux of 36-Cl agrees well with that calculated from the "flip-flop" parameters, strongly suggests that the flux of 36-Cl and "flip-flop" of phosphatidylcholine may be the same process.     

Cholesterol inhibition of the temporary increase of membrane fluidity of lymphocytes induced by mitogenic lectins

The mitogenic response of human peripheral lymphocytes to lectins can be decreased by brief treatment of the cells with lecithin-cholesterol liposomes. This fact indicates that the temporary increase of membrane fluidity, which occurs within 30 min after addition of mitogenic lectins, is an important early event for the subsequent activation of lymphocytes. This temporary increase of membrane fluidity is accompanied by neither a decrease in cellular cholesterol level nor by particular acceleration of the incorporation of polyunsaturated fatty acids into phospholipids. These facts suggest that this change in membrane fluidity is not due to the alteration of membrane lipid composition, but can be regarded as a result of temporary perturbation of membrane lipid bilayers induced by binding of the lectins to their membrane receptors.     

Stereochemistry of Cervicarcin

The relative stereochemistry of cervicarcin, an antitumor antibiotic, was determined as shown in 1, which represents the absolute stereochemistry also.     

Phototropin‐ and photosynthesis‐dependent mitochondrial positioning in Arabidopsis thaliana mesophyll cells

Mitochondria are frequently observed in the vicinity of chloroplasts in photosynthesizing cells, and this association is considered necessary for their metabolic interactions. We previously reported that, in leaf palisade cells of Arabidopsis thaliana, mitochondria exhibit blue‐light‐dependent redistribution together with chloroplasts, which conduct accumulation and avoidance responses under the control of blue‐light receptor phototropins. In this study, precise motility analyses by fluorescent microscopy revealed that the individual mitochondria in palisade cells, labeled with green fluorescent protein, exhibit typical stop‐and‐go movement. When exposed to blue light, the velocity of moving mitochondria increased in 30 min, whereas after 4 h, the frequency of stoppage of mitochondrial movement markedly increased. Using different mutant plants, we concluded that the presence of both phototropin1 and phototropin2 is necessary for the early acceleration of mitochondrial movement. On the contrary, the late enhancement of stoppage of mitochondrial movement occurs only in the presence of phototropin2 and only when intact photosynthesis takes place. A plasma‐membrane ghost assay suggested that the stopped mitochondria are firmly adhered to chloroplasts. These results indicate that the physical interaction between mitochondria and chloroplasts is cooperatively mediated by phototropin2‐ and photosynthesis‐dependent signals. The present study might add novel regulatory mechanism for light‐dependent plant organelle interactions.     

Influences of the priming procedure and saline circulation conditions on polyvinylpyrrolidone in vitro elution from polysulfone membrane dialyzers

In hemodialysis (HD), the patient's blood is purified via circulation in an extracorporeal circuit containing a dialyzer. In the manufacturing process of polysulfone (PSu) membrane dialyzers, the membranes are hydrophilized via the addition of the hydrophilic agent polyvinylpyrrolidone (PVP) to increase their hydraulic permeability. The elution of PVP from the membrane reduces the membrane's hydraulic permeability, and the eluted PVP could cause adverse effects in the human body. Therefore, it is important to identify the factors that induce PVP elution from PSu dialyzer membranes to improve the efficiency and safety of HD. In the present study, experimental circuits connecting each of the three types of PSu membrane dialyzers that had been sterilized, using gamma irradiation, autoclaving, or in-line steam methods, were prepared. After the dialyzers were primed, saline was circulated in the circuits at a flow rate of 100 mL/min or 200 mL/min. At 0, 2, 4, 6, and 8 h after circulation was initiated, the amount of PVP eluted from the PSu membranes in vitro was determined. In this experimental setting, longer the circulation duration, greater the amount of PVP eluted from the PSu membranes of the tested dialyzers; however, the flow rate did not influence the in vitro elution of PVP. Furthermore, the immersion of the dialyzer membranes in saline for 24 h strongly facilitated the in vitro elution of PVP. In sum, these results suggest that the duration of PSu membrane incubation in saline is a determinant of the level of PVP elution from the PSu membrane dialyzers.     

A Highlights from MBoC Selection: Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.