Characterization of liposomes prepared using a microemulsifier

A new type of device can prepare liposomes continuously, in large quantities and with excellent aqueous space capture efficiency. At initial lipid concentration of 300 μmol/ml these liposomes capture approx. 75% of cytosine arabinoside used as an aqueous space marker. Liposome size can be reduced by increasing the number of times the preparations are recycled through the microemulsifier. Liposomes less than 0.1 μm in diameter, as shown by electron microscopy, can be made easily. Liposomes prepared at 300 μmol/ml, composed of phosphatidylglycerol/phosphatidylcholine/cholesterol in a 0.1:0.4:0.5 molar ratio leaked less than 1% of entrapped cytosine arabinoside (Ara-C) at 4°C, and less than 10% Ara-C at 37°C plus serum, over a 48 h period. These liposomes could be useful for a number of applications including diagnostics, therapeutics and model membrane studies.     

Dehalogenation in marine sediments containing natural sources of halophenols

Halophenols such as 2,4-dibromophenol (DBP) occur naturally in some marine sediments, as a consequence of various animal and algal activities. In an earlier study, DBP was observed in the burrow microenvironment of the hemichordate Saccoglossus kowalewskii. At the concentrations found in the burrow lining, aerobic respiration appeared to be inhibited significantly relative to anaerobic catabolism. This effect, as well as factors contributing to the degradation of DBP, has been documented further here. Results from the addition of radiolabeled DBP to oxic and anoxic sediment slurries and growth experiments with aerobic and anaerobic enrichments suggested that aerobes did not significantly metabolize DBP and that concentrations likely to be encountered on the inner surfaces of the burrow wall were inhibitory. In contrast, only minimal inhibition of growth occurred for anaerobes exposed to 1 mM DBP; in addition, DBP was substantially degraded in both enrichments and sediments under anaerobic conditions. Dehalogenation with the consequent production of phenol appeared to initiate anaerobic degradation. Sulfate-reducing bacteria did not dehalogenate DBP but appeared to degrade phenol. Decreased bacterial numbers and marked differences in the concentration and chemical speciation of iron in sediments from S. kowalewskii burrows may be attributed to toxic effects of DBP on aerobic bacteria.     

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing

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Glycerol reorientation during the conversion of phosphatidylglycerol to bis(monoacylglycerol)phosphate in macrophage-like RAW 264.7 cells

Bis(monoacylglycero)phosphate (BMP) has the unique stereoconfiguration of 3-acyl-sn-glycero-1-phosphoryl-1'-sn-[3'-acylglycerol] (Brotherus, J., Renkonen, O., Herrmann, J., and Fischer, W. (1974) Chem. Phys. Lipids 13, 178-182) which differs from other known mammalian phospholipids that have the sn-glycero-3-phosphoryl configuration. This stereochemistry may contribute to its physiologic function. Here we describe studies using the macrophage-like cell line RAW 264.7 designed to determined how this unique stereoconfiguration occurs. These studies show that the stereoconfiguration of BMP produced from exogenous phosphatidylglycerol (PG) by RAW 264.7 cells has the expected stereoconfiguration of 3-acyl-sn-glycero-1-phosphoryl-1'-sn-[3'-acylglycerol]. Experiments using diacyl-sn-[2-3H]glycero-3-phosphoryl-sn-1'-[2-3H]glycerol demonstrate that this unique stereoconfiguration is not produced due to an oxidation/reduction mechanism involving the sn-2-glycerol carbon. When dioleoyl-sn-[1-14C]glycero-3-phosphoryl-rac-glycerol was converted to 14C-labeled BMP, the 14C label was found esterified to the phosphate moiety. These results suggest that a stereospecific enzyme is capable of reorienting the radiolabeled glycerol backbone of this PG substrate, effectively changing the stereochemistry of the lipid. We also show that this enzyme is stereoselective with regard to the base glycerol moiety of the substrate PG used. Finally, we propose a new pathway for the synthesis of BMP from PG.     

The conformation of cross-linked actin.S-1 in the presence and absence of ATP

Electron microscopy studies have shown that the structure of the complex of myosin subfragment 1 (S-1) cross-linked to actin with 1-ethyl-3-[3-(dimethyl-amino) propyl] carbodiimide is very different in the presence and absence of ATP (Craig, R., Greene, L. E., and Eisenberg, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3247-3251). More recent studies have found that the structure of the cross-linked complex between S-1 modified extensively with N-ethylmaleimide (NEM.S-1) and actin resembles that of the rigor complex both in the presence and absence of ATP, whereas the structure of the cross-linked complex between S-1 modified with N',N'-p-phenylenedimaleimide (pPDM.S-1) and actin resembles that of the cross-linked actin.S-1 complex in the presence of ATP. In the present study, we have obtained biochemical evidence supporting these results. The conformation of the different cross-linked actin.S-1 complexes was determined by studying their effect on the troponin-tropomyosin-actin complex (regulated actin). The basis of this probe for conformation is that S-1.ATP, which is in the weak-binding conformation, interacts very differently with regulated actin than S-1 or S-1.ADP, which are in the strong-binding conformation. We find that both in the presence and absence of ATP, cross-linked NEM.S-1 appears to be in the strong-binding conformation, whereas cross-linked pPDM.S-1 appears to be shifted toward the weak-binding conformation. In contrast, cross-linked unmodified S-1 appears to be in the strong-binding conformation in the presence of ADP and the weak-binding conformation in the presence of ATP. Therefore, in agreement with electron microscopy studies, the cross-linked actin.S-1 complex appears to be able to alternate between the weak-binding and strong-binding conformation during the cross-bridge cycle.     

Head group and chain length dependence of phospholipid self-assembly studied by spin-label electron spin resonance

The critical micelle concentrations (cmc's) of a variety of spin-labeled phospholipids, 1-acyl-2-[4-(4,4-dimethyloxazolidine-N-oxyl)valeryl]-sn-glycero-3-pho sph o derivatives, have been determined by electron spin resonance (ESR) spectroscopy. The narrow, three-line ESR spectra of the rapidly tumbling monomers are clearly distinguished from the spin-spin broadened spectra of the micellar aggregates, allowing a direct determination of the concentrations of the two species. The influence of both the hydrocarbon chain length and the polar head group on the energetics of self-assembly has been studied. For phosphatidylcholine, 1n [cmc] decreases linearly with the length of the sn-1 chain. The gradient of this linear dependence corresponds to a free energy of transfer of the monomer from the aqueous phase to the micelle of delta Gtr = -1.1RT per CH2 group. The cmc's of the 1-lauroyl derivatives of both phosphatidylcholine and phosphatidylglycerol have relatively shallow, biphasic temperature dependences with a minimum at approximately 20 degrees C. Both of these properties are characteristic of the hydrophobic effect, with the free energy of transfer being slightly less than that for the solubility of n-hydrocarbons in water, corresponding to the reduced configurational entropy of the lipid chains in the micellar state. The cmc's of the 1-lauroyl derivatives of the phospholipids in 0.15 M NaCl, for their various charge states, are as follows: phosphatidic acid(2-), 0.77 mM; phosphatidic acid(1-), 0.13 mM; phosphatidylserine(1-), 0.24 mM; phosphatidylglycerol(1-), 0.17 mM; phosphatidylcholine, 0.10 mM; phosphatidylethanolamine, 0.05 mM.(ABSTRACT TRUNCATED AT 250 WORDS)